Journal: Molecular human reproduction

Article Title: Sphingosine-1-phosphate restores endothelial barrier integrity in ovarian hyperstimulation syndrome.

doi: 10.1093/molehr/gaw065

Figure Lengend Snippet: Figure 2 Effect of S1P on the expression of endothelial adherens junction proteins in endothelial cells in the presence of FF from controls and patients at risk of OHSS. Densitometric quantification of (A) phospho-vascular endothelial (p-VE)-cadherin, (B) VE-cadherin, (C) N-cadherin and (D) β-catenin. Representative immunoblots are shown in the lower panel. Data are expressed as means ± SEM normalized to β-actin of three independent experiments using 20 control patients and 20 OHSS patients at risk of OHSS (*P < 0.05).

Article Snippet: The blot was preincubated in blocking buffer (5% nonfat milk, 0.05% Tween 20 in 20 mM TBS pH 8.0) for 1 h at room temperature and incubated overnight in blocking buffer at 4°C with appropriate primary antibodies: β-actin 1/3000 (sc-1616-R), N-cadherin 1/200 (sc-7939), vascular endothelial (VE)-cadherin 1/100 (sc-9989) and β-catenin 1/200 (sc-59737) (Santa Cruz Biotechnology, Inc., Santa Cruz, USA), phospho-VE-cadherin 1/1000 (MAB1002) (Millipore, MA, USA), VEGF 1/1000 (ab46154) (Abcam, Cambridge, USA), KDR (VEGFR-2) 1/1000 (D5B1) (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Biomedicines

Article Title: Ivosidenib Confers BRCAness Phenotype and Synthetic Lethality to Poly (ADP-Ribose) Polymerase Inhibition in BRCA1/2-Proficient Cancer Cells

doi: 10.3390/biomedicines13040958

Figure Lengend Snippet: Figure 1. High-throughput screening shows that IDH1 inhibitor ivosidenib greatly sensitizes BRCA1/2-proficient cancer cells to PARPi. (A) The workflow for screening sensitizer drugs for the PARP inhibitor Olaparib in A2780 cells using an FDA-approved drug library (TargetMol, #L1000) that contains 2040 approved small molecule drugs. (B) The relative cell viability of A2780 cells grown in the presence of 10 µM of FDA-approved drugs alone (upper) or FDA-approved drugs plus 10 µM of Olaparib for 72 h; cell viability was analyzed through CCK-8 assay. (C) Ivosidenib increases Olaparib sensitivity in BRCA1/2-WT breast and ovarian cancer cells. Indicated cells were treated with increasing concentrations of Olaparib (ola) in the presence or absence of 5 µM of ivosidenib (Ivo) for 72 h, followed by cell viability analysis. (D) Ivosidenib and Olaparib synergistically suppress cancer cell growth. Indicated cells were co-treated with serial dosages of Olaparib and ivosidenib for 72 h, followed by Bliss synergy analysis. (E) Ivosidenib and Olaparib synergistically inhibit clonogenic survival in BRCA1/2-WT cancer cells. The colony formation analysis indicated cells treated with 2 µM of ivosidenib, 2 µM of Olaparib, or their combination. Data are presented as means ± SD, n = 6. * p < 0.05 and *** p < 0.001 based on the two-tailed t-test.

Article Snippet: High-throughput screening shows that IDH1 inhibi r ivosidenib greatly sensitizes BRCA1/2-proficient cancer cells to PARPi. (A) The workflow for screening sensitizer drugs for the PARP inhibitor Olaparib in A2780 cells using an FDA-approved drug library (TargetMol, #L1000) that contains 2040 approved small molecule drugs. (B) The relative cell viability of A2780 cells grown in the presence of 10 μM of FDA-app oved drugs alone (upper) o FDA-approved drugs plus 10 μM of Olaparib for 72 h; cell viability was analyzed through CCK-8 assay. (C) Ivosidenib increases Olaparib sensitivity in BRCA1/2-WT breast and ovarian cancer cells.

