Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Structural comparison of Cas12k-transposon recruitment and Cas12k-TnsC non-productive complexes, related to Figures 1 , , and (A) Cryo-EM density maps of the Cas12k-transposon recruitment complex (top) and the Cas12k-TnsC non-productive complex (bottom). Side views and structural superpositions are shown. Proteins are shown in surface representation. The DNA in the transposon recruitment complex is bent by ∼56° relative to the non-productive complex. (B) Atomic models, shown in surface representation, of the Cas12k-transposon recruitment complex (top) and the Cas12k-TnsC non-productive complex (bottom).

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: R-loop completion upon complex assembly (A) Detailed views of the R-loop structure in the Cas12k-transposon recruitment complex, comprised of the crRNA portion of the single guide RNA (red cartoon backbone), the TS (blue cartoon backbone), and the NTS (dark gray cartoon backbone). Only the REC lobe, the RuvC domain, and the bridging helix (BH) of Cas12k are shown for clarity. (B) Detailed views of the R-loop structure in the Cas12k-sgRNA-target-DNA complex (PDB: 7PLA ). (C) Zoomed-in view of the PAM-distal end of the R-loop in the Cas12k-transposon recruitment complex. Density corresponding to nucleic acids is shown (contour level of 7.7 σ). TniQ is depicted as surface representation. See also Figure S4 .

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques:

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Structural rearrangements in Cas12k and guide RNA upon R-loop completion, related to Figure 2 (A) Structural models of the Cas12k-sgRNA-target DNA complex (PDB: 7PLA , top) and the Cas12k-transposon recruitment complex bottom), shown in the same orientation. Domain architecture of Cas12k is shown below each model. REC, recognition lobe. WED, wedge domain. PI, PAM interacting domain. BH, bridging helix. TS, target DNA strand; NTS, non-target DNA strand. (B) Structural superpositions of the RuvC and BH domains in the Cas12k-sgRNA-target DNA and Cas12k-transposon recruitment complexes. (C) Structural superposition of the tracrRNA part of the sgRNA in the Cas12k-sgRNA-target DNA (gray) and the Cas12k-transposon recruitment complex (orange).

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques:

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: TniQ recognizes tracrRNA and completed R-loop (A) Overview of TniQ in the Cas12k-transposon recruitment complex, depicting interfaces with the tracrRNA (orange) and the RNA:DNA heteroduplex formed by the crRNA (red) and the TS (blue). NTS is colored in dark gray. The N and C termini of TniQ are indicated. (B) Close-up view of key tracrRNA-interacting residues of TniQ. (C) Site-specific transposition activity in E. coli of ShCAST systems containing structure-based mutations in the tracrRNA or the tracrRNA-interacting interface in TniQ, as determined by droplet digital PCR (ddPCR) analysis. Data are presented as mean ± SD (n = 3 biologically independent replicates). (D) Detailed view of R-loop recognition by TniQ. (E) Site-specific transposition activity in E. coli of ShCAST systems containing structure-based mutations in the R-loop recognition interface of TniQ. Data are presented as mean ± SD (n = 3 biologically independent replicates).

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Activity Assay, Digital PCR

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Cryo-EM analysis of TniQ-capped TnsC filament, related to Figures 5 and and STAR Methods (A) Cryo-EM image processing workflow for the TniQ-capped TnsC filament complex. (B) Fourier Shell Correlation (FSC) of TnsC-DNA-TniQ reconstruction from two independently refined half-maps. The gold-standard cut-off (FSC = 0.143) is marked with a blue line. (C) Final electron density map colored according to the local resolution. (D) Fourier Shell Correlation (FSC) of the reconstruction from two independently refined half-maps.

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Structural comparisons of TniQ-capped TnsC filament and Cas12k-transposon recruitment complex, related to Figures 5 and and Table S1 (A) Side and top views of the TniQ-capped TnsC filament. Proteins are shown in surface representation. (B) Side and top views of the Cas12k-transposon recruitment complex, with TniQ shown in the same orientation as TniQ1 in (A). In the top view, Cas12k, S15 and tracrRNA are omitted to visualize the contacts between TniQ and TnsC. (C) Structural overlay of three consecutive TnsC protomers (TnsC1-TnsC3) in the Cas12k-transposon recruitment complex (colored protomers, white DNA) and in the TniQ-capped TnsC filament (gray protomers and DNA) and the associated DNA (shown in stick representation).

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques:

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: TnsC assembly on PAM-distal end of R-loop DNA (A) Overview of guide-target R-loop structure within the Cas12k-transposon recruitment complex. TS (blue) and NTS (dark gray) are shown in cartoon format. Only the crRNA portion of the single-guide RNA (red) is shown. Proteins are shown in surface representation. Residues 132–254 of Cas12k and the TnsC2 and TnsC3 protomers are omitted from view for clarity. (B) Zoomed-in view of target DNA-binding residues of TniQ. (C) Zoomed-in view of the DNA-binding residues in the TnsC1 protomer. (D) Comparison of DNA binding modes of consecutive TnsC protomers (TnsC1–TnsC3) in the Cas12k-transposon recruitment complex (left) and in the TniQ-capped TnsC filament (right). See also Figure S5 .

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Binding Assay

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: S15 promotes Cas12k-transposon recruitment complex assembly and transposition activity (A) Zoomed-in view of S15 binding in the Cas12k-recruitment complex. (B) Co-precipitation of TnsC and TniQ in presence or absence of S. hofmanni S15 (ShS15) or E. coli S15 (EcS15) by immobilized Cas12k-sgRNA-target DNA complex. (C) In vitro transposition activity of purified ShCAST components in the absence or presence of EcS15 (wild-type or mutant), ShS15, and Homo sapiens RPS13 (HsS13) proteins, as determined by ddPCR analysis. Data are presented as mean ± SD (n = 4 independent replicates). Statistical analysis was conducted using unpaired two-tailed t-tests. P-values: ∗ p < 0.05; ∗∗ p < 0.01; ns, not significant. See also Figure S7 .

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Activity Assay, Binding Assay, In Vitro, Purification, Mutagenesis, Two Tailed Test

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Interactions and conservation of the ribosomal protein S15, related to Figure 6 (A) Zoomed-in view of E. coli S15 interactions with the tracrRNA and crRNA:TS-DNA duplex in the Cas12k-transposon recruitment complex. (B) Zoomed-in view of S15 contacts with the Cas12k REC2 domain. (C) Co-precipitation of E. coli S15 (EcS15) wild-type and mutant, S. hofmanni S15 (ShS15) and Homo sapiens RPS13 (HsS13) proteins by immobilized Cas12k-sgRNA complex. (D) Sequence alignment of the ribosomal proteins EcS15, ShS15 and HsS13. (E) Zoomed-in views of EcS15 interactions with tracrRNA in the Cas12k-transposon recruitment complex (left), 16S rRNA in the E. coli ribosome (middle; PDB: 6Q97 ), and a superposition of both focused on S15 (right). (F) Structural models of the Cas12k-transposon recruitment complex (left), Cas12e-sgRNA-target DNA complex (middle; PDB: 6NY2 ), and their superposition focused on S15 (right).

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Mutagenesis, Sequencing

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet: Mechanism of RNA-guided assembly in type V CASTs Mechanistic model for the recruitment of the transposition machinery by the RNA-guided Cas12k complex in type V-K CASTs. Cas12k in association with a crRNA-tracrRNA dual guide RNA initially binds target DNA to form a partial R-loop structure. Full R-loop formation occurs upon recruitment of S15, TniQ, and TnsC. TniQ recognizes specific regions of the tracrRNA and primes polymerization of a TnsC filament by bridging the first two TnsC protomers. The ribosomal protein S15 facilitates productive complex assembly by interacting with tracrRNA and Cas12k. The resulting TnsC filament provides a recruitment platform for TnsB, which triggers TnsC depolymerization to expose the insertion site and catalyzes transposon DNA insertion.

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques:

Journal: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet:

Article Snippet: The DNA sequences of S. hofmanni (Sh)Cas12k (WP_029636312.1), TnsC (WP_029636336.1), TniQ (WP_029636334.1), TnsB (WP_084763316.1), S15 (WP_029633173.1), E. coli (Ec)S15 (AP009048.1) and the Homo sapiens (Hs)RPS13 (P62277) proteins were codon optimized for heterologous expression in E. coli and synthesized by GeneArt (ThermoFisher Scientific) or IDT.

Techniques: Recombinant, Mass Spectrometry, Electron Microscopy, Plasmid Preparation, Clone Assay, Software

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A, B) STAT5 +/+ , STAT5 ΔIEC−/− , and STAT5 ΔIEC+++ mice were inoculated with 1 × 10 3 CFU C. difficile for 4 d. Histopathology of colonic and cecal inflammation was scored. Results are expressed as mean ± SEM, n ≥ 5 mice per group, ** P < 0.01 versus STAT5 +/+ , * P < 0.05 versus STAT5 +/+ . (C) Mice were inoculated with C. difficile at 1 × 10 4 CFU per mouse. Inducible depletion of STAT5 in IECs or IESCs significantly reduced survival following C. difficile infection. Survival was analyzed with Kaplan–Meier estimates, n = 7 mice per group, ** P < 0.01 versus STAT5 +/+ mice. (D) Lgr5Cre ER; VilCre ER;icS5 mice were treated with C. difficile . Lgr5 + IESCs were counted in 200 crypts in colonic mucosa, n ≥ 3 mice per group. Results are expressed as mean ± SEM, * P < 0.05 versus Lgr5Cre ER mice. Representative images of Lgr5 + IESCs in control (Con) and C. difficle colitis are shown. (E, F) The severity of ileitis was scored as neutrophil infiltration, submucosal edema, IEC necrosis, and Paneth cell or goblet cell depletion. Paneth cell depletion or expansion was semi-quantitated in C. difficile -infected STAT5 +/+ , STAT5 ΔIEC−/− , and STAT5 ΔIEC+++ mice. Histological scores show that STAT5 ∆IEC−/− mice display worse ileal inflammation than STAT5 +/+ mice, while STAT5 ∆IEC+++ mice exhibit IEC protection and more regenerated BrdU + IECs. Results are expressed as mean ± SEM, * P < 0.05 versus STAT5 ΔIEC−/− mice, n ≥ 5 mice per group. All results are expressed as mean ± SEM, and t tests and ANOVA were used to compare the significance of a difference.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Histopathology, Infection, Control

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) STAT5 +/+ and STAT5 ΔIEC−/− mice were inoculated with 1 × 10 3 CFU C. difficile for 4 d. Percentage of weight loss in the individual mice was calculated on day 4 before euthanized. Results are expressed as mean ± SEM, t tests was used to compare the significance of a difference, n ≥ 5 per group. (B) Ileal Lgr5-GFP was immune-stained in Lgr5Cre ER, Lgr5Cre ER; VilCre ER;Stat5 and Lgr5Cre ER; VilCre ER;icS5 mice. Representative images are shown, n ≥ 5 mice per group. Scale bar = 200 μm. (C) STAT5 +/+ mice were orally given with three cycles of 7-d 3% DSS treatment with an interval of 5-d water recovery between each cycle of DSS. Mucosal histology was evaluated as a total score including intestinal epithelial damage (0–3), ulceration (0–3) and transmural lesion (0–3). Colonic tissues were isolated and then immune-stained with pYSTAT5 pointed by arrowheads. Representative images are shown. Results are expressed as mean ± SEM, t tests was used to compare the significance of a difference, n = 4 or 5 mice per group.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Staining, Isolation

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: Stat5 or icS5 floxed mice were crossed with VilCre ER. VilCre ER; Stat5 or icS5 mice, and then treated with Tam for 3, 5, or 7 d followed by 5-d Cre recombination. (A) Paneth cells were determined with Lyso immunohistochemistry (IH) and IF staining. Average numbers of Lyso + Paneth cells were counted in 200 crypts, and the size of Paneth niche was measured as volume with confocal microscopy . (B) Intestinal crypts were isolated, total proteins were extracted, and immunoblotting was performed to determine Lyso and STAT5a protein expression. Densitometry was used to determine the expression of Lyso relative to β-actin. Results are expressed as mean ± SEM, n ≥ 5 mice per group. (C) Jejunal and ileal crypts were isolated and stained with Lyso IH and AB. Lyso + crypt cells were counted in 200 isolated crypts. (D) Real-time PCR was performed to determine anti-microbial peptide expression in STAT5 +/+ and STAT5 ΔIEC+++ mice. (E) Ectopic Paneth cells were counted as the number of migrated Lyso + IECs from crypt bases to villi in 3, 5, and 7 d Tam-treated VilCre ER or 5 d Tam-treated Rs26Cre ER or VilCre ;icS5 mice. Scale = 200 μm. (F) Over 200 villi and crypts were counted. Results are expressed as mean ± SEM, n ≥ 5 mice per group. (G) Colonic crypts were double-stained with anti-Lgr5 (green) and anti-cKit (red), and 3D images were captured with confocal microscopy, n > 3 mice each group. (H) Colonic crypts were disassociated with TrypLE into IECs. Lgr5-GFP − and + IECs were then separated by gating with FACS. PE-Cy7-conjugated pYSTAT5 and APC-conjugated CD44 staining were used to quantitate pYSTAT5 + Lgr5 − CD44 + colonic crypt IECs, n = 4 mice per group, ** P < 0.01 versus STAT5 +/+ . All results are expressed as mean ± SEM, and t tests and ANOVA were used to compare the significance of a difference.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Immunohistochemistry, Staining, Confocal Microscopy, Isolation, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Lgr5Cre ER mice were crossed with LacZ mice to generate a mouse line (Lgr5-LacZ), then crossed with Stat5 floxed mice (Lgr5-LacZ;Stat5). Lgr5-LacZ;Stat5 mice were used for determining the effects of Stat5 on IESC self-renewal upon a single dose of Tam. (B, C) Jejunal and ileal lineage tracing was done and analyzed 14 d after a single dose of Tam (25 mg/kg), n = 3 per group. (D) Whole colon fragments were isolated from Lgr5-LacZ and Lgr5-LacZ;Stat5 mice and then stained with X-gal. These stained colonic fragments were sectioned and counter-stained with eosin. The LacZ + colonic crypts were counted, n = 3 per group. (E) Enteroids prior to or after 4HT induction were fixed and sectioned. Sox9 + cells (red) were immunostained and representative images are shown. (F) Enteroids prior to or after 4HT induction were frozen. Total RNA was extracted from the enteroids, the expression of Sox9 was quantitated by real-time PCR. Results are expressed as mean ± SEM, t tests was used to compare the significance of a difference, n = 4 or 5 wells of enteroids per group. * P < 0.01 versus Lgr5- Rosa mT/mG . (G) Ileal sections were stained with pYSTAT5 (green) and Ki67 (red) IF. n ≥ 5 mice per group. Scale bar = 200 μm

