Journal: American Journal of Translational Research

Article Title: CXCL16 is activated by p-JNK and is involved in H 2 O 2 -induced HK-2 cell injury via p-ERK signaling

doi:

Figure Lengend Snippet: The involvement of JNK signaling in H2O2-mediated HK-2 cell injury. A: H2O2-induced HK-2 cell apoptosis was lessened by treatment with the ROS scavenger, NAC, and the JNK inhibitor, SP600125. B: H2O2-induced CXCL16, IL-6, TNF-α, and TGF-β contents in the supernatant were lessened by NAC and SP600125 treatment. C: The mRNA expression levels of CXCL16 and CXCR6 were increased by H2O2 treatment but were decreased by NAC and SP600125 treatment. D: The protein levels of CXCL16 and CXCR6 were increased by H2O2 treatment but were down-regulated by NAC and SP600125 treatment. *P < 0.05, **P < 0.01 vs. H2O2 (400 μM) treatment group (n = 3).

Article Snippet: An Annexin V-FITC cell apoptosis detection kit was purchased from Beyotime Biotech (Shanghai, China).

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: CXCL16 is activated by p-JNK and is involved in H 2 O 2 -induced HK-2 cell injury via p-ERK signaling

doi:

Figure Lengend Snippet: Effects of different concentrations of CXCL16 on HK-2 cells. (A) Recombinant human CXCL16 protein inhibits the proliferation and (B) induces the apoptosis of HK-2 cells. (C) The protein levels of CXCR6, CXCL16, Bax, and p-ERK1/2 were increased, but the protein level of Bcl2 was decreased by recombinant human CXCL16 protein treatment. *P < 0.05, **P < 0.01 vs. normal control group (n = 3).

Article Snippet: An Annexin V-FITC cell apoptosis detection kit was purchased from Beyotime Biotech (Shanghai, China).

Techniques: Recombinant, Control

Journal: American Journal of Translational Research

Article Title: CXCL16 is activated by p-JNK and is involved in H 2 O 2 -induced HK-2 cell injury via p-ERK signaling

doi:

Figure Lengend Snippet: The involvement of CXCL16 in HK-2 cell injury. A: Recombinant human (rh) CXCL16 protein (200 μg/L)-induced HK-2 cell apoptosis was lessened by PD98059 (10 μM) and CXCR6 siRNA treatment. B: The protein levels of Bax, p-ERK1/2, COL-I, and COL-III were up-regulated, and the protein levels of Bcl2, MMP2, and MMP9 were down-regulated by recombinant human CXCL16 protein treatment. These same effects were weakened by PD98059 and CXCR6 shRNA treatment. EPC: empty plasmid control. *P < 0.05, **P < 0.01 vs. rh CXCL16 + EPC group (n = 3).

Article Snippet: An Annexin V-FITC cell apoptosis detection kit was purchased from Beyotime Biotech (Shanghai, China).

Techniques: Recombinant, shRNA, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: Enhanced Cd276 promoter activity in Tsc2−/− 105K cells expressing empty vector (EV) compared to reconstitution of TSC2 ( a ) and in Tsc2 KO MEFs compared to Tsc2 -WT MEFs ( b ). Relative luciferase activity was determined by a dual-luciferase assay system. psiCHECK2-Cd276 encodes the Cd276 promoter. Empty psiCHECK2 vector was used as the negative control. n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, *** p < 0.001. Raptor, mTOR or S6K knockdown suppresses Cd276 promoter activity in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ). n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01, **** p < 0.0001. siRNA knockdown of YY2 reduces B7-H3 protein expression in Tsc2−/− 105K cells ( e ) and Tsc2 KO MEFs ( f ) ( n = 3). Knockdown of YY2 reduces Cd276 mRNA expression in Tsc2−/− 105K cells ( g ) and Tsc2 KO MEFs ( h ). n = 3, means ± SD, two-tailed unpaired Student’s t -test, * p < 0.05, *** p < 0.001. i Promoter region of Cd276 displaying the location of the YY2 binding site. YY2 ChIP-qPCR analysis showing increased YY2 occupancy on the Cd276 promoter in Tsc2−/− 105K cells ( j ) and Tsc2 KO MEFs ( k ). n = 4, means ± SEM, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Cd276 promoter activity is suppressed by YY2 knockdown in Tsc2−/− 105K cells ( l ) and Tsc2 KO MEFs ( m ). n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, *** p < 0.001. Source data and exact p values are provided in the Source data file.

Article Snippet: The following primary antibodies were used for immunoblotting at 1:1000 dilution unless otherwise indicated: Phospho-p70 S6 Kinase T389 (CST, Cat#9234), p70 S6 Kinase (CST, Cat#2708), TSC2 (CST, Cat#4308), Phospho-S6 S235/236 (CST, Cat# 2211), S6 (CST, Cat#2217), mTOR (CST, Cat# 2983), Raptor (CST, Cat#2280), Rictor (CST, Cat#2114), Phospho-Akt S473 (CST, Cat#4060), Phospho-Akt T308 (CST, Cat#4056), Akt (CST, Cat# 4685), GAPDH (CST, Cat# 2118), CREB (CST, Cat#4820), STAT1 (CST, Cat#14994), p-STAT1 (Tyr 701) (CST, Cat#9167), human B7-H3 (CST, Cat# 14058), YY1 (CST, Cat#46395), HA-tag (CST, Cat#3724), Phospho-substrate (RXXS*/T*) (CST, Cat# 9614), MHC-II (1:500, LSBio Cat#LS-C204829), β-actin (Sigma, Cat#A1978), YY2 (A-5) (1:500, Santa Cruz, Cat#sc-377008), YY2 (C-10) (1:500, Santa Cruz, Cat#sc-374455), mouse B7-H3 (1:500, R&D Systems, Cat#AF1397), and GFP (Abcam, Cat#ab6556).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Luciferase, Negative Control, Knockdown, Two Tailed Test, Binding Assay, ChIP-qPCR