Techniques: High Throughput Screening Assay, Drug discovery, CCK-8 Assay, Two Tailed Test

Journal: Biomedicines

Article Title: Ivosidenib Confers BRCAness Phenotype and Synthetic Lethality to Poly (ADP-Ribose) Polymerase Inhibition in BRCA1/2-Proficient Cancer Cells

doi: 10.3390/biomedicines13040958

Figure Lengend Snippet: Figure 2. Ivosidenib (Ivo) greatly potentiates Olaparib (Ola)-induced DNA damage in BRCA1/2- proficient cells. (A) Ivosidenib enhances Olaparib-induced γH2AX formation in BRCA1/2-WT cancer cells. Indicated cells were treated with 50 µM of Ola, 10 µM of Ivo, or their combination for 48 h, followed by Western blot analysis of γH2AX. (B) Ivosidenib delayed the reduction of γH2AX after Olaparib treatment. A2780 cells were treated with 100 µM for 48 h and then released into normal culture medium in the presence or absence of Ivo for indicated time points, followed by Western blot analysis of γH2AX. (C) Ivosidenib increases Olaparib-caused DNA damage. A2780 cells were treated with 50 µM of Ola, 10 µM of Ivo, or their combination for 48 h, followed by measuring the DNA damage through comet assay. Data are presented as means ± SD.

Article Snippet: High-throughput screening shows that IDH1 inhibi r ivosidenib greatly sensitizes BRCA1/2-proficient cancer cells to PARPi. (A) The workflow for screening sensitizer drugs for the PARP inhibitor Olaparib in A2780 cells using an FDA-approved drug library (TargetMol, #L1000) that contains 2040 approved small molecule drugs. (B) The relative cell viability of A2780 cells grown in the presence of 10 μM of FDA-app oved drugs alone (upper) o FDA-approved drugs plus 10 μM of Olaparib for 72 h; cell viability was analyzed through CCK-8 assay. (C) Ivosidenib increases Olaparib sensitivity in BRCA1/2-WT breast and ovarian cancer cells.

Techniques: Western Blot, Single Cell Gel Electrophoresis

Journal: Biomedicines

Article Title: Ivosidenib Confers BRCAness Phenotype and Synthetic Lethality to Poly (ADP-Ribose) Polymerase Inhibition in BRCA1/2-Proficient Cancer Cells

doi: 10.3390/biomedicines13040958

Figure Lengend Snippet: Figure 3. Ivosidenib (Ivo) treatment suppresses the repair of Olaparib (Ola)-induced DSBs. Ivosidenib inhibits HR repair. (A,B) Ivosidenib A2780 cells were treated with 50 of µM Ola, 10 µM of Ivo, or their combination for 48 h, followed by immunofluorescence analysis of Rad51/γH2AX (A) or RPA2/γH2AX (B). (C) Cells were treated as indicated for 48 h, followed by Western blot analysis of pRPA2 S4/8. Data were are presented as means ± SD. ** p < 0.01 and *** p < 0.001 according to the two2-tailed t-test.

Article Snippet: High-throughput screening shows that IDH1 inhibi r ivosidenib greatly sensitizes BRCA1/2-proficient cancer cells to PARPi. (A) The workflow for screening sensitizer drugs for the PARP inhibitor Olaparib in A2780 cells using an FDA-approved drug library (TargetMol, #L1000) that contains 2040 approved small molecule drugs. (B) The relative cell viability of A2780 cells grown in the presence of 10 μM of FDA-app oved drugs alone (upper) o FDA-approved drugs plus 10 μM of Olaparib for 72 h; cell viability was analyzed through CCK-8 assay. (C) Ivosidenib increases Olaparib sensitivity in BRCA1/2-WT breast and ovarian cancer cells.

Techniques: Immunofluorescence, Western Blot

Journal: Biomedicines

Article Title: Ivosidenib Confers BRCAness Phenotype and Synthetic Lethality to Poly (ADP-Ribose) Polymerase Inhibition in BRCA1/2-Proficient Cancer Cells

doi: 10.3390/biomedicines13040958

Figure Lengend Snippet: Figure 5. YTHDC2 knockdown suppresses HR repair. (A) Schematic diagram of HR repair reporter system (DR-GFP). (B,C) Silencing of YTHDC2 decreases HR repair. QU20S-DRGFP cells were transfected with control (ctrl) or YTHDC2 siRNA and, 24 h later, cells were transfected with pCBASceI plasmid to induce endonuclease-induced DSB, followed by flow cytometry analysis of GFP expression 48 h after pCBASceI expression (B). A2780 cells were transfected with control (Ctrl) or YTHDC2 siRNA; cells were then treated with 50 µM of Olaparib for 48 h, followed by immunofluorescence analysis of Rad51/γH2AX (C). Data are presented as means ± SD. *** p < 0.001 according to the two-tailed t-test.