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Isolation, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: Intestinal crypts were extracted from Lgr5Cre ER; VilCre ER;Stat5 or icS5 and dissociated with TrypLE into IECs. Lgr5 − , low and high IECs were then separated by gating with FACS . (A) APC-conjugated pYSTAT5 and PE-conjugated CD24 staining were used to quantitate pYSTAT5 + Lgr5 − CD24 + cells (P1) or Lgr5 − CD24 + Paneth cells (P2). (B) Lgr5 hi :CD24 + doublets in the dissociated IECs were determined by FACS. n = 4 or 5 mice per group, ** P < 0.01 versus STAT5 +/+ . (C) The dissociated IECs were immunostained with CD24, and co-localization of Lgr5 and CD24 was determined with a confocal microscope. Lgr5:CD24 doublets are shown as circles. (D) Lgr5-GFP crypts from Lgr5Cre ER or Lgr5Cre ER; VilCre ER;icS5 mice were employed for IESC culture for 4 d, and pYSTAT5 was inducibly activated by 4HT (200 nM) after one dose of γ-irradiation (IR). The numbers of Lgr5-GFP buds were counted in each well, and budding curve was created 6 d after 4HT treatment. ** P < 0.01 and * P < 0.05 versus controls without 4HT induction. Representative images of Lgr5GFP + buds (arrows) in the enteroids (circles) are shown, *Autofluorescence. (E, F) Lgr5-GFP enteroids were dissociated into IECs. Lgr5 hi Ki67 + pYSTAT5 + (E) and Lgr5 low Ki67 + pYSTAT5 + (F) were determined. * P < 0.05 versus enteroids from Lgr5Cre ER; VilCre ER, # P < 0.05 versus enteroids from IR-treated Lgr5Cre ER; VilCre ER, n = 4–5 mice per group. (G) Colonic crypts were isolated and differentiated into colonoids. These colonoids were induced by 4HT for 4 d and then irradiated at 4 Gy for 10 min. Lgr5 (green) buds are shown; arrowheads indicate crypt budding. GFP crypt buds were counted per colonoids (n ≥ 20) from each of six wells from three independent experiments. All results are expressed as mean ± SEM, and t tests and ANOVA were used to compare the significance of a difference.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Staining, Microscopy, Irradiation, Isolation

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Intestinal crypts were extracted from Lgr5Cre ER; VilCre ER;Stat5 or icS5 mice, ileal crypt IECs were dissociated. Lgr5 low and high IESCs were then separated by gating for FACS analysis. (B) The dissociated IECs were immune-stained by Lgr5 (green) and CD24 (red) to show Lgr5 singlets (green) and Lgr5:CD24 doublets (orange) as shown. (C) Quantification of enteroid budding: Enteroids were stained with Methylene Blue, the number of crypt buds per enteroids was counted, n ≥ 20 enteroids per well, six wells per mouse, and three mice per group. (D) Colonic crypts were disassociated and cultured into colonoids with conditioned medium (1 μg/ml R-Spondin, 100 ng/ml Noggin, 50 ng/ml EGF and 100 ng/ml Wnt3a). After 7-d growth, colonoids were respectively treated with different doses of SCF: 5, 10 and 20 ng/ml for 10 d. (E) The number of crypt buds per colonoids was counted, n ≥ 20 colonoids per well, six wells per mouse, and three mice per group. Results are expressed as mean ± SEM. One-way ANOVA was used to analyze the difference between groups. ** P ≤ 0.01 versus Vehicle control.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Staining, Cell Culture, Control

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Enteroids were cultured from intact intestinal crypts dissociated from VilCre ER, VilCreCre ER ;Stat5 +/ − and VilCre R;icS5 mice (STAT5 +/+ , STAT5 ΔIEC+/− and STAT5 ΔIEC+++ ). Based on the number of buds, enteroid morphology was categorized as: 1 bud (Org 1 ), 2 buds (Org 2 ), greater than 3 buds (Org3 + ) and no buds (Sphere). Representative images are shown. Results were expressed as mean ± SEM, t tests and ANOVA were used to compare the significance of a difference, n = 4 or 5 mice per group. (B) Enteroids were cultured from intact intestinal crypts dissociated from STAT5 +/+ , STAT5 ΔIEC−/− , STAT5 ΔIEC+/− and STAT5 ΔIEC+++ mice. The number of grown enteroids were counted with or without TNF-α treatment, the percentage of survival enteroids versus initial grown enteroids was calculated. Results were expressed as mean ± SEM, t tests was used to compare the significance of a difference, n = 4 or 5 mice per group. (C) Intestinal crypts were disassociated from Lgr5Cre ER and Lgr5Cre ER;icS5 mice, Lgr5 hi IESCs were sorted and representative FACS and Lgr5-GFP IF image are shown.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Cell Culture

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Inducible STAT5a mutant constructs. STAT5a domains, critical phosphorylation sites and the GOF mutation Ser710/Phe (icS5), which causes enhanced and prolonged tyrosine phosphorylation upon cytokine/growth factor action. Below, Tam inducible STAT5a-ER fusion protein that dimerizes to physiologically activate STAT5 target genes in a Tam dose dependent manner. (B) H9 iPSCs were transduced with a lentiviral GFP construct for STAT5a-ER and icS5-ER mutant. HIOs were matured in vitro for 35 d. Transduced HIOs were then microinjected 250 ng FD 4 (FD4) with IFNγ (10 ng/ml) and TNFα (10 ng/ml), or TcdA (400 ng/ml), or TcdB (400 ng/ml). Trans-membrane permeability was determined as the amount of FD4 diffusion from the HIO lumen to culture media 24-h IFNγ + TNFα or 6-h TcdA or TcdB after 4HT (200 nM) induction. Results are expressed as mean ± SEM, t tests and ANOVA were used to compare the significance of a difference, n ≥ 4 HIOs per group, * P < 0.05 versus control HIOs. (C) Total proteins were extracted from single HIOs, and LGR5, Bmi1, Dclk1, Lyso, inter-cellular junctional proteins (E-Cad, JAMA-A, ZO-1 and 2, claudin-1 and 2, and occludin) and Wnt/Notch pathway markers (β-catenin and intracellular domain of the Notch protein [NICD]) were measured using immunoblotting. n ≥ 4 HIOs per group.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Mutagenesis, Construct, Phospho-proteomics, Transduction, In Vitro, Membrane, Permeability, Diffusion-based Assay, Control, Western Blot

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: iPSCs or LGR5:eGFP BAC reporter iPSCs were transduced with a lentiviral GFP construct for STAT5a-ER and icS5-ER mutant. HIOs were in vitro matured for 35 d. (A) Transduced HIOs were imaged under 3D in vitro culture systems with dark-field and bright-field 3D confocal deconvolution microscopy. IEC types in HIOs were determined by double-IF, E-Cadherin (E-Cad) + Chromogranin A (CHGA) or E-Cad + Muc2, and Lyso IF staining. (B) Transduced HIOs were then microinjected 250 ng FD 4 (FD4) with 0.25 ng TNFα (100 ng/ml) or 25 ng TcdA or 50 ng TcdB after 3-d 4HT induction. HIO integrity was determined as the TEER after 24-h TNFα or 6-h TcdA or TcdB treatment. Results are expressed as mean ± SEM. * P < 0.05 versus control HIOs. n ≥ 4 HIOs per group. (C) NEs were extracted from single HIOs, and pYSTAT5 and STAT5 were measured using immunoblotting. n ≥ 4 HIOs per group. (D) Transduced HIOs were matured and then transplanted beneath the kidney capsule of NSG mice. Tam induction was performed for 5 d 1-mo post-engraftment. (E) Proliferation of transplanted IESCs was determined with anti-Ascl2 and BrdU IH in the presence and absence of icS5 activation, n = 5 per group. (F, G) LGR5:eGFP BAC reporter iPSCs were transduced with lenti-viral STAT5a-ER or icS5-ER. 3, 7, or 14 d after one dose of Tam, transplanted mice were euthanized. Transplanted HIOs were stained with GFP IF, and IESCs were labeled with LGR5-GFP and EdU. icS5 activation increased LGR5 + IESCs and enhanced LGR5 lineage tracing compared with that in STAT5a-ER. n ≥ 3 mice per group. (H) Transplanted HIOs were stained with AB, Alkaline phosphatase (ALP), Lyso, and Muc2 IH. AB + , Lyso + , or Muc2 + crypt cells were counted as average numbers per crypt. 100 well-orientated crypts were chosen from five mice per group, ** P < 0.01 versus STAT5a-ER. (I) Some of the transplanted mice were subjected to 12-Gy irradiation. Regenerated crypts (RC) were counted in the transplanted HIOs. Inserts are the images at a higher-magnification. Intestines from the irradiated mice show no RC. ** P < 0.01 versus STAT5a-ER, n ≥ 5 mice per group, scale = 200 μm. All results are expressed as mean ± SEM, and t tests and ANOVA were used to compare the significance of a difference.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Transduction, Construct, Mutagenesis, In Vitro, Microscopy, Staining, Control, Western Blot, Activation Assay, Labeling, Irradiation

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Hierarchical clustering of 691 genes with ANOVA P < 0.05 and fold change > 2 in at least one pairwise comparison between STAT5 +/+ , STAT5 ΔIEC−/− , and STAT5 ΔIEC+++ mice, using Pearson’s centered distance metric and average linkage rule. (B) Scatterplots of log (FPKM) of differentially regulated genes in STAT5 ΔIEC−/− and STAT5 ΔIEC+++ mice when compared to STAT5 +/++ mice. (C) β-catenin was determined by IH and immunoblotting with NE from intestinal crypts. Results are expressed as mean ± SEM, n ≥ 3 mice per groups, and t tests were used to compare the significant difference. (D) Sox9 was immunostained and quantitated in the 200 crypts. Results are expressed as mean ± SEM, n ≥ 3 mice per groups, and t tests were used to compare the significant difference. (E) icS5-ER-transduced HIOs were in vitro matured for 35 d. icS5 activation in HIOs was induced with different doses of 4HT (0, 10, 50, 100, or 200 nM or 1 mM) for 72 h. pYSTAT5, STAT5A, and β-catenin were determined by immunoblotting. This experiment was repeated three times. (F) Hypothesis model: pYSTAT5 amplifies Lgr5 hi →to Lgr5 Low Ki67 + IESCs; Ca-pYSTAT5 promotes Lgr5 low Ki67 + IESCs to give rise to a sublineage of crypt cells, intestinal pYSTAT5 + Lgr5 − CD24 + Lyso + Paneth cells by activating Wnt/β-catenin/Sox9 pathway.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Comparison, Western Blot, In Vitro, Activation Assay

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Venn diagram to identify differentially regulated the genes shared between and specific to STAT5 ΔIEC−/− and STAT5 ΔIEC+++ as compared to STAT5 +/+ . (B, C) Barchart of significance of pathways and biological processes enriched in genes significantly up- and down-regulated in STAT5 ΔIEC+++ and STAT5 ΔIEC−/− as compared to STAT5 +/+ . Ontologies were identified through ToppGene.cchmc.org . (D) Diagram of ontological enrichments associated with gene lists built through comparisons between STAT5 ΔIEC−/− and STAT5 ΔIEC+++ , when compared to STAT5 +/+ , showing shared and specific ontologies connected to up- and down-regulated genes, generated by Toppcluster.cchmc.org and cytoscape. (E) Hierarchical clustering of averaged normalized expression values of 13 genes in STAT5 ΔIEC−/− and STAT5 ΔIEC+++ mice compared to STAT5 +/+ mice identified significantly enriched canonical Paneth cell genes regulated by STAT5 protein. (F) Representative images of colonic crypts used for RNA-seq.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Generated, Expressing, RNA Sequencing

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A–C) PCR was used to genotype VilCr eER and Stat5 f/f (A), VilCre and icS5 (B), VilCre ER and icS5 mice. Representative PCR gels are shown.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques:

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Overexpression of AKAP95 in breast cancer tissues of 82 TNBC patient samples. From cBioPortal. Top, each box is a patient sample. Bottom, disease-free survival curves of patients with or without AKAP95 alterations. b, Growth assay for MDA-MB-231 cells expressing control or two AKAP95 shRNAs. Left, immunoblotting of total cell lysates and images of cell colonies stained with crystal violet. Right, numbers of cells in growth assays as mean ± SD from n = 3 independent experiments. c, Tumors from xenograft of control or AKAP95-KD MDA-MB-231 cells in immune-deficient mice. Tumor volumes at the indicated days post transplantation are plotted as mean ± SD (n = 9). d,e, RNA-seq analysis in MDA-MB-231 cells expressing control or AKAP95 shRNA #1 and the indicated vector or AKAP95-expressing construct. d, Heatmap showing relative expression levels of genes down- or up-regulated in the indicated cells. It includes 951 and 294 genes down- and up-regulated in KD compared to control cells, respectively. Also see Supplementary Table 1, tab 1. e, GSEA for gene expression profiles of control and AKAP95-KD cells. Plots above and below the broken line show gene sets significantly enriched in up- and down-regulated genes by AKAP95 KD, respectively. f, Heatmap showing relative alternative splicing and clustered by changes in percent-spliced-in (PSI) values in the indicated cells. It includes 807 and 1275 alternative splicing events with decreased or increased PSI in KD cells, respectively. Also see Supplementary Table 1, tab 2. g, Gene ontology analysis for the indicated clusters from the heatmap in f. Blue (n = 807) and red (n = 1275) show functions significantly enriched in genes with PSI increase or decrease by AKAP95 KD, respectively. P values by log-rank test for a, Student’s t -test for b and d, and modified Fisher’s exact test for g. All two-sided. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Over Expression, Growth Assay, Expressing, Western Blot, Staining, Transplantation Assay, RNA Sequencing Assay, shRNA, Plasmid Preparation, Construct, Modification