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: YY2 protein expression in Tsc2−/− 105K cells ( a ) and Tsc2 KO MEFs ( b ) treated with 20 nM rapamycin (Rapa) or vehicle for 24 hr ( n = 3). YY2 protein expression in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ) treated with 10 μM PF-4708671 (S6K1 inhibitor) or vehicle for 24 hr ( n = 3). e Immunoblot analysis of HeLa cells transfected with eGFP-YY2, wild-type S6K (HA-WT-S6K), or kinase-dead S6K (HA-KD-S6K) for 48 hr ( n = 3). f Immunoblot analysis of in vitro kinase assays of recombinant active S6K in the presence or absence of myc-YY2 as a substrate. eIF4B was used as a positive control ( n = 3). g Immunoblot analysis of immunoprecipitated (IP) GFP-YY2 and the whole-cell-lysate (WCL) from HeLa cells expressing GFP-YY2 ( n = 3). h Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing GFP-YY2 ( n = 3). i The evolutionarily conserved putative S6K phosphorylation site in YY2. j Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells stably expressing wild-type (WT) GFP-YY2 or mutant (T336A) GFP-YY2, treated with 40 μM PF-4708671 for 1 hr ( n = 3). k Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing WT GFP-YY2 or T336A GFP-YY2 ( n = 3). l , m Network view of predicted E3 ubiquitin ligase-YY2 interactions by UbiBrowser 2.0. Confidence level of ( m ). n Decreased EGFP-YY2 expression in HeLa cells transfected with Smurf1-FLAG ( n = 3). o Decreased Smurf1 ubiquitination in HeLa cells transfected with S6K. Immunoblot analysis of GFP-immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG or HA-WT-S6K for 24 hr ( n = 3). p Immunoblot analysis of GFP-and IgG control immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG ( n = 3). q Immunoblot analysis of B7-H3 expression in Tsc2−/− 105K cells, transfected with control or myc-YY2 for 48 hr and treated with 20 nM rapamycin or vehicle for the last 24 hr ( n = 3). Source data is provided in the Source data file.

Article Snippet: The following primary antibodies were used for immunoblotting at 1:1000 dilution unless otherwise indicated: Phospho-p70 S6 Kinase T389 (CST, Cat#9234), p70 S6 Kinase (CST, Cat#2708), TSC2 (CST, Cat#4308), Phospho-S6 S235/236 (CST, Cat# 2211), S6 (CST, Cat#2217), mTOR (CST, Cat# 2983), Raptor (CST, Cat#2280), Rictor (CST, Cat#2114), Phospho-Akt S473 (CST, Cat#4060), Phospho-Akt T308 (CST, Cat#4056), Akt (CST, Cat# 4685), GAPDH (CST, Cat# 2118), CREB (CST, Cat#4820), STAT1 (CST, Cat#14994), p-STAT1 (Tyr 701) (CST, Cat#9167), human B7-H3 (CST, Cat# 14058), YY1 (CST, Cat#46395), HA-tag (CST, Cat#3724), Phospho-substrate (RXXS*/T*) (CST, Cat# 9614), MHC-II (1:500, LSBio Cat#LS-C204829), β-actin (Sigma, Cat#A1978), YY2 (A-5) (1:500, Santa Cruz, Cat#sc-377008), YY2 (C-10) (1:500, Santa Cruz, Cat#sc-374455), mouse B7-H3 (1:500, R&D Systems, Cat#AF1397), and GFP (Abcam, Cat#ab6556).

Techniques: Expressing, Western Blot, Transfection, In Vitro, Recombinant, Positive Control, Immunoprecipitation, Phospho-proteomics, Stable Transfection, Mutagenesis, Ubiquitin Proteomics, Control

Journal: The Journal of Cell Biology

Article Title: Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle

doi: 10.1083/jcb.201910059

Figure Lengend Snippet: Determinants for CPC localization in to chromosomes and the anaphase spindle. (A) A ClustalX alignment of INCENP sequences from different species highlighting the survivin–borealin interaction region, the CDK phosphorylation site at T59, and the conserved RRKKRR-motif. (B) HeLa Flp-In T-Rex cells, expressing different doxycycline-inducible GFP-INCENP constructs (full-length [ƒ] WT, T59E ƒ, or T59A ƒ point mutants or truncations 1–58, 1–80, RRKKRR (1–80 RRKKRR to hexa-alanine mutant) were Western blotted before and after doxycycline induction. The INCENP antibody recognizes an epitope in the C terminus and does not detect the N-terminal fragments. (C and D) The induced cells were stained for MKLP2, and representative images of thymidine-treated cells in either anaphase (C) or metaphase (D) are shown. (E and F) GFP-INCENP enrichment (f central_spindle /f cytoplasmic ) at the central spindle (WT n = 29, 1–58 n = 36, 1–80 n = 36, RRKKRR n = 31, T59E ƒ n = 39, T59A ƒ n = 34; E) or metaphase chromatin (f chromatin /f cytoplasmic ; WT n = 50, 1–58 n = 50, 1–80 n = 58, RRKKRR n = 51, T59E ƒ n = 72, T59A ƒ n = 43; F) are plotted in the graphs. Both graphs show the mean with individual data points marked, and error bars indicate SEM. An unpaired t test with Welch’s correction and 99% confidence intervals was performed (*, P < 0.05; ****, P < 0.0001).

Article Snippet: Commercially available polyclonal antibodies (pAbs) or mAbs were used for tubulin (mouse mAb DM1A, T6199; Sigma-Aldrich), cyclin B1 (mouse mAb GNS3, 05-373; Millipore), GFP (rabbit pAb, ab290; Abcam), Histone H3 (rabbit mAb D1H2, 4419; Cell Signaling), Histone H3pT3 (rabbit pAb, 07-424; Millipore), Aurora B (mouse mAb AIM-1, 611083; BD Transduction Laboratories), Survivin (rabbit mAb EP2880Y, ab76424; Abcam), Borealin (mouse mAb A-5, sc-376635; Santa Cruz), Histone H3pS10 (mouse mAb 6G3, 9706; Cell Signaling), and PRC1 pT481 (rabbit mAb EP1514Y, ab62366; Abcam).