Article Snippet: High-throughput screening shows that IDH1 inhibi r ivosidenib greatly sensitizes BRCA1/2-proficient cancer cells to PARPi. (A) The workflow for screening sensitizer drugs for the PARP inhibitor Olaparib in A2780 cells using an FDA-approved drug library (TargetMol, #L1000) that contains 2040 approved small molecule drugs. (B) The relative cell viability of A2780 cells grown in the presence of 10 μM of FDA-app oved drugs alone (upper) o FDA-approved drugs plus 10 μM of Olaparib for 72 h; cell viability was analyzed through CCK-8 assay. (C) Ivosidenib increases Olaparib sensitivity in BRCA1/2-WT breast and ovarian cancer cells.

Techniques: Knockdown, Transfection, Control, Plasmid Preparation, Flow Cytometry, Expressing, Immunofluorescence, Two Tailed Test

Journal: Biomedicines

Article Title: Ivosidenib Confers BRCAness Phenotype and Synthetic Lethality to Poly (ADP-Ribose) Polymerase Inhibition in BRCA1/2-Proficient Cancer Cells

doi: 10.3390/biomedicines13040958

Figure Lengend Snippet: Figure 7. Combination treatment of ivosidenib (Ivo) and Olaparib (Ola) produces synergistic anti- tumor efficacy in BRCA1/2-proficient ovarian cancers. (A–C) Ivosidenib and Olaparib synergistically suppress tumor growth. A2780 xenograft bearing mice treated with Ola (100 mg/kg, orally) and Ivo (100 mg/kg, orally) and tumor growth curve (A), tumor image (B), and tumor weights (C) are shown. (D,E) Co-treatment with ivosidenib and Olaparib greatly induces DSBs and reduces proliferation in A2780 xenograft tumors. Immunohistochemistry analysis of γH2AX (D) and Ki67 (E) in tumors treated as indicated. Data are presented as means ± SD. *** p < 0.001 according to the two-tailed t-test.

Article Snippet: High-throughput screening shows that IDH1 inhibi r ivosidenib greatly sensitizes BRCA1/2-proficient cancer cells to PARPi. (A) The workflow for screening sensitizer drugs for the PARP inhibitor Olaparib in A2780 cells using an FDA-approved drug library (TargetMol, #L1000) that contains 2040 approved small molecule drugs. (B) The relative cell viability of A2780 cells grown in the presence of 10 μM of FDA-app oved drugs alone (upper) o FDA-approved drugs plus 10 μM of Olaparib for 72 h; cell viability was analyzed through CCK-8 assay. (C) Ivosidenib increases Olaparib sensitivity in BRCA1/2-WT breast and ovarian cancer cells.

Techniques: Immunohistochemistry, Two Tailed Test

Journal: Chemical research in toxicology

Article Title: Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing.

doi: 10.1021/acs.chemrestox.2c00172

Figure Lengend Snippet: Figure 7. 3′-terminus produced by hyTDG-lyase cannot be extended by DNA Pol β but is resolved by APE1. In lane 1, A 5′-FAM-labeled 79 base oligonucleotide containing a U in a U:G mispair (2.5 pmol, 0.2 μM). In lane 2, the oligonucleotide was incubated with UDG (2.5 U, 0.84 pmol, 0.07 μM) for 1 h at 37 °C in CutSmart buffer, and subsequently, the abasic site was cleaved by incubating with APE1 (5 U, 0.18 pmol, 0.01 μM) for an additional 30 min. In lane 3, Pol β (6.2 pmol, 0.5 μM) and E. coli ligase (5 U, 4 pmol, 0.32 μM), dCTP (250 pmol, 20 μM), and NAD+ (325 pmol, 26 μM) were added for an additional 1 h to simulate short-patch BER. Lane 4 was otherwise identical to lane 2, but hyTDG-lyase was used instead of APE1 at 37 °C for 30 min. Lane 5 was otherwise identical to lane 3, except hyTDG-lyase (26.9 pmol, 2.15 μM) was used instead of APE1 at 37 °C. In lane 6, we similarly generated an abasic site that was then cleaved by hyTDG-lyase for 30 min at 37 °C. Then APE1, Pol β, dCTP, and E. coli ligase were added and incubated for an additional 1 h. Lane 6 demonstrates that the AP endonuclease activity of APE1 can clean up the PUA-βME 3′-end produced by the hyTDG-lyase. The complementary strand is labeled with a 5′-Cy5 fluorophore (red). The overlap between the full-length 5′-FAM-labeled oligo and its 5′-Cy5 complement is depicted as yellow.