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, CCNA2 expression in MDA-MB-231 cells upon AKAP95 KD. Left, relative mRNA levels of indicated cyclins were determined by RT-qPCR and normalized to GAPDH , and presented as mean ± SD from n = 3 biological repeats. Right, immunoblotting for Cyclin A1/A2 in total cell lysates. b, Co-overexpression of AKAP95 and CCNA2 in breast cancer tissues of 82 TNBC patients. Left, each box represents a patient. Right, correlation of their mRNA levels in the TNBC patients with indicated Pearson correlation coefficient. Both from cBioPortal. c,f, RNA immunoprecipitation-sequencing (RIP-seq) profiles for CCNA2 (c) and SMAD6 (f) based on our previous work . In blue are Anti-FLAG RIP-seq in control or 293 cells expressing the FLAG-HA-tagged AKAP95 WT or mutants. In red are anti-AKAP95 RIP-seq in control or AKAP95-KD 293 cells. In black are profiles of total input RNAs. All profiles have the same Y-axis scale. Red arrows indicate AKAP95-binding sites at intron 1. d, Total RNAs were used for RT-PCR, in the absence (-RT) or presence (+ RT) of reverse transcriptase, for CCNA2 intron 1 in MDA-MB-231 cells with indicated combination of siRNAs. Top, PCR products on agarose gel. Asterisk, an unknown amplification product. Repeated 3 times. Bottom, relative ratios of the signal for the intron 1-retaining transcript over the intron 1-spliced transcript, as mean ± SD from n = 3 independent experiments. e, Assay for CCNA2 mRNA stability. Control (Scr) and AKAP95-KD MDA-MB-231 cells were treated starting from 0 min with Actinomycin D (+A, to block RNA synthesis) and cycloheximide (+C, to block NMD) or not as indicated. Total RNA at indicated times were used for RT-PCR and normalized to ACTB , as mean ± SD from n = 3 biological repeats. g, mRNA-seq profiles for SMAD6 in MDA-MB-231 cells expressing control or AKAP95 shRNA #1 (KD) and vector or AKAP95-expressing construct. Asterisk, a stop codon. P values by two-sided Student’s t -test for a and e and one-way ANOVA followed by Tukey’s post hoc test for d. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Immunoprecipitation, Sequencing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Blocking Assay, shRNA, Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a-f, MDA-MB-231 cells were transduced with control or AKAP95 shRNA #1 (KD) and vector and FLAG-HA-tagged full-length AKAP95 WT or mutants. a, Immunoblotting of total cell lysates. Repeated 3 times. b, Colony formation assays. Left, colony numbers as mean ± SD from n = 3 biological repeats. Right, images of cells stained with crystal violet. c, Growth of cultured cells, as mean ± SD from n = 3 independent experiments. d, Relative SMAD6 mRNA level were determined by RT-qPCR and normalized to GAPDH , as mean ± SD from n = 3 biological repeats. e, RT-PCR for ratios for intron 1-retained over-spliced CCNA2 transcript, as mean ± SD from n = 4 biological repeats. f, RT-PCR for ratios for exon-included over-skipped RPUSD3 transcript, as mean ± SD from n = 3 biological repeats. g-k, MYC-transduced Akap95 KO MEFs were transduced with vector or constructs expressing HA-tagged full-length AKAP95 WT or mutants. g, Immunoblotting of total cell lysates. Repeated 3 times. h, Left, percentage of SA-beta-gal-positive cells as mean ± SD from n = 3 different images of MEFs from two embryos. Right, images from KO MEF 1. i, Relative mRNA levels of indicated genes, with related functions at bottom, were determined by RT-qPCR and normalized to Gapdh , as mean ± SD from n = 3 independent experiments. * or ** between vec and WT, WT and YS, WT and YF, except for Plk1, for which * only between vec and WT, WT and YF. *P<0.05, **P<0.01. j, Heatmap showing relative alternative splicing with PSI changes in MYC-transduced KO MEFs expressing indicated constructs (2 embryos each). Also see Supplementary Table 2, tab 5. k, Sashimi plot showing Aamdc alternative splicing that was rescued by introduction of AKAP95 WT, but not but the mutant, and RT-PCR for the inclusion of the alternative exon as mean ± SD from n = 4 biological replicates pooled from 2 embryos each. l, Diagram showing impact of material properties of AKAP95 WT and mutants on gene regulation and tumorigenesis. P values by two-sided Student’s t -test for c and one-way ANOVA followed by Tukey’s post hoc test for all other analyses. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Transduction, shRNA, Plasmid Preparation, Western Blot, Staining, Cell Culture, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Construct, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a,b, MDA-MB-231 cells virally expressing control or indicated shRNAs ( a ) or shRNA combined with indicated constructs ( b ) were subject to immunoblotting of total cell lysates (top) and colony formation assay. Middle, colony numbers as mean ± SD from n = 3 ( a ) or 2 ( b ) independent experiments. Bottom, images of cells stained with crystal violet. c, Top, mRNA-seq profiles for CCNA2 in control or 293 cells of AKAP95 KD . The numbers of exon junction reads are indicated. The red asterisk at the gene diagram indicates a stop codon 57 bp downstream of exon 1 in the intron. The number of reads for the junction of exons 1 and 2, and for the average neighboring exons, and their ratios are in the tables below for indicated cells. d, Total RNAs were used for RT-PCR for intron 1 region in control and AKAP95-KD MDA-MB-231 cells treated with or without cycloheximide for 6 hours. Repeated > 3 times. e, Relative mRNA levels of UPF in UPF1-KD samples and BTZ in BTZ-KD samples, respectively, each relative to the control samples, as determined by RT-qPCR and normalized to GAPDH . f, Relative expression level of TGF-β pathway genes based on RNA-seq reads from control and AKAP95-KD MDA-MB-231 cells expressing vector or AKAP95. Venn diagram shows numbers of TGF-β pathway genes (from GSEA) downregulated by AKAP95 KD and upregulated by rescue with AKAP95 expression, and the relative expression of the 16 overlapped genes in both categories are plotted. g, RIP-seq profiles showing AKAP95 binding to RPUSD3 and PPM1K pre-mRNAs. Track information is the same as in . Red circles indicate the alternatively included exons (corresponding to the middle exon in the gene diagrams in ( h ), and red boxes show AKAP95 binding at the introns flanking these exons. h, Sashimi plots showing that the alternative splicing of RPUSD3 and PPM1K pre-mRNAs was affected by AKAP95 KD and rescued by restored expression of AKAP95. The numbers of exon junction reads and PSI are indicated. P values by two-sided Student’s t -test for a and b. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Expressing, shRNA, Construct, Western Blot, Colony Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, RNA Sequencing Assay, Plasmid Preparation, Binding Assay

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a,b, MDA-MB-231 cells were virally infected to stably express scramble (control) or AKAP95 shRNA #1 (KD) and the indicated constructs including empty vector (vec) and FLAG-HA-tagged full-length AKAP95 WT or mutants. a, Relative CCNA2 mRNA level as determined by RT-qPCR and normalized to GAPDH , and plotted for each of the 2 biological repeats individually. b, RT-PCR for ratios for exon-included over-skipped PPM1K transcript, as mean ± SD from n = 3 biological repeats. c-f, MYC-transduced Akap95 KO MEFs were transduced with vector or constructs expressing HA-tagged full-length AKAP95 WT or mutants. c, Heatmap showing relative expression levels of genes changed in MYC-transduced KO MEFs (from 2 embryos each) stably expressing indicated rescue constructs. Also see Supplementary Table 2, tab 4. d, Relative mRNA levels of indicated SASP genes as determined by RNA-seq reads from n = 2 biological repeats (KO1 and KO2). e,f, Sashimi plots showing example genes for which the alternative exon inclusion was promoted ( e ) or suppressed ( f ) by introduction of AKAP95 WT, but not as effectively by YS or YF, and RT-PCR for the inclusion of the alternative exon, as mean ± SD from n = 2 embryos each. g, A model for how AKAP95 condensates may regulate gene expression for tumorigenesis. P values by one-way ANOVA followed by Tukey’s post hoc test. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Infection, Stable Transfection, shRNA, Construct, Plasmid Preparation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Transduction, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Growth of MEFs from Akap95 +/− (Het) and Akap95 −/− (KO) embryos (n = 6 each). b, Relative HRAS and MYC mRNA levels were determined by RT-qPCR and normalized to Actb , as mean ± SD from HRAS G12V and MYC transduced MEFs of 2 embryos each. c, HRAS-MYC-transduced MEFs in soft agar colony formation assay. Colony numbers as mean ± SD from n = 6 experiments using MEFs of 2 embryos each. d, Six mice received HRAS-MYC-transduced Het and KO MEFs on each flank. Tumor weights at four weeks are plotted. Each dot represents a tumor. e-i, MEFs derived from 3 KO and 3 Akap95-expressing (containing 1 WT and 2 Het) embryos were transduced with MYC. e, Right, images of cells before and after MYC transduction. Images of SA-beta-galactosidase activity assay are at the bottom. Relative MYC mRNA levels after transduction were determined by RT-qPCR and normalized to Actb (left top). Percentage of SA-beta-gal-positive cells are plotted (left bottom). Both as mean ± SD from MEFs (n = 3 embryos each). f, Heatmap showing relative expression levels of genes and clustered by changes in KO MEFs (2 embryos each). It includes 265 and 742 genes down- or up-regulated in KO, respectively. Also see Supplementary Table 2, tab 1. g, Gene ontology analysis for the indicated gene clusters from the heatmap in f. Blue (n = 265) and red (n = 742) show functions significantly enriched in down- and up-regulated genes, respectively. h, GSEA plots above and below the dashed line show gene sets significantly enriched in genes down- and up-regulated in the MYC-transduced KO compared to Het MEFs, respectively. i, Relative Akap95 and Ccna2 mRNA levels before and after MYC transduction as determined by RT-qPCR and normalized to Actb , as mean ± SD from MEFs from 3 KO and 2 WT/Het embryos. j, A diagram summarizing regulation of tumorigenesis by AKAP95 through gene expression control. P values by two-sided Student’s t -test for all except one-way ANOVA followed by Tukey’s post hoc test for i, and modified Fisher's exact test for g. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Quantitative RT-PCR, Soft Agar Assay, Derivative Assay, Expressing, Transduction, Activity Assay, Modification

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Immunoblotting for AKAP95 in HeLa cell nuclear extract and AKAP95 immunoprecipitation from the extract. Samples were boiled in the presence of DTT and resolved by SDS-PAGE. b, Disorder plot of human AKAP95. c, Turbidity by pictures and OD600 of MBP (none) and MBP fused to AKAP95 truncations at indicated concentrations all in 30 mM NaCl before and after TEV protease treatment for indicated time. OD600 is plotted as mean ± SD from n = 3 biological repeats. d, DIC (top) and fluorescence microscopy (bottom) images for 20 μM MBP-AKAP95 (101-210) and spiked with Oregon-green-labeled same protein (molar ratio 10:1) after TEV protease treatment for 30 min. Changes in NaCl concentration is indicated. Images were taken 5 min after salt adjustment. e, Phase contrast images of 50 μM MBP-AKAP95 (101-210) in 30 mM NaCl in the absence and presence of 10% of PEG6000 after TEV protease treatment for 30 min. f, Fusion of two droplets formed by 50 μM MBP-AKAP95 (101-210) in 30 mM NaCl and 10% of PEG6000 after TEV protease treatment for 30 min. Also see Movie 1. g, DIC and fluorescence microscopy images of 6.25 μM MBP, MBP fused to Δ(101-210) or full-length AKAP95 in 150 mM NaCl, spiked with Oregon-green-labeled AKAP95 (101-210) at a molar ratio of 150:1 after TEV protease treatment for 30 min. Note that the lack of any condensates in the DIC images showed the inability of Δ(101-210) in condensation. Experiments in a, d, e-g were Repeated > 3 times. Scale bar, 5 μm for all. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Western Blot, Immunoprecipitation, SDS Page, Fluorescence, Microscopy, Labeling, Concentration Assay

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, 293T cells were transfected with either empty vector (vec), or indicated AKAP95 construct with FLAG-HA-tag. Following α-Flag IP, the pulldown proteins were boiled and resolved by SDS-PAGE and detected by immunoblotting with α-HA. Blue and red asterisks indicate monomer and dimer, respectively. b, Identification of 1-100 as a probable prion subsequence on AKAP95. By the PLAAC program, using homo sapiens as background and core length of 30. c, Purified MBP and MBP fused to AKAP95 truncations as indicated or full-length AKAP95 (1-692) were resolved on SDS-PAGE and stained with Coomassie blue. d, MBP fused to AKAP95 truncations as indicated or full-length AKAP95 were resolved on SDS-PAGE and stained with Coomassie blue following treatment with TEV protease. Note that the cleaved MBP serves as a better indicator for cleavage efficiency as staining signal various for protein fragments of different sequences and sizes. e, Another event of fusion of two droplets formed by 50 μM MBP-AKAP95 (101-210) in 30 mM NaCl and 10% of PEG6000 after treatment with TEV protease for 30 min. Scale bar, 5 μm. Also see Movie S1. f, Quantification of nuclear AKAP95 concentration by anti-AKAP95 Western blot. Total lysates from indicated number of MDA-MB-231 (M231) and flp-TREx 293 cells (f293, un-induced and dox-induced for FH-AKAP95 expression) were loaded, along with indicated ng of purified MBP-AKAP95. AKAP95 signal of un-induced f293 is similar to that of 25 ng of MBP-AKAP95. All experiments were repeated 2 times. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Transfection, Plasmid Preparation, Construct, SDS Page, Western Blot, Purification, Staining, Concentration Assay, Expressing

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Fluorescence microscopy images of Oregon-green-labeled AKAP95 (101-210) WT and YF at indicated protein and NaCl concentrations after TEV protease treatment for 30 min. Scale bar, 10 μm. Repeated > 3 times. b, Fluorescence microscopy images of 50 μM AKAP95 (101-210) WT and YF in 30 mM NaCl both spiked with Oregon-green-labeled (101-210) WT (molar ratio 150:1) after TEV protease treatment for 30’ and imaged immediately (30’) or after incubation for 60 (90’) or 120 (150’) more minutes. Scale bar, 10 μm. Repeated > 3 times. c, FRAP of 10 μM GFP-AKAP95 (101-210) WT and YF after 30 min of MBP cleavage in 150 mM NaCl and 10% of PEG6000. FRAP was performed immediately (30’) or after incubation for 60 min more (90’). Left, fluorescence microscopy images of droplets at indicated times. Middle, FRAP recovery curves as mean ± SD. Right, mean ± SD of recovery (relative to minimal level) at the final time. n = 7 droplets each. Scale bar, 2 μm. d, FRAP of Full-length AKAP95 WT and YF fused to GFP in HeLa cell nuclei. Left, fluorescence microscopy images of foci. The photobleached focus was boxed and amplified for the indicated time points. Middle, FRAP recovery curves as mean ± SD. Right, mean ± SD of recovery (relative to minimal level) at the final time. n = 7 cells each. Scale bar, 5 μm. e,f, Diffusion of full length AKAP95 WT and YF fused to GFP in HeLa cell nuclei, showing Line RICS normalized autocorrelation curves G(Ψ) as a function of the Spatial Lag (Ψ) ( e ) and diffusion coefficients ( f ), both as mean ± SD (n = 20 for WT, n = 22 for YF). g,h, Diffusion coefficients of purified GFP-AKAP95 (101-210) WT and YF, showing Line RICS normalized autocorrelation curves G(Ψ) ( g ) and diffusion coefficients ( h ), both as mean ± SD (n = 22 for WT, n = 13 for YF). P values by two-sided Student’s t -test (c, d) or Mann-Whitney U test (f, h). For box and whisker plots, data are median (line), 25–75th percentiles (box) and minimum-maximum values recorded (whiskers). Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Fluorescence, Microscopy, Labeling, Incubation, Amplification, Diffusion-based Assay, Purification, MANN-WHITNEY, Whisker Assay

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Immunostaining of endogenous AKAP95 (red) and DNA (DAPI, blue) in indicated cancer cell lines and primary MEFs from WT and Akap95 KO embryos. b, Confocal microscopy images of AKAP95 WT or ZF C-S fused to GFP in nuclei following transfection into HeLa cells. c, Fluorescence microscopy images of HeLa cells transiently expressing AKAP95 WT or Δ(101-210 fused to GFP. d, HeLa cells were transfected with AKAP95-GFP, and two nuclei were imaged at different time points. Time 0 was 24 hr after transfection. Note the growth and merge of the foci, especially those in the red circle. e, Rapid fusion of AKAP95 (ZF C-S )-GFP foci in a HeLa cell nucleus. The white oval and arrow show two different fusion events. These images are from movie 2. All experiments were Repeated > 3 times. Scale bar, 5 μm for all.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Immunostaining, Confocal Microscopy, Transfection, Fluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Alignment of human and mouse AKAP95 (101-210). Middle row shows identical residues (by letter) and conservative mutations (by “+”). Tyr, red; Phe, blue and tall. Box, Tyr and Phe swapping. b, MBP alone (none) or MBP-AKAP95 (101-210) WT or mutants all at 50 μM and in 30 mM NaCl after TEV protease treatment for 30 min. Turbidity of each reaction was shown in pictures, and by OD600 as mean ± SD from n = 3 (for YA, YS) or 4 (the rest) independent assays. Samples taken after mixing (“mixed”) and from supernatant after centrifugation were resolved by SDS-PAGE followed by coomassie blue staining. c, DIC and fluorescence microscopy images for 10 μM Oregon-green-labeled MBP-AKAP95 (101-210) WT and mutants in 30 mM NaCl after TEV protease treatment for 30 min. Plots from left to right at bottom show the relative protein amount in droplet, number of droplets in a field, and the ratio of protein concentration inside droplets over sum of inside and outside droplets, respectively. Calculated as mean ± SD from n = 24 randomly picked droplets for WT or YF, except for number of droplets from n = 3 randomly picked fields. NA, not applicable. d, Fluorescence microscopy images of HeLa cells transfected with (top) and Flp-In T-Rex 293 cell lines expressing (bottom) GFP fusions with full-length AKAP95 WT or mutants. Repeated > 3 times. e,h, HEK293 cells co-transfected with indicated siRNAs and plasmids were subject to splice reporter assay (top) and immunoblotting with α-AKAP95 (bottom). Δ = Δ(101-210). Mean ± SD from n = 8 [except 5 for Δ(101-210) and 13 for YF and 2 nd WT] independent transfections are plotted in e and 7 independent transfections in h. f, Schematic of AKAP95 chimeras. g, Fluorescence microscopy images of 293T cells transfected with indicated AKAP95 chimeras fused to GFP. Repeated > 3 times. P values by two-sided Student’s t -test for b and one-way ANOVA followed by Tukey’s post hoc test for e and h. Scale bar, 5 μm for all. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Centrifugation, SDS Page, Staining, Fluorescence, Microscopy, Labeling, Protein Concentration, Transfection, Expressing, Reporter Assay, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Tumor Necrosis Factor Receptor 2 Inhibits HIV-1 Infection by Blocking the Binding of gp120 to CD4

doi: 10.7150/ijbs.124330

Figure Lengend Snippet: The TNFR2 protein blocks the binding of gp120 to CD4. (A) ELISA was used to assess whether TNFR2(2 µM) can inhibit the interaction between gp120 and immobilized CD4 on the plate. The final OD450 measurement statistics are shown. (B) Flow cytometry was utilized to evaluate cell-bound His-tag, indicating the binding of gp120-His to the Jurkat cells, and also assessed if TNFR2 (1 µM) can block gp120-cell binding. (C) The percentage of positive cells was determined from flow cytometry analysis results. (D) The statistical analysis of mean fluorescence intensity. (E) Further examination assessed whether the blocking effect of TNFR2 (50 nM, 100 nM, 200 nM, 400 nM, 800 nM) exhibited dose-dependency. (F) The half-maximal inhibitory concentration (IC50) was calculated through the dose-dependent inhibition curve. (G) Flow cytometry was utilized to detect the binding of gp120 to human peripheral blood mononuclear cells and to evaluate the effect of TNFR2. (H) The statistical analysis of the double positive cells. One-way ANOVA was used to analyze group differences. Significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns no significant difference.