Techniques: Phospho-proteomics, Expressing, Construct, Mutagenesis, Western Blot, Staining

Journal: The Journal of Cell Biology

Article Title: Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle

doi: 10.1083/jcb.201910059

Figure Lengend Snippet: Structure of CPI58 and CPI80 peptide complexes. (A) CPI NT (survivin in green, borealin in light blue, and INCENP in pink) crystallized as a dimer, inhibiting H3pT3 peptide binding. INCENP N-terminal region of the symmetry-related molecule is interacting with survivin, occupying the same region where the peptide should have bound. Survivin H80 and K62 are shown. (B and C) Electrostatic potential maps of the CPI58 (B) and CPI80 (C) surfaces shows the negatively charged (red) peptide-binding groove and the positively charged (blue) cleft where the T3 phosphate sits. The green density map indicates the presence of H3pT3 peptide bound to survivin of both CPI58 (B) and CPI80 (C). Superimposition of CPI58 (B) and CPI80 (C) with PDB accession no. 2QFA (yellow) highlights the different orientations of the side chain of Lys62 of survivin (green) in the presence of H3pT3, confirming its role in phosphate recognition, as previously observed for survivin . (D–G) ITC data plots of survivin E65A-H80A CPI58 (D) and CPI80 (E) mutants in the presence of H3 or H3pT3. ITC data plots of CPI58 (F) and CPI80 (G) in the presence of H2A or H2ApT121. ΔH, enthalpy; DP, differential power.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or mAbs were used for tubulin (mouse mAb DM1A, T6199; Sigma-Aldrich), cyclin B1 (mouse mAb GNS3, 05-373; Millipore), GFP (rabbit pAb, ab290; Abcam), Histone H3 (rabbit mAb D1H2, 4419; Cell Signaling), Histone H3pT3 (rabbit pAb, 07-424; Millipore), Aurora B (mouse mAb AIM-1, 611083; BD Transduction Laboratories), Survivin (rabbit mAb EP2880Y, ab76424; Abcam), Borealin (mouse mAb A-5, sc-376635; Santa Cruz), Histone H3pS10 (mouse mAb 6G3, 9706; Cell Signaling), and PRC1 pT481 (rabbit mAb EP1514Y, ab62366; Abcam).

Techniques: Binding Assay

Journal: The Journal of Cell Biology

Article Title: Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle

doi: 10.1083/jcb.201910059

Figure Lengend Snippet: Structure of the CPC bound to T3-phosphorylated histone H3. ( A and B ) Crystal structures of CPI58 (chromosomal passenger with INCENP 7-58 ; A) and CPI80 (chromosomal passenger with INCENP 7-80 ; B) in the presence of the H3pT3 peptide (survivin in green, borealin in light blue, INCENP in pink, and H3pT3 in orange) are shown. The close-up view highlights the intermolecular interactions between the BIR domain of survivin and H3pT3 (amino acids 1–5) for CPI58 and survivin and H3pT3 (amino acids 1–7) for CPI80. The free N-terminal A1 of H3pT3 contacts the negatively charged side chains of survivin D71 and E76. Hydrogen bonds also mediate interactions between amino acids A1, R2, and K4 of the H3pT3 peptide and amino acids H80, E65, and E63 of survivin, respectively. Recognition of phospho-threonine 3 in H3pT3 peptide is mediated by amino acids K62 and H80 of survivin. Hydrogen bonds are indicated with red dashed lines; survivin amino acids are labeled in black, and peptide amino acids are labeled in red. (C–E) ITC binding curves for complex formation between CPI58 (C), CPI80 (D), and CPI80 RRKKRR (E) and either H3 or H3pT3. (F) Histograms show K d values, and error bars represent the K d calculation uncertainty, obtained using MicroCal PEAQ-ITC analysis software. Cartoons represent the protein complexes used for each ITC experiment, where survivin is in green, borealin in blue, and INCENP in purple (1–58, 1–80, and 1–80 RRKKRR). ΔH, enthalpy; DP, differential power.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or mAbs were used for tubulin (mouse mAb DM1A, T6199; Sigma-Aldrich), cyclin B1 (mouse mAb GNS3, 05-373; Millipore), GFP (rabbit pAb, ab290; Abcam), Histone H3 (rabbit mAb D1H2, 4419; Cell Signaling), Histone H3pT3 (rabbit pAb, 07-424; Millipore), Aurora B (mouse mAb AIM-1, 611083; BD Transduction Laboratories), Survivin (rabbit mAb EP2880Y, ab76424; Abcam), Borealin (mouse mAb A-5, sc-376635; Santa Cruz), Histone H3pS10 (mouse mAb 6G3, 9706; Cell Signaling), and PRC1 pT481 (rabbit mAb EP1514Y, ab62366; Abcam).