Article Snippet: Human 8-oxoguanine DNA glycosylase (hOGG1, #NBP1-453180.1mg) and DNA polymerase β (Pol β, #NBP1-72434-0.5mg) were purchased from Novus Biologicals.

Techniques: Produced, Labeling, Incubation, Generated, Activity Assay

Journal: Advanced Science

Article Title: G3BP1 and SLU7 Jointly Promote Immune Evasion by Downregulating MHC‐I via PI3K/Akt Activation in Bladder Cancer

doi: 10.1002/advs.202305922

Figure Lengend Snippet: G3BP1 binds to the mRNAs of class IA PI3Ks and promotes their expression. A) Cell lysates from UMUC3 cells with stable expression of HA‐G3BP1 were immunoprecipitated with anti‐HA antibody, and the purified RNA from the immunoprecipitants was subjected to RNA‐seq analysis. Motif spacing analysis was used to identify the significant RNA motifs of HA‐G3BP1 on RNAs. B) The RIP assay with anti‐HA antibodies was performed using UMUC3 cells with stable expression of HA‐G3BP1. qRT–PCR analysis was used to evaluate the mRNA levels of class IA PI3Ks in the immunoprecipitants. C) The locations of G3BP1 and the mRNAs of class IA PI3Ks in UMUC3 cells were analyzed using immuno‐FISH. Scale bar, 10 µm. The fluorescence intensity curves of DAPI (blue), G3BP1 (green), and mRNA (purple) that crossed the arrows were analyzed. D and E) qRT–PCR analysis was used to evaluate the mRNA levels of G3BP1 and the class IA PI3Ks in UMUC3 and T24 cells with G3BP1 knockdown or overexpression. F,G) Western blotting was used to detect the protein expression levels of class IA PI3Ks in UMUC3 and T24 cells with G3BP1 knockdown or overexpression. GAPDH was used as the internal control. Error bars represent SD ( n = 3 in B, D, and E). P ‐values are presented and were calculated using unpaired two‐tailed Student's t ‐test (B) and two‐way ANOVA (D and E).

Article Snippet: For re‐IP, UMUC3 cell lysates with simultaneous stable expression of SLU7‐Flag and HA‐G3BP1 were primarily immunoprecipitated with anti‐Flag antibody, after which they were blocked with 15 μg mL −1 Flag peptide (TP1273, TargetMol, USA) for 2 h at 4°C under gentle rotation.

Techniques: Expressing, Immunoprecipitation, Purification, RNA Sequencing, Quantitative RT-PCR, Fluorescence, Knockdown, Over Expression, Western Blot, Control, Two Tailed Test

Journal: Advanced Science

Article Title: G3BP1 and SLU7 Jointly Promote Immune Evasion by Downregulating MHC‐I via PI3K/Akt Activation in Bladder Cancer