Article Snippet: CD4 protein (DNA sequence: NP_000607 , Catalog No: TP306453) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Blocking Assay, Fluorescence, Concentration Assay, Inhibition

Journal: International Journal of Biological Sciences

Article Title: Tumor Necrosis Factor Receptor 2 Inhibits HIV-1 Infection by Blocking the Binding of gp120 to CD4

doi: 10.7150/ijbs.124330

Figure Lengend Snippet: The TNFR2 protein can inhibit HIV pseudovirus infection of CD4+CCR5+ cells. (A) HIV pseudovirus carrying green fluorescent protein genes was used to infect CD4+CCR5+293T cells (MOI=10). The flow cytometry was used to analyze the rate of GFP-positive cells to illustrate the virus infection efficiency. (B) The statistical analysis for the ratio of GFP-positive cells in each group. (C) Further examination assessed whether the blocking effect of TNFR2 (0.1 µM、0.25 µM、0.5 µM、1 µM) exhibited dose-dependency. (D) The statistical analysis for the dose-dependent effects. (E) Infected CD4+CCR5+ 293T cells were observed by fluorescence microscopy on the 12h post infection. (F) Flow cytometry analysis of Jurkat-CCR5 cells infected with HIV-1 pseudovirus, with or without pre-incubation of the virus with 10 µg/mL TNFR2 protein. (G) Quantification of infection efficiency. One-way ANOVA was used to analyze group differences. Significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns no significant difference.

Article Snippet: CD4 protein (DNA sequence: NP_000607 , Catalog No: TP306453) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Infection, Flow Cytometry, Virus, Blocking Assay, Fluorescence, Microscopy, Incubation

Journal: International Journal of Biological Sciences

Article Title: Tumor Necrosis Factor Receptor 2 Inhibits HIV-1 Infection by Blocking the Binding of gp120 to CD4

doi: 10.7150/ijbs.124330

Figure Lengend Snippet: TNFR1 impacts the binding of gp120 to CD4 but promotes the virus infection. (A) The binding interaction between gp120 and TNFR1 was assessed using an ELISA assay with varying concentrations of gp120 (5 μM, 0.5 μM, 0.05 μM, blank). (B) Flow cytometry was utilized to evaluate cell-bound His-tag, indicating the binding of gp120 to the Jurkat cells, and also assess if TNFR1 (0.4µM, 0.6µM, 1µM) can block gp120-cell binding. (C) The percentage of positive cells was determined from flow cytometry analysis results. (D) Flow cytometry was utilized to detect the binding of gp120 to human peripheral blood mononuclear cells and to evaluate the blocking effect of TNFR1. (E) The statistical analysis of the double-positive cells. (F) HIV pseudovirus carrying green fluorescent protein genes was used to infect CD4 + CCR5 + 293T cells (MOI=5, MOI=10). Flow cytometry was used to analyze the rate of GFP-positive cells, illustrating the virus infection efficiency. (G) The percentage of GFP-positive cells was determined from flow cytometry analysis results. (H) His-tagged gp120 was pre-incubated with recombinant TNFR1 or TNFR2 and applied to CCR5⁺CD4⁻ 293T cells. (I) CD4⁻CCR5⁺ 293T, TNFR1 + CD4⁻CCR5⁺ 293T, and TNFR2 + CD4⁻CCR5⁺ 293T cells were infected with GFP-expressing HIV-1 pseudovirus. Infection efficiency was assessed by flow cytometry based on the percentage of GFP⁺ cells. Significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns no significant difference.

Article Snippet: CD4 protein (DNA sequence: NP_000607 , Catalog No: TP306453) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Binding Assay, Virus, Infection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Blocking Assay, Incubation, Recombinant, Expressing

Journal: bioRxiv

Article Title: Dynamics of SARS-CoV-2 host cell interactions inferred from transcriptome analyses

doi: 10.1101/2021.07.04.450986

Figure Lengend Snippet: ( A ) Experimental setup. Caco-2 cells were infected with SARS-CoV-2 at an MOI of 5. Cells were lysed and RNA-seq was performed at the indicated time-points post infection. ( B ) Microscopy analysis of SARS-CoV-2 replication in Caco-2 cells infected with SARS-CoV-2 (top) or non-infected control cells (bottom). Representative images of three biological replicates are shown (blue, DAPI staining of nuclei; red, immunostaining of SARS-CoV-2 N protein; scale bar, 50 μm). ( C ) Analysis of absolute (top) and relative (bottom) host cell and virus transcript counts. At 12 hpi, virus transcripts peaked, constituting 41% of all transcripts (means of n=3 replicates; error bars, SEM). ( D ) Virus transcript read counts increased from ORF1 to ORF10, reflecting the nested RNA architecture of SARS-CoV-2 (bars: average read counts of n=3 replicates normalized by CDS length at 24 hpi; error bars, SEM; CDS, coding sequence). ( E , F ) Microscopy analysis of the fraction of cells with detectable expression of SARS-CoV-2 N protein ( E ) and the normalized total cell count ( F ) at the indicated time points.( G ) Quantification of the released virus particles by endpoint dilution assay (TCID50, 50% tissue culture infective dose). (E-G) n=3, error bars indicate standard deviation.

Article Snippet: We used antibodies to detect the SARS-CoV-2 nucleoprotein (mouse monoclonal; Sino Biologicals, Hong Kong, China) and GFP (mouse monoclonal; Roche Diagnostics).

Techniques: Infection, RNA Sequencing Assay, Microscopy, Staining, Immunostaining, Sequencing, Expressing, Cell Counting, Endpoint Dilution Assay, Standard Deviation

Journal: bioRxiv

Article Title: Dynamics of SARS-CoV-2 host cell interactions inferred from transcriptome analyses

doi: 10.1101/2021.07.04.450986

Figure Lengend Snippet: ( A ) Distributions of log2 fold changes of normalized and background-corrected gene transcript reads relative to initial transcript counts, and percentages of genes with log2 fold changes L ≥ 1 or L ≤ −1 (dashed lines, log2 fold changes of ±1; vertical lines, 2.5 and 97.5 percentiles). ( B ) Clusters of p-values from GO term enrichment at interpolated time intervals indicate the dynamics of cellular processes in response to SARS-CoV-2 infection. The following qualitative patterns can be distinguished: early, transient response defined by transient expression changes within 12 hpi (cluster I), late, sustained response characterized by expression changes that take place after 12 hpi and are maintained (cluster II), and an early, sustained response indicated by maintained expression changes that already start at few hours post infection (cluster III).

Article Snippet: We used antibodies to detect the SARS-CoV-2 nucleoprotein (mouse monoclonal; Sino Biologicals, Hong Kong, China) and GFP (mouse monoclonal; Roche Diagnostics).

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: Dynamics of SARS-CoV-2 host cell interactions inferred from transcriptome analyses

doi: 10.1101/2021.07.04.450986

Figure Lengend Snippet: ( A ) Scatter plot showing the number of regulated genes in KEGG pathways affected by SARS-CoV-2 infection. Pathways comprising strongly up- or downregulated genes, defined by log2 fold changes of at least 1, were selected. ( B ) Scatter plot of KEGG pathways affected by SARS-CoV-2 infection. Dots indicate the percentage of strongly regulated genes relative to the total number of expressed genes within a pathway. ( C ) 25 cellular pathways with the largest fractions of upregulated genes, selected from pathways with at least 10 strongly regulated genes. Pathways were sorted according to times at which 50% of all strongly regulated genes were up- or downregulated (top to bottom). ( D ) Top 25 cellular processes with largest fractions of downregulated processes as in C . Transcription was downregulated, delayed relative to the observed upregulation, in various processes involved in metabolism (N up/down , number of up- or downregulated genes; N exp , number of expressed genes detected by RNA sequencing; N tot , total number of genes within KEGG pathways).

Article Snippet: We used antibodies to detect the SARS-CoV-2 nucleoprotein (mouse monoclonal; Sino Biologicals, Hong Kong, China) and GFP (mouse monoclonal; Roche Diagnostics).

Techniques: Infection, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Dynamics of SARS-CoV-2 host cell interactions inferred from transcriptome analyses

doi: 10.1101/2021.07.04.450986

Figure Lengend Snippet: ( A ) Experimental setup. Cleavage probes consisting of a nuclear export sequence (NES), a 3CL pro cleavage site (NS4-NS5) and GFP were expressed in HEK293T cells. Uncleaved probes are continuously exported from the nucleus and hence located in the cytoplasm. Following 3CL pro -mediated cleavage, GFP enters the nucleus by diffusion. The cytoplasmic and nuclear fluorescence intensities (I cpl , I ncl ) were used to calculate the concentration of the uncleaved probe. ( B , C ) HEK293T cells co-expressing a cleavage probe containing the cleavage site between NSP4 and NSP5 and 3CL pro were incubated in presence of 20 mM GC376, the drug was removed at time point 0 and re-added at 250 minutes (scale bar, 10 μm). Representative microscopy images ( B ) and corresponding quantification of the concentration of the uncleaved probe over time ( C ). Nuclear signal increase following GC376 removal indicates probe cleavage (data in C ,means of n=10 cells, error bars: SEM). ( D ) Caco-2 cells were infected in the presence or absence of inhibitors. Virus replication was assessed at 24 hpi via immunofluorescence of SARS-CoV-2 N protein (means of n=3 replicates, error bars: SEM).

Article Snippet: We used antibodies to detect the SARS-CoV-2 nucleoprotein (mouse monoclonal; Sino Biologicals, Hong Kong, China) and GFP (mouse monoclonal; Roche Diagnostics).

Techniques: Sequencing, Diffusion-based Assay, Fluorescence, Concentration Assay, Expressing, Incubation, Microscopy, Infection, Immunofluorescence

Journal: Cancers

Article Title: Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer

doi: 10.3390/cancers11121910

Figure Lengend Snippet: Single-cell RNA-seq showing upregulation of Src and Eph-ephrin signaling in advanced prostate cancer cells. ( A ) Dot plot of differentially expressed (DE) genes (n = 171) in Src signaling. LNCaP, LNCaP-Abl, and PC3 are clusters distinctly separated from the other clusters. ( B ) Dot plot of DE gene expression (n = 20) in Eph-Ephrin signaling. Unpaired t -test, ** p < 0.01, *** p < 0.0001. ( C ) The expression of 16 representative genes from EPH Transcripts of 16 genes in four cell lines were validated using bulk RNA qRT-PCR. ( D ) mRNA levels of EPHB2 and SRC predict poor prognosis in The Cancer Genome Atlas (TCGA) prostate cancer patient cohort using Kaplan–Myer estimation analysis. The red line represents the patients with a high expression of the genes with more than 2 SD as compared to the patients presented in the blue line.

Article Snippet: Antibodies against SRC1 (2109, Cell Signaling Biotechnology, Danvers, MA, USA), EPHB2 (AF467-SP, R&D Systems), EGFR (MS-311, Thermo Fisher Scientific), and secondary antibodies with FITC and Alexa 530 (712-585-153, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were purchased commercially.

Techniques: RNA Sequencing, Gene Expression, Expressing, Quantitative RT-PCR

Journal: Cancers

Article Title: Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer

doi: 10.3390/cancers11121910

Figure Lengend Snippet: Dasatinib inhibits proliferation, migration, invasion, and EGFR diffusivity in advanced prostate cancer cells. ( A ) Src is highly upregulated in PC3 (2.5x) and DU145 (1.6x) as compared to LNCaP shown in Western blots. EphB2 is overexpressed in both PC3 and DU145. Both proteins are almost not expressed in LNCaP-Abl. ( B ) Src is present in these cell lines. There is an intense level of Src on the PC3 cell membrane. ( C ) Immunostaining of EphB2 protein is present in plasma and membrane. ( D ) Image-based IncuCyte assays allow us to conduct the time-lapse analysis of cell proliferation, migration, and invasion of the cells treated with or without dasatinib. The dasatinib significantly inhibits the proliferation, migration, and invasion of DU145 and PC3 cells but reduces the EGFR diffusivity in only PC3 cells. The mean value of each bar was measured at the end time of each assay or at the 48th hour. All statistical analyses were performed using the unpaired t -test. The asterisk represents the level of statistical significance for t -test: *** p < 0.001, ** p < 0.01, * p < 0.05. The error bar represents the standard error of the mean. The RWD stands for relative wound density.

Article Snippet: Antibodies against SRC1 (2109, Cell Signaling Biotechnology, Danvers, MA, USA), EPHB2 (AF467-SP, R&D Systems), EGFR (MS-311, Thermo Fisher Scientific), and secondary antibodies with FITC and Alexa 530 (712-585-153, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were purchased commercially.

Techniques: Migration, Western Blot, Membrane, Immunostaining, Clinical Proteomics

Journal: Cancers

Article Title: Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer

doi: 10.3390/cancers11121910

Figure Lengend Snippet: Disruption of EphB2/Src pathways leads to attenuated cell motility, invasion, and EGFR diffusion in advanced prostate cancer cells. ( A ) Effective gene knockdowns in siRNA-treated DU145 and PC3. ( B ) Structured Illumination Microscopy (SIM) images of siRNA treated cells. Maximum intensity projection on the xy plane and orthogonal cross-sections (xz and yz) of DU145 and PC3 siRNA treated cells. ( C ) Quantification of cortical actin based on fluorescence intensities of xz and yz orthogonal projections along the apical plasma membrane. The number of projections analyzed is labeled on each bar. ( D ) EGFR diffusivities of the siRNAs treated cells. The error bar represents the standard error of the mean. ( E , F ) The image-based assays allow us to conduct the time-lapse analysis of cell migration and invasion on the siRNA-treated cells. The error bar represents the standard deviation. All statistical analyses were performed using the unpaired t -test. The asterisk represents the level of statistical significance for t -test: *** p < 0.001, ** p < 0.01, * p < 0.05. ( G ) Schematic shows the effects of EMT-induced actin reorganization on EGFR dynamics and the Src/EphB2 induced signaling from the plasma membrane that controls cell behavior.

Article Snippet: Antibodies against SRC1 (2109, Cell Signaling Biotechnology, Danvers, MA, USA), EPHB2 (AF467-SP, R&D Systems), EGFR (MS-311, Thermo Fisher Scientific), and secondary antibodies with FITC and Alexa 530 (712-585-153, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were purchased commercially.