Techniques: Labeling, Binding Assay, Software

Journal: The Journal of Cell Biology

Article Title: Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle

doi: 10.1083/jcb.201910059

Figure Lengend Snippet: CPC interacts with DNA through the INCENP RRKKRR-motif. (A) EMSA with linear plasmid DNA incubated with 3.6 µM CPI58, CPI80, or CPI80 RRKKRR . Buffer indicates no protein was added, whereas control is a negative control protein in the same buffer that does not bind DNA (HRV 3C). (B) EMSA of linear plasmid DNA incubated with increasing concentrations of CPI80 from 0 to 3.6 µM. (C) EMSA of linear plasmid DNA incubated with 3.6 µM CPI58, CPI80, CPI80 T59E , or CPI80 RRKKRR buffer. (D–G) MST of 0.1–200 µM CPI80 (D), CPI58 (E), CPI80 RRKKRR (F), or CPI80 T59E (G) with centromeric α-satellite (pink) or non–α-satellite (gray) DNA duplexes. K d was calculated for CPI80, where graph shows the mean and error bars indicate SEM ( n = 4) using NanoTemper analysis software. A two-way ANOVA was performed, considering dilution factor and DNA sequence (centromeric α-satellite or non–α-satellite) as independent variables (****, P < 0.0001). It was not possible to calculate a K d value for the other conditions. Cartoons inset into the panels represent protein complexes used for each EMSA or MST experiment, where survivin is in green, borealin in blue, and INCENP in purple (1–80, 1–58, 1–80 RRKKRR, 1–80 T59E , respectively). F norm , normalized fluorescence; na, not applicable.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or mAbs were used for tubulin (mouse mAb DM1A, T6199; Sigma-Aldrich), cyclin B1 (mouse mAb GNS3, 05-373; Millipore), GFP (rabbit pAb, ab290; Abcam), Histone H3 (rabbit mAb D1H2, 4419; Cell Signaling), Histone H3pT3 (rabbit pAb, 07-424; Millipore), Aurora B (mouse mAb AIM-1, 611083; BD Transduction Laboratories), Survivin (rabbit mAb EP2880Y, ab76424; Abcam), Borealin (mouse mAb A-5, sc-376635; Santa Cruz), Histone H3pS10 (mouse mAb 6G3, 9706; Cell Signaling), and PRC1 pT481 (rabbit mAb EP1514Y, ab62366; Abcam).

Techniques: Plasmid Preparation, Incubation, Control, Negative Control, Software, Sequencing, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle

doi: 10.1083/jcb.201910059

Figure Lengend Snippet: INCENP RRKKRR-motif dependant interaction of CPI80 with MKLP2. (A) SEC-MALS chromatograms and molecular weight of MKLP2 557-668 (yellow), CPI80 (orange), and MKLP2:CPI80 (blue) are shown. MKLP2:CPI80 has a single peak and unique mass showing MKLP2 binds CPC in a 1:1 ratio. (B and C) SEC of MKLP2 557-668 (yellow) with CPI80 RRKKRR (orange; B) or CPI58 (orange; C) was performed to test for MKLP2:CPI80 RRKKRR or MKLP2:CPI58 (blue) complexes, respectively. Dotted lines indicate the fraction numbers. InstantBlue-stained acrylamide gels are shown for all proteins, where M, S, and B stand for MKLP2, survivin, and borealin, respectively. For INCENP, I 80 is amino acids 1–80, I RRKKRR is amino acids 1–80 with the RRKKRR to hexa-alanine mutation, and I 58 is amino acids 1–58. In the cartoon, depictions of protein complexes are as follows: survivin is in green, borealin in blue, INCENP in purple, and MKLP2 in yellow. mAU, milli-absorbance unit.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or mAbs were used for tubulin (mouse mAb DM1A, T6199; Sigma-Aldrich), cyclin B1 (mouse mAb GNS3, 05-373; Millipore), GFP (rabbit pAb, ab290; Abcam), Histone H3 (rabbit mAb D1H2, 4419; Cell Signaling), Histone H3pT3 (rabbit pAb, 07-424; Millipore), Aurora B (mouse mAb AIM-1, 611083; BD Transduction Laboratories), Survivin (rabbit mAb EP2880Y, ab76424; Abcam), Borealin (mouse mAb A-5, sc-376635; Santa Cruz), Histone H3pS10 (mouse mAb 6G3, 9706; Cell Signaling), and PRC1 pT481 (rabbit mAb EP1514Y, ab62366; Abcam).

Techniques: Molecular Weight, Staining, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle

doi: 10.1083/jcb.201910059

Figure Lengend Snippet: Survivin-dependent centromere targeting in metaphase is a prerequisite for MKLP2-dependent CPC localization in anaphase. (A and B) HeLa cells expressing different survivin-GFP constructs (WT, K62A, E65A, H80A, K62A-H80A, and E65A-H80A) were depleted of endogenous survivin using a 3′ UTR siRNA and then stained for MKLP2. Representative images of cells in metaphase (A) and anaphase (B) are shown. (C) SEC of MKLP2 557-668 (yellow) with CPI80 Survivin E65A-H80A (orange) was performed to test for MKLP2:CPI80 Survivin E65A-H80A complexes (blue). In the cartoon, depictions of protein complexes are as follows: survivin is in green, borealin in blue, INCENP in purple, and MKLP2 in yellow. AA denotes the survivin E65A, H80A mutant. mAU, milli-absorbance unit. M, MKLP2; S E65,H80 , survivin E65,H80 mutant; B, borealin; I 80 , INCENP amino acids, 1–80.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or mAbs were used for tubulin (mouse mAb DM1A, T6199; Sigma-Aldrich), cyclin B1 (mouse mAb GNS3, 05-373; Millipore), GFP (rabbit pAb, ab290; Abcam), Histone H3 (rabbit mAb D1H2, 4419; Cell Signaling), Histone H3pT3 (rabbit pAb, 07-424; Millipore), Aurora B (mouse mAb AIM-1, 611083; BD Transduction Laboratories), Survivin (rabbit mAb EP2880Y, ab76424; Abcam), Borealin (mouse mAb A-5, sc-376635; Santa Cruz), Histone H3pS10 (mouse mAb 6G3, 9706; Cell Signaling), and PRC1 pT481 (rabbit mAb EP1514Y, ab62366; Abcam).