doi: 10.1002/advs.202305922

Figure Lengend Snippet: G3BP1 interacts with SLU7 to promote PI3K/Akt signaling activation, downregulating MHC‐I and promoting immune evasion. A,B) qRT–PCR analysis was used to evaluate the mRNA levels of SLU7 and the class IA PI3Ks in UMUC3 and T24 cells with SLU7 knockdown or overexpression. C,D) Western blotting was used to detect the protein expression levels of class IA PI3Ks in UMUC3 and T24 cells with SLU7 knockdown or overexpression. E) Cell lysates from UMUC3 cells with stable expression of SLU7‐Flag or HA‐G3BP1 were immunoprecipitated with anti‐Flag or anti‐HA antibodies, and immunoblotting was conducted with the indicated antibodies. F) The PLA was used to evaluate the interaction between G3BP1 and SLU7 in UMUC3 cells. Scale bar, 10 µm. G) IF was used to analyze the locations of G3BP1 and SLU7 in UMUC3 cells. Scale bar, 10 µm. Fluorescence intensity curves of DAPI (blue), G3BP1 (green), and SLU7 (red) that crossed the arrow were analyzed. H,I) The protein expression levels of SLU7, total Akt, p‐Akt Thr308 , and p‐Akt Ser473 in UMUC3 and MB49 cells with stable SLU7 knockdown were detected using western blotting. J,K) Western blotting was used to detect the protein expression levels of G3BP1, SLU7, total Akt, p‐Akt Thr308 , p‐Akt Ser473 , and/or MHC‐I in G3BP1‐overexpressing UMUC3 and MB49 cells with SLU7 knockdown. L) Flow cytometry was used to analyze the expression of MHC‐I on the surface of MB49 cells in (K). MHC‐I expression was quantified based on the mean fluorescence intensity (MFI). M) mCherry‐expressing MB49‐OVA cells with or without G3bp1 overexpression and Slu7 knockdown were cocultured with antigen‐activated CTLs for 24 h. Cytotoxicity was determined by analyzing the percentage of 7‐AAD+ cells among mCherry+ cells. Error bars represent SD ( n = 3 in A, B, L, and M). P ‐values are presented and were calculated using two‐way ANOVA (A and B) and one‐way ANOVA (L and M).

Article Snippet: For re‐IP, UMUC3 cell lysates with simultaneous stable expression of SLU7‐Flag and HA‐G3BP1 were primarily immunoprecipitated with anti‐Flag antibody, after which they were blocked with 15 μg mL −1 Flag peptide (TP1273, TargetMol, USA) for 2 h at 4°C under gentle rotation.

Techniques: Activation Assay, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Expressing, Immunoprecipitation, Fluorescence, Flow Cytometry

Journal: Advanced Science

Article Title: G3BP1 and SLU7 Jointly Promote Immune Evasion by Downregulating MHC‐I via PI3K/Akt Activation in Bladder Cancer

doi: 10.1002/advs.202305922

Figure Lengend Snippet: G3BP1 and SLU7 form a complex with PABPC1 and eIF4G1 to stabilize the closed‐loop structure of the mRNAs of class IA PI3Ks. A) Scatter plot of the G3BP1‐interacting proteins identified using MS. B and C) Cell lysates from UMUC3 cells with stable expression of SLU7‐Flag or HA‐G3BP1 were immunoprecipitated with anti‐Flag or anti‐HA antibodies, and immunoblotting was performed with the indicated antibodies. D) Cell lysates from UMUC3 cells with simultaneous stable expression of SLU7‐Flag and HA‐G3BP1 were primarily immunoprecipitated with anti‐Flag antibody and then blocked with Flag peptide. The resultant immunoprecipitants were subjected to re‐IP with anti‐HA antibodies and immunoblotted with the indicated antibodies. E) IF was used to analyze the locations of eIF4G1 or PABPC1 with SLU7 and G3BP1 in UMUC3 cells stably expressing eIF4G1‐GFP or PABPC1‐GFP. Scale bar, 10 µm. The fluorescence intensity curves of DAPI (blue), eIF4G1‐GFP (green), PABPC1‐GFP (green), SLU7 (red), and G3BP1 (purple) that crossed the arrows were analyzed. F) Cell lysates from UMUC3 cells with PABPC1‐GFP stable expression and SLU7 or G3BP1 knockdown were immunoprecipitated with anti‐GFP antibody. Western blotting was used to detect the expression levels of PABPC1‐GFP, SLU7, G3BP1, and eIF4G1 in whole cell lysates and immunoprecipitants. G) qRT–PCR analysis was used to evaluate the mRNA expression levels of class IA PI3Ks in the immunoprecipitants. H) Western blotting was used to detect the expression levels of G3BP1 and SLU7 in UMUC3 cells with SLU7 knockdown or G3BP1 knockdown. I) FISH–FRET was performed in UMUC3 cells with SLU7 or G3BP1 knockdown using Cy3‐labeled 5′‐UTR probes and Cy5‐labeled 3′‐UTR probes targeting the mRNAs of PI3Ks. The mean values of FRET efficiency are shown. Error bars represent SD ( n = 3 in G; n = 5 in I). P ‐values are presented and were calculated using one‐way ANOVA (G and I).