Techniques: Disruption, Diffusion-based Assay, Microscopy, Fluorescence, Clinical Proteomics, Membrane, Labeling, Migration, Standard Deviation

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. In theory, acute inhibition of transcriptional CDKs, particularly those involved in phosphorylating RPB1 CTD Ser2 phosphorylation, is expected to globally suppress gene expression, predominantly affecting mRNAs with short half-lives. B. Volcano plots of gene expression derived from 4-6 hours treatment of CDK7 inhibitor THZ1 (250 nM) in ovarian cancer cell line Kuramochi , CDK9 inhibitor HH1 (10 µM) in a cell line (YB5) derived from the SW48 colon cancer cell line , or CDK12 inhibitor SR-4835 (90 nM) in triple-negative breast cancer line MDA-MB-231 . RNA-seq data were downloaded from the Gene Expression Omnibus (GEO) and analyzed. C. A volcano plot of nascent RNA expression from neuroblastoma cells (IMR32) treated with 400 nM THZ531 for 2 hours . Note that the sequencing involved 4-thiouridine-pulse labeling and included RNA spike-in control. D. (Top) selection of TCGA ovarian serous adenocarcinoma samples with low or high expression of the indicated CDK genes (the top and bottom 5% in terms of CDK7, CDK9 or CDK12 mRNA expression in ovarian cancer samples with expression data; n = 17 each group). (Bottom) volcano plots of differential gene expression in tumors with low expressing CDKs compared to those with high expressing CDKs. For all volcano plots, genes significantly upregulated or downregulated (absolute log 2 fold change (FC) ≥ 1, p < 0.1) are colored in red and blue, respectively.

Article Snippet: The primary antibodies used include the following: Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523); mouse monoclonal Anti-EMD/Emerin antibody, clone CL0203 (Sigma-Aldrich, #AMAB90562).

Techniques: Inhibition, Phospho-proteomics, Gene Expression, Derivative Assay, RNA Sequencing, RNA Expression, Sequencing, Labeling, Control, Selection, Expressing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. Fluorescent immunoblotting of whole cell lysates. HER2+ cells HCC1954 were treated for 4 hours with vehicle, 200 nM THZ531, or NVP2. Following the treatment, cells were lysed with 1x SDS sample buffer to generate whole cell lysates for fluorescent immunoblotting. Each lane was loaded with lysates representing the same number of cells. The molecular weights of the fluorescent protein markers and clone identities for monoclonal antibodies are indicated. Merged images show signals from two primary antibodies raised in different species. B. HER2+ breast cancer cells (SKBR3) were treated as in (A), with total cell lysates subjected to fluorescent immunoblotting. C. The primary culture of HER2+ metastatic breast cancer DFBC-1407 were treated with THZ531 at the indicated concentrations for 4 hours, followed by total cell lysate preparation and fluorescent immunoblotting. D. Fluorescent images of cells stained with anti-phospho-RPB1 CTD (Ser2) (clone E1Z3G). HCC1954 cells growing on glass coverslips were treated with vehicle (0.8% DMSO, v/v), 400 nM THZ531, 200 nM NVP2, or 400 nM flavopiridol, fixed with 4% paraformaldehyde, and subjected to staining using the rabbit monoclonal anti-phospho-RPB1 CTD (Ser2) antibody (clone E1Z3G) together with the mouse monoclonal anti-Emerin antibody (clone CL0203). Images were acquired using identical parameters including exposure time and laser intensity on a spinning disk confocal microscope. The images displayed have the identical minimum and maximum displayed values among different treatment groups. Scale bar is 10 micrometers. E. Fluorescent staining for phospho-RPB1 CTD (Ser5) (clone D9N5I) alongside Emerin for nuclear membrane visualization. Cells were treated and stained as in (C). Confocal images were acquired using a 60x objective lens. Note that phospho-RPB1 (Ser5) signal persists in cells treated with THZ531, but diminished in cells exposed to NVP2 or flavopiridol.

Article Snippet: The primary antibodies used include the following: Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523); mouse monoclonal Anti-EMD/Emerin antibody, clone CL0203 (Sigma-Aldrich, #AMAB90562).

Techniques: Western Blot, Bioprocessing, Staining, Microscopy, Membrane

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. Traces of RNA-seq reads for MYC in SKBR3 cells treated with vehicle control (0.4% DMSO, v/v) or THZ531 (200 nM) for 4 h. B. SKBR3 cells were as in (A) followed by total RNA extraction and reverse transcription. Quantitative PCR was performed for the indicated genes. Note that MYC expression is increased meanwhile other selected genes demonstrated significant downregulation. * p<0.05, ** p<0.01, and *** p<0.001 (Student’s t test). C. SKBR3 cells were treated with increasing doses of THZ531 for 4 hours. Cell lysates were prepared in SDS sample buffer and subjected to fluorescent immunoblotting. Clone numbers for monoclonal antibodies used are indicated. D. (top) chemical structures of MFH290 and THZ531. (bottom) HER2+ breast cancer cells HCC1954 were treated with vehicle or increasing doses of MHF-290 for 4 hours. Cells were then lysed with 1x SDS sample buffer, and cell lysates were subjected to fluorescent immunoblotting using the indicated antibodies. Note that 50 nM MFH-290 treatment reduced Ser2 CTD phosphorylation, but increased Ser5 CTD phosphorylation as well the protein abundance of MYC and JUNB. E. HCC1954 cells were treated as in (E) except the use of a-amanitin. The molecular weights of the fluorescent protein markers and clone identities for monoclonal antibodies are indicated. Merged images show signals from two primary antibodies raised in different species. F. THZ531-induced MYC expression relies on de novo protein synthesis. SKBR3 cells were treated with cycloheximide (100 μg/ml), or THZ531 (200 nM), either individually or in combination for three hours. Cell lysates were harvested for fluorescent immunoblotting.

Article Snippet: The primary antibodies used include the following: Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523); mouse monoclonal Anti-EMD/Emerin antibody, clone CL0203 (Sigma-Aldrich, #AMAB90562).

Techniques: RNA Sequencing, Control, RNA Extraction, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Bioprocessing, Phospho-proteomics, Quantitative Proteomics

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. Boxplots depict PRO-seq read density in mRNAs upregulated (N = 117) or downregulated (N = 805) upon THZ531 treatment, as defined in . Reads were counted from +250 nt downstream of the TSS to the transcript end site (TES), and normalized for gene length (reads/kb). Boxes show 25th–75th percentiles and whiskers depict 1.5 times the interquartile range. p values from Wilcoxon matched-pairs signed rank test. B. Metagene plot of average PRO-seq signal in vehicle and THZ531-treated HCC1954 cells across all active mRNA genes > 400 nt (N=11,876). Bins from TSS to TES are scaled to gene length, with 100 bins/gene, and data outside gene bodies are shown as average reads per gene in 200-nt bins. C. Cumulative distribution plot comparing the pausing indices of all genes shown in (B), comparing vehicle to THZ531 treatment. D-E. Metagene analysis of PRO-seq signal at (D) uaRNA loci (N = 9,617) or (E) eRNA loci (N=24,790) upon 2 h of indicated treatment. Data are shown as average reads per TSS in 25-nt bins. F. The distribution of the number of enhancers associated with the 13,530 active genes in HCC1954 cells. Association was based on the nearest active gene to each enhancer TSS. A subset of genes (n = 152; marked by dotted line) contains ≥ 100 associated eTSSs. G. Heat maps rank-ordered by decreasing size of each enhancer region. Shown are histone modifications (H3K27ac), and PRO-seq reads on both strands, with data centered around the dominant eTSS in each enhancer cluster (N = 7,631). H. Metagene plots of average PRO-seq signal in vehicle and THZ531-treated HCC1954 cells across genes defined as upregulated or downregulated in RNA-seq. Data are shown as in (B). I. Top cartoon: Measurements of Pol II diffusion kinetics determined by fluorescence recovery after photobleaching of a narrow strip spanning the nucleus (Strip-FRAP). (Bottom Left) FRAP was performed in untreated MRC-5 GFP-RPB1 KI cells and GFP-RPB1 fluorescence in the strip was background-corrected and normalized to pre-bleach fluorescence intensity and set at 1. The three dotted lines indicate kinetically distinct Pol II fractions. (Bottom Right) Pol II mobility in untreated cells or upon treatment with 400 nM THZ531 for 90 min, or 2 µM THZ1 for 90 min. Mean values from >13 nuclei of two independent experiments were plotted. RFI, Relative fluorescence intensity.

Article Snippet: The primary antibodies used include the following: Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523); mouse monoclonal Anti-EMD/Emerin antibody, clone CL0203 (Sigma-Aldrich, #AMAB90562).

Techniques: RNA Sequencing, Diffusion-based Assay, Fluorescence, Stripping Membranes

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A, B) STAT5 +/+ , STAT5 ΔIEC−/− , and STAT5 ΔIEC+++ mice were inoculated with 1 × 10 3 CFU C. difficile for 4 d. Histopathology of colonic and cecal inflammation was scored. Results are expressed as mean ± SEM, n ≥ 5 mice per group, ** P < 0.01 versus STAT5 +/+ , * P < 0.05 versus STAT5 +/+ . (C) Mice were inoculated with C. difficile at 1 × 10 4 CFU per mouse. Inducible depletion of STAT5 in IECs or IESCs significantly reduced survival following C. difficile infection. Survival was analyzed with Kaplan–Meier estimates, n = 7 mice per group, ** P < 0.01 versus STAT5 +/+ mice. (D) Lgr5Cre ER; VilCre ER;icS5 mice were treated with C. difficile . Lgr5 + IESCs were counted in 200 crypts in colonic mucosa, n ≥ 3 mice per group. Results are expressed as mean ± SEM, * P < 0.05 versus Lgr5Cre ER mice. Representative images of Lgr5 + IESCs in control (Con) and C. difficle colitis are shown. (E, F) The severity of ileitis was scored as neutrophil infiltration, submucosal edema, IEC necrosis, and Paneth cell or goblet cell depletion. Paneth cell depletion or expansion was semi-quantitated in C. difficile -infected STAT5 +/+ , STAT5 ΔIEC−/− , and STAT5 ΔIEC+++ mice. Histological scores show that STAT5 ∆IEC−/− mice display worse ileal inflammation than STAT5 +/+ mice, while STAT5 ∆IEC+++ mice exhibit IEC protection and more regenerated BrdU + IECs. Results are expressed as mean ± SEM, * P < 0.05 versus STAT5 ΔIEC−/− mice, n ≥ 5 mice per group. All results are expressed as mean ± SEM, and t tests and ANOVA were used to compare the significance of a difference.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Histopathology, Infection, Control

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) STAT5 +/+ and STAT5 ΔIEC−/− mice were inoculated with 1 × 10 3 CFU C. difficile for 4 d. Percentage of weight loss in the individual mice was calculated on day 4 before euthanized. Results are expressed as mean ± SEM, t tests was used to compare the significance of a difference, n ≥ 5 per group. (B) Ileal Lgr5-GFP was immune-stained in Lgr5Cre ER, Lgr5Cre ER; VilCre ER;Stat5 and Lgr5Cre ER; VilCre ER;icS5 mice. Representative images are shown, n ≥ 5 mice per group. Scale bar = 200 μm. (C) STAT5 +/+ mice were orally given with three cycles of 7-d 3% DSS treatment with an interval of 5-d water recovery between each cycle of DSS. Mucosal histology was evaluated as a total score including intestinal epithelial damage (0–3), ulceration (0–3) and transmural lesion (0–3). Colonic tissues were isolated and then immune-stained with pYSTAT5 pointed by arrowheads. Representative images are shown. Results are expressed as mean ± SEM, t tests was used to compare the significance of a difference, n = 4 or 5 mice per group.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Staining, Isolation

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: Stat5 or icS5 floxed mice were crossed with VilCre ER. VilCre ER; Stat5 or icS5 mice, and then treated with Tam for 3, 5, or 7 d followed by 5-d Cre recombination. (A) Paneth cells were determined with Lyso immunohistochemistry (IH) and IF staining. Average numbers of Lyso + Paneth cells were counted in 200 crypts, and the size of Paneth niche was measured as volume with confocal microscopy . (B) Intestinal crypts were isolated, total proteins were extracted, and immunoblotting was performed to determine Lyso and STAT5a protein expression. Densitometry was used to determine the expression of Lyso relative to β-actin. Results are expressed as mean ± SEM, n ≥ 5 mice per group. (C) Jejunal and ileal crypts were isolated and stained with Lyso IH and AB. Lyso + crypt cells were counted in 200 isolated crypts. (D) Real-time PCR was performed to determine anti-microbial peptide expression in STAT5 +/+ and STAT5 ΔIEC+++ mice. (E) Ectopic Paneth cells were counted as the number of migrated Lyso + IECs from crypt bases to villi in 3, 5, and 7 d Tam-treated VilCre ER or 5 d Tam-treated Rs26Cre ER or VilCre ;icS5 mice. Scale = 200 μm. (F) Over 200 villi and crypts were counted. Results are expressed as mean ± SEM, n ≥ 5 mice per group. (G) Colonic crypts were double-stained with anti-Lgr5 (green) and anti-cKit (red), and 3D images were captured with confocal microscopy, n > 3 mice each group. (H) Colonic crypts were disassociated with TrypLE into IECs. Lgr5-GFP − and + IECs were then separated by gating with FACS. PE-Cy7-conjugated pYSTAT5 and APC-conjugated CD44 staining were used to quantitate pYSTAT5 + Lgr5 − CD44 + colonic crypt IECs, n = 4 mice per group, ** P < 0.01 versus STAT5 +/+ . All results are expressed as mean ± SEM, and t tests and ANOVA were used to compare the significance of a difference.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Immunohistochemistry, Staining, Confocal Microscopy, Isolation, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Lgr5Cre ER mice were crossed with LacZ mice to generate a mouse line (Lgr5-LacZ), then crossed with Stat5 floxed mice (Lgr5-LacZ;Stat5). Lgr5-LacZ;Stat5 mice were used for determining the effects of Stat5 on IESC self-renewal upon a single dose of Tam. (B, C) Jejunal and ileal lineage tracing was done and analyzed 14 d after a single dose of Tam (25 mg/kg), n = 3 per group. (D) Whole colon fragments were isolated from Lgr5-LacZ and Lgr5-LacZ;Stat5 mice and then stained with X-gal. These stained colonic fragments were sectioned and counter-stained with eosin. The LacZ + colonic crypts were counted, n = 3 per group. (E) Enteroids prior to or after 4HT induction were fixed and sectioned. Sox9 + cells (red) were immunostained and representative images are shown. (F) Enteroids prior to or after 4HT induction were frozen. Total RNA was extracted from the enteroids, the expression of Sox9 was quantitated by real-time PCR. Results are expressed as mean ± SEM, t tests was used to compare the significance of a difference, n = 4 or 5 wells of enteroids per group. * P < 0.01 versus Lgr5- Rosa mT/mG . (G) Ileal sections were stained with pYSTAT5 (green) and Ki67 (red) IF. n ≥ 5 mice per group. Scale bar = 200 μm