Techniques: Expressing, Construct, Staining, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle

doi: 10.1083/jcb.201910059

Figure Lengend Snippet: MKLP2 competes with DNA not H3pT3 for CPC binding. (A) ITC binding curves for complex formation between MKLP2 557-668 :CPI80 (survivin, INCENP 1-80 , borealin 10-109 ) and H3 or H3pT3. Histogram bars represent K d values and error bars represent K d calculation uncertainty, obtained using MicroCal PEAQ-ITC analysis software. ΔH, enthalpy; DP, differential power. (B) EMSA of plasmid DNA incubated with 3.6 µM CPI58, CPI80, CPI80 RRKKRR , MKLP2:CPI80, MKLP2 only, or buffer. (C) MST of 0.1–200 µM MKLP2 557-668 :CPI80 (survivin, INCENP 1-80 , borealin 10-109 ) after incubation with centromeric α-satellite or non–α-satellite DNA duplexes. The graph shows the mean and error bars indicate SEM ( n = 3), and it was not possible to calculate a K d values for these conditions. In the cartoon depictions of protein complexes: survivin is in green, borealin in blue, INCENP in purple, MKLP2 in yellow. DNA molecules are colored gray. F norm , normalized fluorescence; na, not applicable.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or mAbs were used for tubulin (mouse mAb DM1A, T6199; Sigma-Aldrich), cyclin B1 (mouse mAb GNS3, 05-373; Millipore), GFP (rabbit pAb, ab290; Abcam), Histone H3 (rabbit mAb D1H2, 4419; Cell Signaling), Histone H3pT3 (rabbit pAb, 07-424; Millipore), Aurora B (mouse mAb AIM-1, 611083; BD Transduction Laboratories), Survivin (rabbit mAb EP2880Y, ab76424; Abcam), Borealin (mouse mAb A-5, sc-376635; Santa Cruz), Histone H3pS10 (mouse mAb 6G3, 9706; Cell Signaling), and PRC1 pT481 (rabbit mAb EP1514Y, ab62366; Abcam).

Techniques: Binding Assay, Software, Plasmid Preparation, Incubation, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle

doi: 10.1083/jcb.201910059

Figure Lengend Snippet: A model for MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle. CPC (light green) binding to the centromeres is mediated through multiple weak interactions between survivin (dark green) and phosphorylated histone H3, borealin (blue) with nucleosomes, and the INCENP (purple) RRKKRR-motif with DNA. For simplicity, the borealin–nucleosome interaction is not depicted. Phosphorylation of INCENP at T59 and MKLP2 (yellow) at multiple sites prevents interaction of MKLP2 with the CPC. In anaphase, dephosphorylation of INCENP and MKLP2 results in binding of these two proteins, competing for interaction to the DNA. We propose a transient intermediate state where the CPC interacts with MKLP2 but still remains bound to phosphorylated histone H3. CPC-stimulated MKLP2 motor activity then drives movement of MKLP2 with bound CPC away from the chromosome to the anaphase spindle.

Article Snippet: Commercially available polyclonal antibodies (pAbs) or mAbs were used for tubulin (mouse mAb DM1A, T6199; Sigma-Aldrich), cyclin B1 (mouse mAb GNS3, 05-373; Millipore), GFP (rabbit pAb, ab290; Abcam), Histone H3 (rabbit mAb D1H2, 4419; Cell Signaling), Histone H3pT3 (rabbit pAb, 07-424; Millipore), Aurora B (mouse mAb AIM-1, 611083; BD Transduction Laboratories), Survivin (rabbit mAb EP2880Y, ab76424; Abcam), Borealin (mouse mAb A-5, sc-376635; Santa Cruz), Histone H3pS10 (mouse mAb 6G3, 9706; Cell Signaling), and PRC1 pT481 (rabbit mAb EP1514Y, ab62366; Abcam).

Techniques: Binding Assay, Phospho-proteomics, De-Phosphorylation Assay, Activity Assay

Journal: BMC Musculoskeletal Disorders

Article Title: The relationship between abnormal Core binding factor-β expression in human cartilage and osteoarthritis

doi: 10.1186/s12891-021-04043-9

Figure Lengend Snippet: A. Showing the PCR amplification band of the positively cloned CBFB/pCDH plasmid vector was 564 bp, which was consistent with the sequence map. B. CBFB/ plkko.1 plasmid vector positive clones were 5 kbp and 2 kbp after double enzymatic digestion, and the sequencing results were consistent. C. Validating Western immunoblot results of plasmid transfection efficiency; D. cell proliferation in each group as detected by the CCK-8 assay; E, F. cellular apoptosis in each group as detected by Annexin V/PI double staining (*: p < 0.05, **: p < 0.01, ***: p < 0.001)

Article Snippet: Annexin V/PI flow cytometry apoptosis (CyFlow® Space; SysmexPartec GmbH, Görlitz, Germany): after trypsin digestion and collection of cells in each group by centrifugation, the experiment was conducted following the annexin V-FITC apoptosis detection kit (Cat. No. c1062l, Beyotime Institute of Biotechnology, Shanghai, China) operation manual guidelines.

Techniques: Amplification, Clone Assay, Plasmid Preparation, Sequencing, Western Blot, Transfection, CCK-8 Assay, Double Staining

Journal: Cell Reports

Article Title: HIV-1 Envelope Recognition by Polyreactive and Cross-Reactive Intestinal B Cells

doi: 10.1016/j.celrep.2019.03.032

Figure Lengend Snippet:

Article Snippet: Mouse anti-human IgA FITC (IS11-8E10) , Miltenyi Biotec , Cat#130-113-475.