Article Snippet: For re‐IP, UMUC3 cell lysates with simultaneous stable expression of SLU7‐Flag and HA‐G3BP1 were primarily immunoprecipitated with anti‐Flag antibody, after which they were blocked with 15 μg mL −1 Flag peptide (TP1273, TargetMol, USA) for 2 h at 4°C under gentle rotation.

Techniques: Expressing, Immunoprecipitation, Western Blot, Stable Transfection, Fluorescence, Knockdown, Quantitative RT-PCR, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The noncanonical RNA-binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

doi: 10.1016/j.jbc.2024.107964

Figure Lengend Snippet: RAN, TDP43, and G3BP1 mRNA form a complex in the nucleus. A, diagrams of full-length G3BP1 transcript consist of CDS and 3′ UTR, as well as its deletion fragments. B and C, the in vitro –transcribed full-length G3BP1 transcript and deletion fragments with the correct sizes were indicated ( upper ). RNA pull-down assay and Western blotting showed whether these biotin-labeled G3BP1 fragments could pull down RAN in cell lysates ( lower ). D, the motif discovery algorithm DREME was used to recognize the top consensus motif based on the RIP-seq data. E, construction of mutant fragments based on predicted motifs. F, the in vitro –transcribed D2 fragments and mutant fragments with the correct sizes are indicated ( upper ). RNA pull-down assay and Western blotting indicated whether these biotin-labeled fragments could pull down RAN in cell lysates ( lower ). G, the interaction between endogenous RAN and TDP43 was evaluated by Co-IP assays using anti-RAN antibody or normal rabbit IgG in HONE-1 and SUNE-1 cell lysates. H, the interaction between endogenous TDP43 and RAN was evaluated by Co-IP assays using anti-TDP43 antibody or normal rabbit IgG in HONE-1 and SUNE-1 cell lysates. I, RAN ( red ) or TDP43 ( green ) protein distribution in cells was recognized by IF, and cell nuclei were stained with DAPI ( blue ). The scale bar represents 10 μm. J, relative enrichment of G3BP1 mRNA immunoprecipitated by anti-TDP43 or anti-IgG antibody was indicated by RT-qPCR. Data are presented as the mean ± SD (n = 3). K, enrichment of TDP43 pulled down by biotin-labeled G3BP1 probes from in vitro transcription or control antisense probes was detected by Western blotting. L, RNA pull-down assay and Western blotting showed whether biotin-labeled full-length G3BP1 transcript or deletion fragments could pull down TDP43 in cell lysates. M, TDP43 protein distribution in cells was recognized by IF ( green ), G3BP1 mRNA distribution in cells was recognized by FISH ( red ), and cell nuclei were stained with DAPI ( blue ). The scale bar represents 10 μm. N, in vitro purified RAN-GST, TDP43, eEF2-His (positive control), and GST (negative control) proteins were used to perform a cell-free RNA pull-down experiment. Coomassie brilliant blue staining showed the purification of these proteins ( left ). Enrichment of proteins pulled down by biotin-labeled G3BP1 probes from in vitro transcription was detected by Western blotting ( right ). O, in vitro purified RAN-GST, TDP43, and GST (negative control) proteins were used to perform a cell-free co-IP experiment. Coomassie brilliant blue staining showed the purification of these proteins ( left ). Enrichment of proteins immunoprecipitated by anti-GST magnetic beads was detected by Western blotting ( right ). ∗ p < 0.05 and ∗∗ p < 0.01. The significant differences were assessed using t test ( J ). RT-qPCR, reverse transcription quantitative real-time PCR; ; NPC, nasopharyngeal carcinoma; Co-IP, coimmunoprecipitation; GST, glutathione- S -transferase; IF, immunofluorescence; DAPI, 4′, 6-diamino-2-phenylindole; CDS, coding sequence.

Article Snippet: For a cell-free RNA pull-down experiment, purified proteins were purchased from TargetMOl: RAN-GST ( Escherichia coli protein purification system, Human, TMPH-01419), TDP43 ( E. coli protein purification system, Human, TMPH-02178), eEF2 ( E. coli protein purification system, Human, TMPH-01286), and GST (Baculovirus Insect Cells protein purification system, Schistosoma japonicum, TMPY-02157).