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Isolation, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: Intestinal crypts were extracted from Lgr5Cre ER; VilCre ER;Stat5 or icS5 and dissociated with TrypLE into IECs. Lgr5 − , low and high IECs were then separated by gating with FACS . (A) APC-conjugated pYSTAT5 and PE-conjugated CD24 staining were used to quantitate pYSTAT5 + Lgr5 − CD24 + cells (P1) or Lgr5 − CD24 + Paneth cells (P2). (B) Lgr5 hi :CD24 + doublets in the dissociated IECs were determined by FACS. n = 4 or 5 mice per group, ** P < 0.01 versus STAT5 +/+ . (C) The dissociated IECs were immunostained with CD24, and co-localization of Lgr5 and CD24 was determined with a confocal microscope. Lgr5:CD24 doublets are shown as circles. (D) Lgr5-GFP crypts from Lgr5Cre ER or Lgr5Cre ER; VilCre ER;icS5 mice were employed for IESC culture for 4 d, and pYSTAT5 was inducibly activated by 4HT (200 nM) after one dose of γ-irradiation (IR). The numbers of Lgr5-GFP buds were counted in each well, and budding curve was created 6 d after 4HT treatment. ** P < 0.01 and * P < 0.05 versus controls without 4HT induction. Representative images of Lgr5GFP + buds (arrows) in the enteroids (circles) are shown, *Autofluorescence. (E, F) Lgr5-GFP enteroids were dissociated into IECs. Lgr5 hi Ki67 + pYSTAT5 + (E) and Lgr5 low Ki67 + pYSTAT5 + (F) were determined. * P < 0.05 versus enteroids from Lgr5Cre ER; VilCre ER, # P < 0.05 versus enteroids from IR-treated Lgr5Cre ER; VilCre ER, n = 4–5 mice per group. (G) Colonic crypts were isolated and differentiated into colonoids. These colonoids were induced by 4HT for 4 d and then irradiated at 4 Gy for 10 min. Lgr5 (green) buds are shown; arrowheads indicate crypt budding. GFP crypt buds were counted per colonoids (n ≥ 20) from each of six wells from three independent experiments. All results are expressed as mean ± SEM, and t tests and ANOVA were used to compare the significance of a difference.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Staining, Microscopy, Irradiation, Isolation

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Intestinal crypts were extracted from Lgr5Cre ER; VilCre ER;Stat5 or icS5 mice, ileal crypt IECs were dissociated. Lgr5 low and high IESCs were then separated by gating for FACS analysis. (B) The dissociated IECs were immune-stained by Lgr5 (green) and CD24 (red) to show Lgr5 singlets (green) and Lgr5:CD24 doublets (orange) as shown. (C) Quantification of enteroid budding: Enteroids were stained with Methylene Blue, the number of crypt buds per enteroids was counted, n ≥ 20 enteroids per well, six wells per mouse, and three mice per group. (D) Colonic crypts were disassociated and cultured into colonoids with conditioned medium (1 μg/ml R-Spondin, 100 ng/ml Noggin, 50 ng/ml EGF and 100 ng/ml Wnt3a). After 7-d growth, colonoids were respectively treated with different doses of SCF: 5, 10 and 20 ng/ml for 10 d. (E) The number of crypt buds per colonoids was counted, n ≥ 20 colonoids per well, six wells per mouse, and three mice per group. Results are expressed as mean ± SEM. One-way ANOVA was used to analyze the difference between groups. ** P ≤ 0.01 versus Vehicle control.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Staining, Cell Culture, Control

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Enteroids were cultured from intact intestinal crypts dissociated from VilCre ER, VilCreCre ER ;Stat5 +/ − and VilCre R;icS5 mice (STAT5 +/+ , STAT5 ΔIEC+/− and STAT5 ΔIEC+++ ). Based on the number of buds, enteroid morphology was categorized as: 1 bud (Org 1 ), 2 buds (Org 2 ), greater than 3 buds (Org3 + ) and no buds (Sphere). Representative images are shown. Results were expressed as mean ± SEM, t tests and ANOVA were used to compare the significance of a difference, n = 4 or 5 mice per group. (B) Enteroids were cultured from intact intestinal crypts dissociated from STAT5 +/+ , STAT5 ΔIEC−/− , STAT5 ΔIEC+/− and STAT5 ΔIEC+++ mice. The number of grown enteroids were counted with or without TNF-α treatment, the percentage of survival enteroids versus initial grown enteroids was calculated. Results were expressed as mean ± SEM, t tests was used to compare the significance of a difference, n = 4 or 5 mice per group. (C) Intestinal crypts were disassociated from Lgr5Cre ER and Lgr5Cre ER;icS5 mice, Lgr5 hi IESCs were sorted and representative FACS and Lgr5-GFP IF image are shown.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Cell Culture

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Inducible STAT5a mutant constructs. STAT5a domains, critical phosphorylation sites and the GOF mutation Ser710/Phe (icS5), which causes enhanced and prolonged tyrosine phosphorylation upon cytokine/growth factor action. Below, Tam inducible STAT5a-ER fusion protein that dimerizes to physiologically activate STAT5 target genes in a Tam dose dependent manner. (B) H9 iPSCs were transduced with a lentiviral GFP construct for STAT5a-ER and icS5-ER mutant. HIOs were matured in vitro for 35 d. Transduced HIOs were then microinjected 250 ng FD 4 (FD4) with IFNγ (10 ng/ml) and TNFα (10 ng/ml), or TcdA (400 ng/ml), or TcdB (400 ng/ml). Trans-membrane permeability was determined as the amount of FD4 diffusion from the HIO lumen to culture media 24-h IFNγ + TNFα or 6-h TcdA or TcdB after 4HT (200 nM) induction. Results are expressed as mean ± SEM, t tests and ANOVA were used to compare the significance of a difference, n ≥ 4 HIOs per group, * P < 0.05 versus control HIOs. (C) Total proteins were extracted from single HIOs, and LGR5, Bmi1, Dclk1, Lyso, inter-cellular junctional proteins (E-Cad, JAMA-A, ZO-1 and 2, claudin-1 and 2, and occludin) and Wnt/Notch pathway markers (β-catenin and intracellular domain of the Notch protein [NICD]) were measured using immunoblotting. n ≥ 4 HIOs per group.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Mutagenesis, Construct, Phospho-proteomics, Transduction, In Vitro, Membrane, Permeability, Diffusion-based Assay, Control, Western Blot

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: iPSCs or LGR5:eGFP BAC reporter iPSCs were transduced with a lentiviral GFP construct for STAT5a-ER and icS5-ER mutant. HIOs were in vitro matured for 35 d. (A) Transduced HIOs were imaged under 3D in vitro culture systems with dark-field and bright-field 3D confocal deconvolution microscopy. IEC types in HIOs were determined by double-IF, E-Cadherin (E-Cad) + Chromogranin A (CHGA) or E-Cad + Muc2, and Lyso IF staining. (B) Transduced HIOs were then microinjected 250 ng FD 4 (FD4) with 0.25 ng TNFα (100 ng/ml) or 25 ng TcdA or 50 ng TcdB after 3-d 4HT induction. HIO integrity was determined as the TEER after 24-h TNFα or 6-h TcdA or TcdB treatment. Results are expressed as mean ± SEM. * P < 0.05 versus control HIOs. n ≥ 4 HIOs per group. (C) NEs were extracted from single HIOs, and pYSTAT5 and STAT5 were measured using immunoblotting. n ≥ 4 HIOs per group. (D) Transduced HIOs were matured and then transplanted beneath the kidney capsule of NSG mice. Tam induction was performed for 5 d 1-mo post-engraftment. (E) Proliferation of transplanted IESCs was determined with anti-Ascl2 and BrdU IH in the presence and absence of icS5 activation, n = 5 per group. (F, G) LGR5:eGFP BAC reporter iPSCs were transduced with lenti-viral STAT5a-ER or icS5-ER. 3, 7, or 14 d after one dose of Tam, transplanted mice were euthanized. Transplanted HIOs were stained with GFP IF, and IESCs were labeled with LGR5-GFP and EdU. icS5 activation increased LGR5 + IESCs and enhanced LGR5 lineage tracing compared with that in STAT5a-ER. n ≥ 3 mice per group. (H) Transplanted HIOs were stained with AB, Alkaline phosphatase (ALP), Lyso, and Muc2 IH. AB + , Lyso + , or Muc2 + crypt cells were counted as average numbers per crypt. 100 well-orientated crypts were chosen from five mice per group, ** P < 0.01 versus STAT5a-ER. (I) Some of the transplanted mice were subjected to 12-Gy irradiation. Regenerated crypts (RC) were counted in the transplanted HIOs. Inserts are the images at a higher-magnification. Intestines from the irradiated mice show no RC. ** P < 0.01 versus STAT5a-ER, n ≥ 5 mice per group, scale = 200 μm. All results are expressed as mean ± SEM, and t tests and ANOVA were used to compare the significance of a difference.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Transduction, Construct, Mutagenesis, In Vitro, Microscopy, Staining, Control, Western Blot, Activation Assay, Labeling, Irradiation

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Hierarchical clustering of 691 genes with ANOVA P < 0.05 and fold change > 2 in at least one pairwise comparison between STAT5 +/+ , STAT5 ΔIEC−/− , and STAT5 ΔIEC+++ mice, using Pearson’s centered distance metric and average linkage rule. (B) Scatterplots of log (FPKM) of differentially regulated genes in STAT5 ΔIEC−/− and STAT5 ΔIEC+++ mice when compared to STAT5 +/++ mice. (C) β-catenin was determined by IH and immunoblotting with NE from intestinal crypts. Results are expressed as mean ± SEM, n ≥ 3 mice per groups, and t tests were used to compare the significant difference. (D) Sox9 was immunostained and quantitated in the 200 crypts. Results are expressed as mean ± SEM, n ≥ 3 mice per groups, and t tests were used to compare the significant difference. (E) icS5-ER-transduced HIOs were in vitro matured for 35 d. icS5 activation in HIOs was induced with different doses of 4HT (0, 10, 50, 100, or 200 nM or 1 mM) for 72 h. pYSTAT5, STAT5A, and β-catenin were determined by immunoblotting. This experiment was repeated three times. (F) Hypothesis model: pYSTAT5 amplifies Lgr5 hi →to Lgr5 Low Ki67 + IESCs; Ca-pYSTAT5 promotes Lgr5 low Ki67 + IESCs to give rise to a sublineage of crypt cells, intestinal pYSTAT5 + Lgr5 − CD24 + Lyso + Paneth cells by activating Wnt/β-catenin/Sox9 pathway.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Comparison, Western Blot, In Vitro, Activation Assay

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A) Venn diagram to identify differentially regulated the genes shared between and specific to STAT5 ΔIEC−/− and STAT5 ΔIEC+++ as compared to STAT5 +/+ . (B, C) Barchart of significance of pathways and biological processes enriched in genes significantly up- and down-regulated in STAT5 ΔIEC+++ and STAT5 ΔIEC−/− as compared to STAT5 +/+ . Ontologies were identified through ToppGene.cchmc.org . (D) Diagram of ontological enrichments associated with gene lists built through comparisons between STAT5 ΔIEC−/− and STAT5 ΔIEC+++ , when compared to STAT5 +/+ , showing shared and specific ontologies connected to up- and down-regulated genes, generated by Toppcluster.cchmc.org and cytoscape. (E) Hierarchical clustering of averaged normalized expression values of 13 genes in STAT5 ΔIEC−/− and STAT5 ΔIEC+++ mice compared to STAT5 +/+ mice identified significantly enriched canonical Paneth cell genes regulated by STAT5 protein. (F) Representative images of colonic crypts used for RNA-seq.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques: Generated, Expressing, RNA Sequencing

Journal: Life Science Alliance

Article Title: Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis

doi: 10.26508/lsa.201900296

Figure Lengend Snippet: (A–C) PCR was used to genotype VilCr eER and Stat5 f/f (A), VilCre and icS5 (B), VilCre ER and icS5 mice. Representative PCR gels are shown.

Article Snippet: Antibodies specific for tyrosine phosphorylation–specific STAT5 antibodies (pYSTAT5, Rabbit mAb #9314), cKit (#3074), and Phospho-cKit (pckit, #3391) were purchased from Cell Signaling Technology, antibodies specific for Lgr5 from OriGene (TA503316), antibodies specific for Bmi1 (MAB33341) and ASCL2 (AF6539) from R&D, antibodies specific for Lysozyme (A0099) and Ki67 (M7249) from Dako, antibodies specific for GFP (ab13970), Dclk1 (ab31704), and another Lgr5 antibodies (ab75850) from Abcam, antibodies specific for STAT5a (71-2400) and STAT5b (71-2500) from Zymed Laboratories (Life Technologies), and antibodies specific for STAT5 and β-tubulin from Santa Cruz Biotechnology (sc-835).

Techniques:

Journal: PLoS ONE

Article Title: Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis

doi: 10.1371/journal.pone.0137217

Figure Lengend Snippet: (A) Scheme of the DNA construct of profilin 1 mutants. (B) Restriction analysis of the DNA construct ligated into the pcDNA3 cloning vector using EcoRI and BamHI. Line 1: GeneRuler 1 kb DNA Ladder (Promega), line 2: restriction of Pfn1-Tyr139 mutant, line 3: restriction of Pfn1-Q138P mutant. (C) Western blot analysis of transfected PC-3 cell lysates expressing different profilin 1 mutants. Cell lysates were prepared 48 h post transfection. Line 1: size marker (SeeBlue® Pre-stained Protein Standard, Life Technologies), line 2: transfected empty pcDNA3 vector, line 3: transfected Pfn1-Tyr139/pcDNA3, line 4: transfected Pfn1-Q138P/pcDNA3. Mutants were detected with anti-FLAG antibodies. Detection of profilin 1 (native and mutated) and β-actin on the same membrane is also shown. Due to the chemiluminescent detection, size marker is added as a separate strip.

Article Snippet: Mutated profilin 1 nucleotide sequences were excised with EcoRI (New England Biolabs) and Bam HI (New England Biolabs), ligated into pcDNA3 vector (Invitrogen) and transformed in E . coli TOP10 cells.

Techniques: Construct, Clone Assay, Plasmid Preparation, Mutagenesis, Western Blot, Transfection, Expressing, Marker, Staining, Stripping Membranes

Journal: PLoS ONE

Article Title: Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis

doi: 10.1371/journal.pone.0137217

Figure Lengend Snippet: The level of cell migration/invasion, assessed by increases in the curve slopes (1/h), is shown. All results were normalized to cells transfected with empty pcDNA3 vector (the control). Assays were carried out in triplicate. (A) Results from migration were calculated for the interval between the 45 hour and 98 hour time points, during which the slopes of the curves were linear. (B) Results from invasion were calculated for the interval between the 49 hour and 107 hour time points. (C) The influence of AMS36 inhibitor (10 μM) and recombinant cathepsin X (2 μM) was tested on the cells transfected with Pfn1-Tyr139 mutant. Results of migration were calculated for the interval between the 29 hour and 38 hour time points. *P≤0.05 **P<0.01; ***P<0.001.

Article Snippet: Mutated profilin 1 nucleotide sequences were excised with EcoRI (New England Biolabs) and Bam HI (New England Biolabs), ligated into pcDNA3 vector (Invitrogen) and transformed in E . coli TOP10 cells.

Techniques: Migration, Transfection, Plasmid Preparation, Recombinant, Mutagenesis

Journal: PLoS ONE

Article Title: Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis

doi: 10.1371/journal.pone.0137217

Figure Lengend Snippet: Cells were treated with cathepsin X inhibitor AMS36 (10 μM) or DMSO. Profilin 1—clathrin complexes were detected with a proximity ligation assay and analyzed with confocal microscopy. Each red dot represents a Texas red signal present on the spot with the complex. Cell nuclei were stained with DAPI. Bar, 20 μm.

Article Snippet: Mutated profilin 1 nucleotide sequences were excised with EcoRI (New England Biolabs) and Bam HI (New England Biolabs), ligated into pcDNA3 vector (Invitrogen) and transformed in E . coli TOP10 cells.