Techniques: Virus, Recombinant, Purification, Staining, Reverse Transcription, Transfection, Plasmid Preparation, Expressing, Software, Imaging

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies

doi: 10.1172/JCI99257

Figure Lengend Snippet: (A) Different melanoma cells were subjected to SOX10 knockdown (KD) by inducible shRNA expression, and SOX10, NRP1, and EGFR mRNA levels were analyzed by qPCR (n = 4). The graph shows log2 fold change variations in SOX10-KD cells versus respective controls. (B) Box plot showing SOX10, NRP1, and EGFR mRNA expression variations in 12 paired melanoma samples (indicated by different symbols) obtained from the same patients before and during treatment with BRAF inhibitors (log2 ratio treated/untreated). (C) qPCR analysis of SOX10, NRP1, and EGFR expression in A375 and SK-MEL-28 melanoma cells that developed acquired resistance to 2 μM PLX-4720 (log2 ratio drug resistant versus parental cells; n = 3). (D) SOX10, NRP1, and EGFR protein expression in the same cells shown in C; vinculin and β-tubulin levels provided loading controls (1 representative experiment of 3 repetitions; duplicate samples were run on parallel gels for staining with different antibodies). (E) The viability of parental or drug-resistant A375 cells was assessed upon NRP1 or EGFR KD with targeted siRNAs (n = 5). (F) A375 parental cells were subjected to NRP1 KD (by targeted shRNAs) before exposure to escalating concentrations of PLX-4720 aimed at establishing drug resistance. NRP1 and EGFR expression was analyzed after 5 weeks of treatment. The statistical analysis compared NRP1-KD samples with respective controls (carrying plkO empty vector) (n = 3). (G) The growth rate of control or NRP1-KD A375, either untreated or exposed to 0.5 μM PLX-4720 for 5 weeks, is scored as the percentage of increase of viable cells across 72 hours (n > 3). (H) The growth rate of control or NRP1-KD A375 cells on therapy with 0.5 μM PLX-4720 for 5 weeks was tracked in culture over 7 days (n > 3). (I) Mice implanted with either mock-transduced or NRP1-KD drug-resistant A375 cells were treated daily with 20 mg/kg PLX-4720 (or vehicle only), and the tumor burden was periodically measured. The statistical analysis compared treated tumors with respective controls (n = 5, per condition). For E, F, G, statistical significance was determined using a Student’s t test with Bonferroni’s correction (*P < 0.01; **P < 0.001; ***P < 0.0001); for H and I, a 2-way ANOVA test with Bonferroni’s correction was used (***P < 0.0001).

Article Snippet: An alpaca (Vicugna pacos) was immunized via an alternating immunization schedule: an injection of 100 μg recombinant human NRP1 (R&D Systems) was followed by 100 μg recombinant mouse NRP1 protein (R&D Systems) 1 week later.

Techniques: Knockdown, shRNA, Expressing, Staining, Plasmid Preparation, Control

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies

doi: 10.1172/JCI99257

Figure Lengend Snippet: (A) The expression of miRNA-338-3p was assessed by quantitative real-time PCR in PLX-4720–resistant A375 and SK-MEL-28 melanoma cells, and normalized versus the respective parental cells (n = 5). Student’s t test, ***P < 0.0001. (B) qPCR analysis of NRP1 (left graph) and EGFR (right graph) expression in drug-resistant A375 and SK-MEL-28 cells, transfected in order to reexpress miR-338 (values normalized to respective mock-transfected cells) (n = 4). (C) NRP1 expression assessed by immunoblotting in parental and PLX-4720–resistant A375 cells transfected with pre–miRNA-338 (or mock transfected); vinculin provided a protein loading control (1 representative experiment of 5 repetitions). (D) Viability of drug-resistant (or parental) A375 and SK-MEL-28 cells, transfected with miR-338 or mock (as shown in B; values normalized to respective mock-transfected cells) (n = 3).

Article Snippet: An alpaca (Vicugna pacos) was immunized via an alternating immunization schedule: an injection of 100 μg recombinant human NRP1 (R&D Systems) was followed by 100 μg recombinant mouse NRP1 protein (R&D Systems) 1 week later.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies

doi: 10.1172/JCI99257

Figure Lengend Snippet: (A) NRP1 expression was assessed by immunoblotting in GLT16 (gastric cancer), EBC1 (lung cancer), and H1993 (lung cancer) Met-addicted cells; β-actin provided a protein loading control (1 representative experiment of 2 repetitions). (B) The viability of the Met-addicted cells described in A was assessed by Cell Titer Glo Viability Assay upon NRP1 knockdown by RNA interference (n = 3). (C–H) The viability of GTL16 (C and D), EBC1 (E–G), and H1993 cells (H), either NRP1-depleted or mock-transfected (same cells as in B), was assessed in the presence of increasing concentrations of the Met inhibitors indicated below the graphs: JnJ-38877605 (JNJ-605), PHA-665752, or crizotinib (n > 5). IC50 values were calculated by Graphpad. (I and J) The growth of NRP1-depleted or mock-transfected GTL16 (I) and EBC1 (J) (same cells as in previous panels) was assessed by staining with crystal violet and reading Abs 595 nm after 2 weeks in culture in the presence of the indicated concentrations of the Met inhibitor JNJ-605 and normalized to untreated control cells condition (n = 3). In C–J, the statistical analysis was done by 2-way ANOVA with Bonferroni’s correction, comparing the behavior of NRP1-silenced and control cells.

Article Snippet: An alpaca (Vicugna pacos) was immunized via an alternating immunization schedule: an injection of 100 μg recombinant human NRP1 (R&D Systems) was followed by 100 μg recombinant mouse NRP1 protein (R&D Systems) 1 week later.