Techniques: In Vitro, Pull Down Assay, Western Blot, Labeling, Mutagenesis, Co-Immunoprecipitation Assay, Staining, Immunoprecipitation, Quantitative RT-PCR, Control, Purification, Positive Control, Negative Control, Magnetic Beads, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunofluorescence, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: The noncanonical RNA-binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

doi: 10.1016/j.jbc.2024.107964

Figure Lengend Snippet: RAN increases TDP43 nucleus import and facilitates TDP43 binding G3BP1 mRNA. A, after treatment with actinomycin D (10 μg/ml), G3BP1 mRNA level was quantified at indicated times in control, RAN-silenced, TDP43-silenced, and both RAN and TDP43-silenced cells. The half-life of G3BP1 mRNA was analyzed by plotting degradation curves. Data are presented as the mean ± 95%CI (n = 3). B, TDP43 protein levels with knockdown of RAN were detected by Western blotting. The blots are the representation of three independent experiments. Data are presented as the mean ± SD (n = 3). C, relative enrichment of G3BP1 mRNA immunoprecipitated by anti-TDP43 or anti-IgG antibody was indicated by RT-qPCR upon knockdown of RAN. Data are presented as the mean ± SD (n = 3). D, the nucleus and cytoplasm proteins in HONE-1 and SUNE-1 cells were isolated by nuclear/cytosol fractionation assays. TDP43 distribution in the nucleus or cytoplasm was identified by Western blotting with or without RAN silencing, GAPDH as the cytoplasm marker, and Lamin B as the nucleus marker. The blots are the representation of three independent experiments. Data are presented as the mean ± SD (n = 3). E, TDP43-GFP overexpression vectors were transfected in HONE-1 and SUNE-1 cells after RAN silencing. Recognizing TDP43-GFP protein distribution ( green ) in HONE-1 and SUNE-1 cells after 24 h of TDP43-GFP overexpression vectors transfected, range indicator was performed to show clearer distribution of TDP43-GFP in the nucleus or cytoplasm, cell nuclei were stained with DAPI ( blue ). The scale bar represents 10 μm. F, the average fluorescence intensity of TDP43-GFP in the nucleus ( lower ) and cytoplasm ( upper ) of each cell was calculated. Data are presented as the mean ± SD (n = 20). ∗ p < 0.05 and ∗∗ p < 0.01. The significant differences were assessed using one-way ANOVA ( B , D , and F ), two-way ANOVA ( A ), and t test ( C ). RT-qPCR, reverse transcription quantitative real-time PCR; DAPI, 4′, 6-diamino-2-phenylindole; CI, confidence interval.

Article Snippet: For a cell-free RNA pull-down experiment, purified proteins were purchased from TargetMOl: RAN-GST ( Escherichia coli protein purification system, Human, TMPH-01419), TDP43 ( E. coli protein purification system, Human, TMPH-02178), eEF2 ( E. coli protein purification system, Human, TMPH-01286), and GST (Baculovirus Insect Cells protein purification system, Schistosoma japonicum, TMPY-02157).

Techniques: Binding Assay, Control, Knockdown, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Isolation, Fractionation, Marker, Over Expression, Transfection, Staining, Fluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The noncanonical RNA-binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

doi: 10.1016/j.jbc.2024.107964

Figure Lengend Snippet: Example diagram of RAN–TDP43–G3BP1 axis modulation. RAN increases the nucleus import of TDP43 and acts as an adapter to facilitate TDP43 direct binding to G3BP1 mRNA in the nucleus, thereby increasing G3BP1 mRNA stability and leading to upregulation of G3BP1, which further enhances AKT and ERK signaling and ultimately facilitates NPC proliferation and metastasis. NPC, nasopharyngeal carcinoma.

Article Snippet: For a cell-free RNA pull-down experiment, purified proteins were purchased from TargetMOl: RAN-GST ( Escherichia coli protein purification system, Human, TMPH-01419), TDP43 ( E. coli protein purification system, Human, TMPH-02178), eEF2 ( E. coli protein purification system, Human, TMPH-01286), and GST (Baculovirus Insect Cells protein purification system, Schistosoma japonicum, TMPY-02157).

Techniques: Binding Assay