Techniques: Proximity Ligation Assay, Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis

doi: 10.1371/journal.pone.0137217

Figure Lengend Snippet: (A) Cells were transfected with two plasmids carrying different profilin 1 mutants and empty plasmid (control). Clathrin-mediated endocytosis of fluorescein-labeled dextran (10 kDa) was followed with flow cytometry. Median values are representative of two independent experiments (one in triplicate and one in duplicate). *P<0.05 (B) Cells were transfected with different profilin 1 mutant constructs. After 48 hours filamentous actin was stained with phalloidin conjugate and analyzed with flow cytometry. Mean values are representative of two independent experiments (one in triplicate and one in duplicate). *P<0.05.

Article Snippet: Mutated profilin 1 nucleotide sequences were excised with EcoRI (New England Biolabs) and Bam HI (New England Biolabs), ligated into pcDNA3 vector (Invitrogen) and transformed in E . coli TOP10 cells.

Techniques: Transfection, Plasmid Preparation, Labeling, Flow Cytometry, Mutagenesis, Construct, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Primers Used for qRT-PCR

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Generation of RPE-specific PNPLA 2-cKO mice. ( A ) Scheme of Pnpla 2 floxed and Cre-mediated recombined allele. The loxP sites flank exon 1. P1 and P2 are the primers homologous to sequences outside the floxed region (flanked by the loxP sites) used to detect Cre-mediated recombination (generating recombined alleles) on genomic DNA. The sizes of the amplicons obtained by PCR using P1 and P2 are indicated. ( B ) Gel electrophoresis of PCR reaction products obtained using primers P1 and P2 and genomic DNA isolated from mouse eyecups from either cKO or control (Ctr) mice ( Pnpla 2 f/+ ); lane 1 (MW) corresponds to molecular weight markers (GeneRuler DNA Ladder Mix). One eyecup per lane from a 4-month-old mouse ( n = 2 cKO, n = 2 Ctr). ( C ) Pnpla 2 expression (vs. HPRT ) in RPE from 1-month-old cKO mice ( Pnpla 2 f/f/Cre ) relative to control littermates ( Pnpla 2 f/f ). Each data point corresponds to the average of six PCR reactions per eyecup, six eyes from three cKO mice and six eyes from three control mice at 5 to 7 months old. ( D ) Cre ( red ) and phalloidin ( yellow ) labeling of RPE/choroid flatmounts from control ( Pnpla2 f/f ) ( left ) and littermate cKO (Pnpla2 f/f/Cre ) ( right ) mice ( n = 2 images from individual mouse eyecup at 11–14 months old). Scale bar : 20 µm. (E) Plot of percentage of Cre-positive RPE cells in cKO animals ( Pnpla2 f/f/Cre ; n = 10; age, 10.5–18.5 months old) as indicated in the x -axis. Each data point corresponds to percentage of Cre-positive RPE cells from an ROI, each bar corresponds to a flatmount of an individual cKO mouse, and the bar for control ( Pnpla2 f/f ) has data from 10 mice.

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Nucleic Acid Electrophoresis, Isolation, Control, Molecular Weight, Expressing, Labeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Lipid accumulation in the RPE of Pnpla2 -cKO mice. Electron microscopy micrographs showing the RPE structure of 3-month-old ( A ) and 13-month-old ( B ) cKO mice and control animals. Scale bar : 2 µm. The representative images were selected among examinations of micrographs from eight eyes of cKO mice ( PNPLA2 f/f/Cre+ ), from seven eyes of PNPLA2 f/f control mice at 1.75 to 3.75 months old, and from three eyes of cKO mice and three eyes of control mice at 12.5 to 13 months old. LD, lipid droplets; BI, basal infoldings.

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Electron Microscopy, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Phagocytosis and β-hydroxybutyrate production in the RPE of Pnpla2 -cKO mice. ( A ) Representative ROIs of the eyecup from one control and one cKO animal isolated at 2 hours (8 AM) and 5 hours (11 AM) after light onset (6 AM) after immunolabeling for rhodopsin ( green ), phalloidin ( yellow ), and Cre ( red ). The column to the right shows magnification of an area. The mean of rhodopsin immunolabel intensity in micrographs ( n ≥ 6 ROIs) from flatmounts (as indicated in the x -axis) relative to control at 2 hours was determined among three mice per condition and is shown in the plot. Age of mice was 10.5 to 18.5 months. ( B ) Ex vivo β-HB release by the RPE of Pnpla 2-cKO eyecups upon ingestion of OSs in comparison to that of controls. Eyecups were isolated at 5 hours (11 AM) and 8 hours (2 PM) after light onset (6 AM). Statistical significance was calculated using two-way ANOVA for the two groups (controls and cKO mice) with and without treatment (second variance) for each time after light onset. * P = 0.02, ** P = 0.006, *** P = 0.0001; ns, not significant; n = 6 eyecups from three control (f/+) mice at 3.5 months; n = 4 eyecups from two control (f/f/Cre–) mice at 3.5 months; n = 10 eyecups from five mice (f/f/Cre+) at 2.75 to 3.5 months. ( C ) The OS-mediated increase in β-HB release above base levels of the cKO RPE/choroid explants was calculated from the data in panel C and plotted.

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Control, Isolation, Immunolabeling, Ex Vivo, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: RPE and retinal functionality in RPE- Pnpla2- cKO mice. ( A ) ERG amplitude graphs of scotopic a- and b-waves and photopic b-waves, as a function of light intensity ( x -axis) of 3- and 12-month-old cKO mice ( open circles ) and littermate controls ( Pnpla2 f/f , closed circles ) ( n = 3 per genotype). ( B ) Bar graph showing the amplitude (mean, SD) of the c-wave, fast oscillation (FO), light peak (LP), and off-response (OFF) measured by DC-ERG of 11-week-old cKO mice ( n = 4, open bars ) and Pnpla2 f/f and Pnpla2 f/+ control mice ( n = 5, closed bars ).

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Knockdown of PNPLA2 in ARPE-19 cells. ARPE-19 cells were transfected with Scr or siRNAs targeting PNPLA2 , and mRNA levels and protein were tested. ( A ) RT-qPCR to measure PNPLA2 mRNA levels in ARPE-19 cells 72 hours after transfection with Scr and six different siRNAs (as indicated on the x -axis) was performed, and a plot is shown. PNPLA2 mRNA levels were normalized to 18S. All siRNA are represented as the percentage of the scrambled siRNA control ( n = 3). ( B ) A plot is shown for a time course of PNPLA2 mRNA levels following transfection with Scr and siPNPLA2-C ( n = 3 ( C ) RT-qPCR of mock-transfected cells, cells transfected with Scr, and siPNPLA2-C ( x -axis) at 72 hours after transfection. mRNA levels were normalized to the 18S RNA ( y -axis) ( n = 3). ( D ) Total protein was obtained from cells harvested 72 hours after transfection and resolved by SDS-PAGE followed by western blotting with anti-PNPLA2 and anti-GAPDH (loading control). The siRNAs used in the transfections are indicated at the top, and migration positions for PEDF-R and GAPDH are to the right of the blot. Data are presented as mean ± SD. ** P < 0.01, *** P < 0.001, *** P < 0.001.

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Control, SDS Page, Western Blot, Migration

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Phagocytosis and fatty acid metabolism in si PNPLA2 cells. ARPE-19 cells were transfected with Scr or siRNAs targeting PNPLA2. At 72 hours after transfection, ARPE-19 cells were incubated with POSs (1 × 10 7 units/mL) in 24-well tissue culture plates for pulse–chase experiments. ( A ) Representative immunoblot of total lysates of ARPE-19 cells at 0.5 hour, 1 hour, and 2.5 hours of POS pulse and at 16-hour and 24-hour chase periods, as indicated at the top of the blot. Proteins in cell lysates were subjected to immunoblotting with anti-rhodopsin followed by reprobing with anti-GAPDH as the loading control. ( B ) Quantification of rhodopsin from duplicate samples and three blots of cell lysates from pulse–chase experiments and time periods (indicated in the x -axis) as from panel. Data are presented as mean ± S.D; ** P < 0.01; ns, not significant. Intensities of the immunoreactive bands were determined, and the percentages of the remaining rhodopsin after 16-hour and 24-hour chase periods relative to rhodopsin at 2.5-hour pulse are plotted ( y -axis). ( C , D ) Levels of secreted free fatty acids ( C ) and β-HB ( D ) were measured in culture media of cells transfected with Scr or siPNPLA2 following incubation with POS for the indicated periods of times ( x -axis). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01 ( n = 3). Duplex si PNPLA2 C was used to generate the data (see Table 3 for sequences of duplexes).

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Transfection, Incubation, Pulse Chase, Western Blot, Control

Journal: Journal of Innate Immunity

Article Title: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

doi: 10.1159/000524461

Figure Lengend Snippet: LPS-mediated temporal expression changes of TLR10. a Time-dependent expression of TLR10 (in green) in human neutrophils (1 × 10 6 ). Nuclei stained in blue with DAPI. Neutrophils were treated with 1-μg/mL LPS for 60, 90, and 120 min ( n = 3). Imaged the temporal expression changes and localization of TLR10 expression using confocal microscopy (magnification, 630; scale bar, 6 μm). b Neutrophils stimulated with LPS (1 μg/mL) for 60, 90, and 120 min and stained with antibodies against TLR10 and isotype-matching antibody for flow cytometry analysis. FITC-TLR10 fluorescent spectrum shift (qualitative) was used to analyze TLR10 surface expression changes. c Immunoblots lysates of neutrophils (2 × 10 6 ). Cells were stimulated with LPS (1 μg/mL) for 60 min, 90 min, and 120 min. Molecular weight is depicted on the left side of the blots. β-Actin showed in the lower panel referred as loading control. d Densitometry analysis showed the downregulation of TLR10 expression in neutrophils treated with 90 min and increased gene expression in LPS 120 min (* p < 0.05, compared with control; ** p < 0.05, compared with LPS 60 min). One representative experiment of three in the above experiments is shown.

Article Snippet: In brief, 80 nM of siRNA for TLR10 (sc-40272; Santa Cruz Biotechnology) and missense oligonucleotide negative control (sc-36869; Santa Cruz Biotechnology) were incubated with transfection reagent for 45 min to form transfection reagent-siRNA complex and added to the cells washed with transfection medium.

Techniques: Expressing, Staining, Confocal Microscopy, Flow Cytometry, Western Blot, Molecular Weight, Control, Gene Expression

Journal: Journal of Innate Immunity

Article Title: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

doi: 10.1159/000524461

Figure Lengend Snippet: Lipid raft-mediated endocytosis of TLR10. a , b Human neutrophils (1 × 10 6 ) adhered to FBS-coated coverslips were activated by LPS (1 μg/mL). Colocalization of TLR10 (red) and early endosomal antigen, EEA1 (green), are shown in merge panel. Treatment time points were 60 min, 90 min, and 120 min (magnification, 630; scale bar, 6 μm). Lower panel shows the graphical representation of quantification of colocalization in terms of Pearson's coefficient (* p < 0.05), analyzed by Imaris 7.4 (Bitplane Inc., Concord, MA, USA) using ImarisColoc module. c , d Colocalization of TLR10 (green) and flotillin-1, lipid raft marker (red) in LPS (1 μg/mL)-treated human neutrophils (1 × 10 6 ) adhered to FBS-coated coverslips. Intact plasma membrane in control cells and membrane rearrangement during 60–120 min was observed (magnification, 630; scale bar, 6 μm). Lower panel shows the graphical representation of quantification of degree of colocalization in terms of Pearson's coefficient (* p < 0.05), analyzed by Imaris 7.4 (Bitplane Inc., USA) using ImarisColoc module. Results in ( a–d ) show representative data of three independent experiments.

Article Snippet: In brief, 80 nM of siRNA for TLR10 (sc-40272; Santa Cruz Biotechnology) and missense oligonucleotide negative control (sc-36869; Santa Cruz Biotechnology) were incubated with transfection reagent for 45 min to form transfection reagent-siRNA complex and added to the cells washed with transfection medium.

Techniques: Marker, Clinical Proteomics, Membrane, Control

Journal: Journal of Innate Immunity

Article Title: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

doi: 10.1159/000524461

Figure Lengend Snippet: Ultra-localization of TLR10 in human neutrophils. a–c Immuno-gold electron microscopy for TLR10 in human neutrophils shows the presence of TLR10 in nucleus, cytoplasm, as well as in the plasma membrane (red arrows). Note the TLR10 localization in pseudopodia of E. coli LPS (1 μg/mL)-activated neutrophils in panels ( b , c ). Magnification, 13,000.

Article Snippet: In brief, 80 nM of siRNA for TLR10 (sc-40272; Santa Cruz Biotechnology) and missense oligonucleotide negative control (sc-36869; Santa Cruz Biotechnology) were incubated with transfection reagent for 45 min to form transfection reagent-siRNA complex and added to the cells washed with transfection medium.

Techniques: Electron Microscopy, Clinical Proteomics, Membrane

Journal: Journal of Innate Immunity

Article Title: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

doi: 10.1159/000524461

Figure Lengend Snippet: TLR10 colocalized with TLR4 on LPS challenge. a Isolated human neutrophils (1 × 10 6 ) adhered on FBS-coated coverslips were challenged with LPS (1 μg/mL) for 60 min, 90 min, and 120 min and examined by confocal microscopy for the colocalization of TLR10 (in green) and TLR4 (in red). Merged channel indicates the overlapping signals from TLR10 and TLR4 along with nuclear stain DAPI (magnification, 630; scale bar, 5 μm). b Quantification of colocalization in terms of Pearson's coefficient analyzed by Imaris 7.4 (Bitplane Inc.) using ImarisColoc module (* p < 0.05, compared with the control, and ** p < 0.05, compared with LPS 60 min, and all data shown in terms of mean ± SEM). One representative of three in the above different experiments is shown.

Article Snippet: In brief, 80 nM of siRNA for TLR10 (sc-40272; Santa Cruz Biotechnology) and missense oligonucleotide negative control (sc-36869; Santa Cruz Biotechnology) were incubated with transfection reagent for 45 min to form transfection reagent-siRNA complex and added to the cells washed with transfection medium.

Techniques: Isolation, Confocal Microscopy, Staining, Control

Journal: Journal of Innate Immunity

Article Title: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

doi: 10.1159/000524461

Figure Lengend Snippet: TLR4 neutralization, ROS depletion, and NF-κB inhibition reduced TLR10 expression. a Isolated human neutrophils (1 × 10 6 ) treated for TLR4 neutralization and activated using bacterial LPS (1 μg/mL). TLR10 (in red) was imaged using confocal microscopy (magnification, 630; scale bar, 5 μm). b Quantification of fluorescence in terms of corrected total-cell fluorescence analyzed by Image J v1.47 (nih.gov, Bethesda, MD, USA) using grey scale intensity analysis. (* p < 0.05, compared with the control and all data shown in terms of mean ± SEM). c Human neutrophils (1 × 10 6 ) pretreated with FCCP , 5 μg/mL, for 1 h at 37°C to deplete ROS production. FCCP pretreated cells were challenged with LPS (1 μg/mL) for 60 min and imaged for TLR10 (in red) using confocal microscopy (magnification, 630; scale bar, 5 μm). d Isolated human neutrophils (1 × 10 6 ) treated for NF-κB inhibitor and treated using bacterial LPS. TLR10 (in green) was imaged using confocal microscopy and merged image in the third column indicates the expression and cytoplasmic localization of TLR10 in control, LPS 60-min treated as well as NF-κB inhibitor pretreated cells challenged with bacterial LPS for 60 min (magnification, 630; scale bar, 4 μm).

Article Snippet: In brief, 80 nM of siRNA for TLR10 (sc-40272; Santa Cruz Biotechnology) and missense oligonucleotide negative control (sc-36869; Santa Cruz Biotechnology) were incubated with transfection reagent for 45 min to form transfection reagent-siRNA complex and added to the cells washed with transfection medium.