Techniques: Expressing, Western Blot, Control, Viability Assay, Knockdown, Transfection, Staining

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies

doi: 10.1172/JCI99257

Figure Lengend Snippet: (A and B) The viability of EBC1 (A) or GTL16 (B) Met-addicted cells, either NRP1 overexpressing or mock transfected, was assessed in the presence of increasing concentrations of the indicated Met inhibitors (n > 6). IC50 values were calculated by Graphpad. (C) NOD-SCID mice were injected subcutaneously with a heterogeneous mix of EBC1 cells, half control and half overexpressing NRP1 (n = 5, per experimental condition). In 2 complementary settings, one of the cell populations was previously marked by fluorescent Turbo-RFP, featuring a mock-RFP–labeled mix and a NRP1-RFP–labeled mix, which allowed us to follow the growth of intermingled fluorescent cells expressing different NRP1 levels by in vivo imaging with the IVIS Spectrum CT system (y axis). As expected, mouse treatment with the Met-inhibitor JNJ-605 (2.5 mg/kg by daily oral gavage) impaired the growth of control cells (revealed by fluorescent signal in mock-RFP mix tumors in this bar graph, and by gene marker–specific qPCR in Supplemental Figure 4, A and B). However, NRP1-overexpressing cells (revealed by fluorescence in NRP1-RFP mix tumors in this bar graph, and by gene marker–specific qPCR in Supplemental Figure 4B) were relatively refractory to targeted therapy. Indeed, due to overgrowth of NRP1hi cells, all tumors developed irrespective of targeted therapy (see Supplemental Figure 4, C and D). In all panels, the statistical analysis was done by 2-way ANOVA with Bonferroni’s correction, comparing the behavior of NRP1hi and mock-transfected control cells; ***P < 0.0001.

Article Snippet: An alpaca (Vicugna pacos) was immunized via an alternating immunization schedule: an injection of 100 μg recombinant human NRP1 (R&D Systems) was followed by 100 μg recombinant mouse NRP1 protein (R&D Systems) 1 week later.

Techniques: Transfection, Injection, Control, Labeling, Expressing, In Vivo Imaging, Marker, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies

doi: 10.1172/JCI99257

Figure Lengend Snippet: (A) NRP1 and EGFR expression was analyzed by immunoblotting in the indicated Met-addicted cells, either control or transduced to overexpress NRP1; vinculin provided a protein loading control (1 representative experiment of 5 repetitions). (B) EBC1 cells overexpressing NRP1 (or mock controls) were analyzed by Western blotting to reveal the expression and the phosphorylation levels of Met and EGFR tyrosine kinases, as well as their intracellular effector AKT (1 representative experiment of 4 repetitions; duplicate samples were run on parallel gels). (C) The viability of EBC1 cells overexpressing NRP1 (or mock controls) was assessed in the presence of the Met-inhibitor JNJ-605 (15 nM), either alone or in combination with the EGFR inhibitors cetuximab (1 μg/ml) or erlotinib (1 μM). Statistical significance was calculated by Student’s t test with Bonferroni’s correction, comparing each cetuximab- or erlotinib-treated condition versus respective vehicle-treated conditions (n = 6); **P < 0.001.

Article Snippet: An alpaca (Vicugna pacos) was immunized via an alternating immunization schedule: an injection of 100 μg recombinant human NRP1 (R&D Systems) was followed by 100 μg recombinant mouse NRP1 protein (R&D Systems) 1 week later.

Techniques: Expressing, Western Blot, Control, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies

doi: 10.1172/JCI99257

Figure Lengend Snippet: (A) Immunoblotting analysis of NRP1 expression in the indicated breast cancer cells, either parental naive or with acquired resistance to the HER2-inhibitor lapatinib (Lap) (250 nM for BT474 and 120 nM for SKBR3); vinculin provided a protein loading control (1 representative experiment of at least 3 repetitions). (B) The same cells described in A were subjected to NRP1 knockdown by shRNAs (or treated with plkO empty vector); the fraction of cells resistant to lapatinib (same concentrations as indicated above) was defined as residual viability in presence of the drug versus untreated conditions (n > 4). (C) Immunoblotting analysis of IGF1R expression and tyrosine phosphorylation in lapatinib-resistant BT474 cells; vinculin provided a protein loading control (1 representative experiment of 3 repetitions). (D) IGF1R mRNA expression levels were measured by qPCR in parental or lapatinib-resistant BT474 cells, either control or upon NRP1 silencing (n = 3). Statistical significance was assessed by Student’s t test; ***P < 0.0001.

Article Snippet: An alpaca (Vicugna pacos) was immunized via an alternating immunization schedule: an injection of 100 μg recombinant human NRP1 (R&D Systems) was followed by 100 μg recombinant mouse NRP1 protein (R&D Systems) 1 week later.

Techniques: Western Blot, Expressing, Control, Knockdown, Plasmid Preparation, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies

doi: 10.1172/JCI99257

Figure Lengend Snippet: (A) p27 mRNA levels were measured by qPCR (n > 3) in the indicated cancer cell lines: A375 and SK-MEL-28 (parental or resistant to PLX-4720), BT474 (parental or resistant to lapatinib), and EBC1 (NRP1 overexpressing or mock transfected). (B) p27-Kip1, activated phospho-JNK, and total JNK protein levels in cancer cells described in A (1 representative experiment of 4 repetitions; duplicate samples were run on parallel gels). The values at the bottom indicate densitometric measurement of relative p-JNK versus total JNK band intensity. (C) NRP1, p27, p-JNK, and total JNK levels in EBC1 cells subjected to NRP1 silencing (1 representative experiment of 3 repetitions; duplicate samples were run on parallel gels). (D) p-JNK and EGFR levels in EBC1 cells (shown in B), subjected to p27 overexpression (1 representative experiment of 3 repetitions; duplicate samples were run on parallel gels). (E) qPCR analysis of EGFR expression in SK-MEL-28 melanoma cells, parental or PLX-4720 resistant, upon treatment with the JNK kinase inhibitor SP600125 (25 μM; JNK-i) or with vehicle alone (n = 5). (F) EGFR expression in EBC1 cells (shown in B), either in basal conditions (Veh) or in the presence of 25 μM SP600125 (1 representative experiment of 6 repetitions). (G) IGF1R levels in BT474 cells (shown in B), either in basal conditions or in the presence of 25 μM SP600125 (1 representative experiment of 5 repetitions). **P < 0.001, ***P < 0.0001.

Article Snippet: An alpaca (Vicugna pacos) was immunized via an alternating immunization schedule: an injection of 100 μg recombinant human NRP1 (R&D Systems) was followed by 100 μg recombinant mouse NRP1 protein (R&D Systems) 1 week later.