Techniques: Neutralization, Inhibition, Expressing, Isolation, Confocal Microscopy, Fluorescence, Control

Journal: Journal of Innate Immunity

Article Title: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

doi: 10.1159/000524461

Figure Lengend Snippet: Role of TLR10 in neutrophil chemotaxis. a Frames captured from live single-cell imaging of TLR10 (labeled in green) staining in human neutrophils. Neutrophils were activated with concentration gradient of LPS (1 μg/mL) attained by pipette tip diffusion and imaged for 100 min (magnification, 630; scale bar, 6 μm). b HL-60 cell line was transfected with 80 nM of TLR10 siRNA (sc-40272; Santa Cruz Biotechnology) using liposome-mediated transfection (Santa Cruz Biotechnology, 10410 Dallas, TX, USA) after obtaining 80% confluence in culture. HL-60 differentiation into neutrophils was achieved by the incubation with 1.3% DMSO for 5 days. Isolated mRNA used for quantitative real time PCR and calculated the fold change using ΔΔCt method. c Immunoblots lysates of HL60-differentiated neutrophils (2 × 10 6 ). Cells were treated with 20 nM, 40 nM, and 80 nM of TLR10 siRNA and total protein isolate was hybridized against anti-TLR10 antibody. Molecular weight is depicted on the left side of the blots. d Chemotaxis experiment was performed using Boyden chamber with control, TLR10 knockdown, LPS treated (60 min; 1 μg/mL), and TLR10 knockdown + LPS treated (60 min; 1 μg/mL). Migrated cells from at least 5 different fields were counted and tallied. One representative of two in the above experiments was shown.

Article Snippet: In brief, 80 nM of siRNA for TLR10 (sc-40272; Santa Cruz Biotechnology) and missense oligonucleotide negative control (sc-36869; Santa Cruz Biotechnology) were incubated with transfection reagent for 45 min to form transfection reagent-siRNA complex and added to the cells washed with transfection medium.

Techniques: Chemotaxis Assay, Imaging, Labeling, Staining, Concentration Assay, Transferring, Diffusion-based Assay, Transfection, Incubation, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Molecular Weight, Control, Knockdown

Journal: Journal of Innate Immunity

Article Title: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

doi: 10.1159/000524461

Figure Lengend Snippet: TLR10 does not affect actin nucleation. a Immunoblots lysates of HL-60-derived neutrophils (2 × 10 6 ). TLR10 gene knockdown was performed as above and cells were stimulated with LPS (1 μg/mL) for 60 min. Actin nucleation proteins ARP3 (1:500) and Diap1 (1:400) were detected in the blot corresponding to the specific molecular weight. Molecular weight is depicted on the left side of the blots. β-Actin showed in the lower panel referred as loading control. b , c Densitometry analysis showed no significant change in actin nucleation proteins.

Article Snippet: In brief, 80 nM of siRNA for TLR10 (sc-40272; Santa Cruz Biotechnology) and missense oligonucleotide negative control (sc-36869; Santa Cruz Biotechnology) were incubated with transfection reagent for 45 min to form transfection reagent-siRNA complex and added to the cells washed with transfection medium.

Techniques: Western Blot, Derivative Assay, Knockdown, Molecular Weight, Control

Journal: Journal of Innate Immunity

Article Title: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

doi: 10.1159/000524461

Figure Lengend Snippet: TLR10 knockdown decreased formation of pseudopodia. Control ( a ) and TLR10-silenced ( b ) HL-60-derived neutrophils (1 × 10 6 ) were challenged with 1-μM fMLP for 1 min. F-actin (165 nM) stained in green and nuclei (blue) stained with DAPI. Arrows (red) indicate pseudopodia formation (scale bar, 12 μm). c , d Control and TLR10 knockdown HL-60-derived neutrophils (1 × 10 6 ) were challenged with 1-μM fMLP for 1 min. TLR10 stained in green and nuclei in blue stained with DAPI (scale bar, 10 μm). e Difference between the cells with pseudopodia in control and TLR10 knockdown groups. HL-60-derived neutrophils (1 × 10 6 ) were challenged with 1-μM fMLP for 1 min, imaged under confocal microscope and counted 8 different fields (double-blinded counting).

Article Snippet: In brief, 80 nM of siRNA for TLR10 (sc-40272; Santa Cruz Biotechnology) and missense oligonucleotide negative control (sc-36869; Santa Cruz Biotechnology) were incubated with transfection reagent for 45 min to form transfection reagent-siRNA complex and added to the cells washed with transfection medium.

Techniques: Knockdown, Control, Derivative Assay, Staining, Microscopy

Journal: Genes & Development

Article Title: TFEB controls expression of human syncytins during cell–cell fusion

doi: 10.1101/gad.351633.124

Figure Lengend Snippet: Perturbation of TFEB and TFE3 in BeWo cells using CRISPR KO. ( A – C ) Western blotting of TFEB ( A ), TFE3 ( B ), and hCG ( C ) expression in wild-type and CRISPR KO BeWo cells. ( D ) Volcano plot of RNA-seq data comparing Forskolin-treated wild-type BeWo cells with Forskolin-treated TFEB/TFE3 DKO BeWo cells. Genes that are significantly higher in the Forskolin-treated DKO cells are shown in red, and genes that are significantly lower in the Forskolin-treated DKO cells are shown in blue. ( E , F ) Normalized CPM values from the RNA-seq data showing expression of ERVFRD-1 (Syncytin-2) ( E ) or ERVW-1 ( F ) across samples. Data points indicate separate RNA-seq replicates, and error bars show standard deviation. ( G ) GO analysis of genes upregulated in Forskolin-treated WT BeWo compared with Forskolin-treated DKO BeWo cells. The top biological process GO terms are shown. ( H ) Expression of ERVFRD-1 transcripts measured by RT-qPCR and quantified by the ΔΔ Ct method. Statistical significance from an ordinary one-way ANOVA with Tukey's multiple comparisons test is shown. (ns) Not significant, (***) P < 0.001, (****) P < 0.0001.

Article Snippet: Human placental choriocarcinoma BeWo cells (ATCC CCL-98) were cultured at 37°C and 5% CO 2 in F-12K medium (Corning or ATCC) supplemented with 10% fetal bovine serum and 10 U/mL penicillin–streptomycin.

Techniques: CRISPR, Western Blot, Expressing, RNA Sequencing, Standard Deviation, Quantitative RT-PCR

Journal: Genes & Development

Article Title: TFEB controls expression of human syncytins during cell–cell fusion

doi: 10.1101/gad.351633.124

Figure Lengend Snippet: KO of TFEB/TFE3 in BeWo cells causes a functional defect in cell–cell fusion. ( A ) Two-color cell–cell fusion experiments coculturing two populations of mCherry- and GFP-expressing BeWo cells were imaged on a spinning-disk confocal microscope. Individual channels are shown in grayscale. In the merged composite image, the GFP channel is represented in cyan, and the mCherry channel is represented in magenta. White arrowheads indicate fused syncytial areas in the Forskolin-treated wild-type cells. Scale bar, 200 μm. ( B ) Split-GFP cell–cell fusion experiments coculturing GFP1–10-expressing BeWo cells with GFP11-expressing 293T cells and acquiring images on a spinning-disk confocal microscope. In the merged composite image, the GFP channel is represented in green, and the Hoechst channel is represented in magenta. Fused syncytial areas are shown by the reconstitution of GFP fluorescence, shown in green. Scale bar, 200 μm. ( C ) Quantification of the two-color cell–cell fusion experiments shown in A . ( D ) Quantification of the split-GFP cell–cell fusion experiments shown in B . For C and D , statistical significance from an ordinary one-way ANOVA with Tukey's multiple comparisons test is shown. (ns) Not significant, (****) P < 0.0001.

Article Snippet: Human placental choriocarcinoma BeWo cells (ATCC CCL-98) were cultured at 37°C and 5% CO 2 in F-12K medium (Corning or ATCC) supplemented with 10% fetal bovine serum and 10 U/mL penicillin–streptomycin.

Techniques: Functional Assay, Expressing, Microscopy, Fluorescence

Journal: Genes & Development

Article Title: TFEB controls expression of human syncytins during cell–cell fusion

doi: 10.1101/gad.351633.124

Figure Lengend Snippet: TFEB/3 KO in BeWo cells has minimal effects on lysosomal biogenesis or lysosomal gene expression. ( A ) Differential gene expression analysis of previously identified lysosomal TFEB target genes (yellow bars) and selected syncytiotrophoblast marker genes (purple bars). The −log 10 false discovery rate (FDR) of the comparison between Forskolin-treated wild-type BeWo cells and Forskolin-treated TFEB/3 DKO BeWo cells is shown with a significance cutoff of FDR = 0.05, equivalent to −log 10 (FDR) = 1.30. ( B ) Live lysosomes were imaged in DMSO-treated or Forskolin-treated BeWo cells by staining with Lysoview-540 and imaging on a spinning-disk confocal microscope. In the merged composite image, the Hoechst channel is represented in magenta, and the Lysoview staining is shown in yellow. Scale bar, 50 μm. ( C ) The normalized number of lysosomes was quantified by the per-well mean of total lysosomal spots detected per image divided by the total number of nuclei per image. ( D ) The normalized total lysosomal area is quantified by the per-well mean of 540 nm-positive thresholded region summed area per image divided by the total number of nuclei per image. For C and D , statistical significance from Kruskal–Wallis ANOVA test with Dunn's multiple comparison test is shown. (ns) Not significant, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.

Article Snippet: Human placental choriocarcinoma BeWo cells (ATCC CCL-98) were cultured at 37°C and 5% CO 2 in F-12K medium (Corning or ATCC) supplemented with 10% fetal bovine serum and 10 U/mL penicillin–streptomycin.

Techniques: Gene Expression, Marker, Comparison, Staining, Imaging, Microscopy

Journal: Genes & Development

Article Title: TFEB controls expression of human syncytins during cell–cell fusion

doi: 10.1101/gad.351633.124

Figure Lengend Snippet: TFEB translocates into the nucleus and binds chromatin during syncytiotrophoblast differentiation. ( A ) Live-cell spinning-disk confocal imaging of Halo-TFEB after treatment with Torin1 for 2 h or treatment with Forskolin for 48 h. The Halo-TFEB channel alone is shown in grayscale ( left ), followed by a composite image showing Halo-TFEB (yellow) with the membrane stain Biotium MembraneSteady-488 nm (magenta) ( middle ), and Halo-TFEB (yellow) with Hoechst-stained nuclei (cyan). Scale bar, 200 μm. ( B ) Quantification of the nuclear/cytoplasmic ratio of Halo-TFEB in A . Statistical significance from an ordinary one-way ANOVA with Tukey's multiple comparisons test is shown. (ns) Not significant, (****) P < 0.0001. ( C , top ) Diagram of single-molecule tracking of Halo-TFEB performed in BeWo cells. An example JF549 mask image ( left ) and a single frame from a JF646-tracking movie ( right ) are shown, with the binarized mask outline used for segmenting nuclear versus external trajectories shown overlaid in yellow. Scale bar, 10 μm. The bottom diagram shows how image acquisition was performed by acquiring a single JF549 image used for masking followed by stroboscopic illumination and activation of dark JF646 molecules using 405 nm activation in the camera readout time. ( D ) Heat maps of diffusion coefficients following Bayesian analysis of single-molecule trajectory data of Halo-TFEB in BeWo cells in different drug conditions. Each row corresponds to the distribution of posterior occupations for a single movie file. ( E ) The distribution of diffusion coefficient occupancies for nuclear segmented trajectories of Halo-TFEB in different drug conditions. The fraction bound (calculated by the fraction of the distribution with a diffusion coefficient of <0.1 μm 2 /sec) is shown highlighted with the blue region and the quantified values displayed as black percentages. The cumulative distribution function (CDF) of this same distribution is shown below .

Article Snippet: Human placental choriocarcinoma BeWo cells (ATCC CCL-98) were cultured at 37°C and 5% CO 2 in F-12K medium (Corning or ATCC) supplemented with 10% fetal bovine serum and 10 U/mL penicillin–streptomycin.

Techniques: Imaging, Membrane, Staining, Activation Assay, Diffusion-based Assay

Journal: Genes & Development

Article Title: TFEB controls expression of human syncytins during cell–cell fusion

doi: 10.1101/gad.351633.124

Figure Lengend Snippet: TFEB directly binds the ERVFRD-1 and ERVW-1 promoters to regulate their expression. ( A , B ) Gene tracks displaying binned reads of 3xFLAG-Halo-TFEB ChIP-seq and RNA-seq in DMSO, 2 h Torin1 treatment, or 48 h Forskolin treatment for the ERVFRD-1 ( A ) and ERVW-1 ( B ) loci. Merged replicates 1 and 2 are shown. ( C ) Venn diagram of statistically significant 3xFLAG-Halo-TFEB ChIP-seq peaks, called with IDR analysis (IDR score < 0.05) in each treatment condition. ( D ) GO enrichment analysis of all annotated genes associated with at least one significant 3xFLAG-Halo-TFEB ChIP-seq peak (IDR score < 0.05). Genes with higher Torin1 (2 h) binding or with equivalent Forskolin and Torin1 (common) enrichment (determined by MACS2 bdgdiff) were analyzed separately. The top biological process GO terms are shown. ( E ) The top significant motif from MEME-ChIP analysis of significant 3xFLAG-Halo-TFEB ChIP-seq peak summits (IDR score < 0.05), expanded by 250 bp in both directions and pooled from all treatment conditions. ( F ) Distribution analysis of the top MEME motif “DRTCACGTGAYH” in the analyzed sequences. ( G ) Heat maps of differentially enriched peaks analyzed with MACS2 bdgdiff showing ±3000 bp centered around each peak. Replicates 1 and 2 were merged, and IDR robust peaks (IDR score < 0.05) are shown (see for expanded plots of replicates). ( H ) Volcano plot of WT versus DKO BeWo RNA-seq expression (as shown in A, gray dots) overlaid with annotated TFEB target genes as identified by ChIP (green dots indicate all annotated genes associated with at least one significant 3xFLAG-Halo-TFEB ChIP-seq peak) (IDR score < 0.05). The top regulated TFEB target genes are labeled with their gene name and include ERVFRD-1 and ERVW-1 .

Article Snippet: Human placental choriocarcinoma BeWo cells (ATCC CCL-98) were cultured at 37°C and 5% CO 2 in F-12K medium (Corning or ATCC) supplemented with 10% fetal bovine serum and 10 U/mL penicillin–streptomycin.

Techniques: Expressing, ChIP-sequencing, RNA Sequencing, Binding Assay, Labeling

Journal: Genes & Development

Article Title: TFEB controls expression of human syncytins during cell–cell fusion

doi: 10.1101/gad.351633.124

Figure Lengend Snippet: Torin1 treatment increases the rate of syncytiotrophoblast fusion. ( A ) Two-color cell–cell fusion experiments coculturing two populations of mCherry- and GFP-expressing BeWo cells were treated with the indicated compounds for 24 h and then imaged on a spinning-disk confocal microscope. In the merged composite image, the GFP channel is represented in cyan, and the mCherry channel is represented in magenta. White arrowheads indicate fused syncytial areas in the Torin1- and Forskolin-treated wild-type cells. Scale bar, 200 μm. ( B ) Quantification of the two-color cell–cell fusion experiments shown in A . ( C ) Same as A , except cells were treated with the indicated compounds for 48 h. ( D ) Quantification of the two-color cell–cell fusion experiments shown in C . Statistical significance from an ordinary one-way ANOVA with Tukey's multiple comparisons test is shown. (ns) Not significant, (*) P < 0.05, (****) P < 0.0001.

Article Snippet: Human placental choriocarcinoma BeWo cells (ATCC CCL-98) were cultured at 37°C and 5% CO 2 in F-12K medium (Corning or ATCC) supplemented with 10% fetal bovine serum and 10 U/mL penicillin–streptomycin.

Techniques: Expressing, Microscopy