Techniques: Transfection, Over Expression, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies

doi: 10.1172/JCI99257

Figure Lengend Snippet: (A) qPCR analysis of SOX2 expression in parental and drug-resistant A375 and EBC1 cells (shown in B), either in basal conditions or in the presence of 25 μM SP600125; values normalized to untreated controls (n = 5). (B) qPCR analysis of EGFR expression in parental and drug-resistant A375 cells subjected to SOX2 silencing by targeted siRNAs (validated in Supplemental Figure 7F) (n = 4). (C) EGFR expression in EBC1 cells (shown in B), subjected to SOX2 silencing (validated in Supplemental Figure 7F) (1 representative experiment of 4 repetitions). (D) qPCR analysis of JUN expression in parental and drug-resistant BT474 cells, either in basal conditions or in the presence of 25 μM SP600125; values normalized to untreated parental cells (n = 3). (E) IGF1R expression in BT474 cells (same as above), subjected to JUN silencing by RNAi (validated in Supplemental Figure 7G) (1 representative experiment of 4 repetitions). (F) Schematic working model of the proposed signaling cascade leading to the onset of resistance to oncogene-targeted therapy in different cancer cells. The upstream regulation of NRP1 levels by miR-338-3p (marked in red) was specifically observed in melanoma cells, whereas in carcinoma cells NRP1 expression might depend on RTK-Ras signaling cascade (marked in blue). (G) SK-MEL-28 cell viability was assessed upon treatment with 1 μM PLX-4720, alone or in association with the indicated concentrations of JNK-i SP600125. While naive cells were initially sensitive to PLX-4720, after 3–5 weeks in culture they progressively developed drug resistance, but this was prevented in the presence of JNK-i (n > 3). In all panels, statistical significance was assessed by Student’s t test with Bonferroni’s correction; *P < 0.01, **P < 0.001, ***P < 0.0001.

Article Snippet: An alpaca (Vicugna pacos) was immunized via an alternating immunization schedule: an injection of 100 μg recombinant human NRP1 (R&D Systems) was followed by 100 μg recombinant mouse NRP1 protein (R&D Systems) 1 week later.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies

doi: 10.1172/JCI99257

Figure Lengend Snippet: (A) The viability of EBC1 Met-addicted cells (n = 5) was assessed upon treatment with increasing concentrations of the Met inhibitor JNJ-605, either alone or in combination with the NRP1-targeted molecule EG00229 (12.5 μM) or the nanobody HS45 (5 μg/ml). (B) HER2-addicted BT474 breast cancer cells, either parental naive or resistant to the targeted inhibitor lapatinib (250 nM), were exposed to lapatinib alone or in combination with EG00229 (12.5 μM); the fraction of drug-resistant cells was calculated based on residual viability versus untreated conditions (n = 4). (C) EGFR mRNA levels were measured in SOX10-depleted A375 melanoma cells (or controls) (n = 4), treated with the NRP1-targeted molecule EG00229 (12.5 μM) or with vehicle alone. (D) Viability of SOX10-depleted A375 melanoma cells (or controls) (n > 4) exposed to 0.25 μM PLX-4720, either alone or in combination with the small molecule EG00229 (12.5 μM) or HS45 nanobody (5 μg/ml). (E) The growth of A375 melanoma cells, parental or PLX-4720 resistant (n = 3), was assessed after 2 weeks of culture in the presence of the BRAF inhibitor PLX-4720, either alone or in combination with 12.5 μM EG00229 (representative images at the bottom). (F) Residual viability of BRAF inhibitor–resistant A375 melanoma cells subjected to different therapeutic associations of PLX-4720 (2 μM) with cetuximab (1 μg/ml) and/or EG00229 (12.5 μM); the statistical analysis compared combinatorial treatments with PLX-4720 alone (n = 5). We applied ANOVA tests with Bonferroni’s correction to analyze multiple sample comparisons in A, D, F; in the other cases, we used Bonferroni-corrected Student’s t tests; *P < 0.01, **P < 0.001, ***P < 0.0001.

Article Snippet: An alpaca (Vicugna pacos) was immunized via an alternating immunization schedule: an injection of 100 μg recombinant human NRP1 (R&D Systems) was followed by 100 μg recombinant mouse NRP1 protein (R&D Systems) 1 week later.

Techniques:

Journal: iScience

Article Title: CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells

doi: 10.1016/j.isci.2023.108287

Figure Lengend Snippet: Differentiation of monoclonal iPSC lines to lymphoid progenitor cells (LPCs) and NK cells (A and B) Parental and edited iPSC clones were differentiated to LPCs through initial CD34 + HSPC stage. HSPCs were reseeded and LPCs harvested on day 14 and stained with the pairwise antibody combinations indicated in Figure S6 A. Quantification of cell subtypes from pairwise antibody staining. Data are from biological replica (n) = 1 and technical replica (t) = 1 per sample. (C and D) Parental KOLF2 iPSCs were differentiated to NK cells through the HSPC and LPC stages. The indicated samples obtained relatively sufficient NK number for analysis. Data are related to Figure S6 C (n = 1; t = 1). (E and F) Parental KOLF2 iPSCs and iPSC lines CXs313_4 GFP (cl. A8) and ROSA26_3 CAR-GFP (cl. A4) were differentiated to NK cells which were analyzed by immunostaining and flow cytometry (n = 1; t = 1). (G) Parental KOLF2 iPSCs and the iPSC clones C7s257 CAR-GFP (cl. B2 and cl. D4) and C8s325 GFP (cl. H6) were initially differentiated to CD34 + HSPCs (see Figures 3 F and 3G), followed by differentiation to NK cells (abbreviated here iNK) (n = 1; t = 1). Cells were harvested at 29 days and stained with antibodies in pairwise combinations. CD56 + CD45 +bright are considered as NK cells. (H and I) Quantification of cells positive for key markers (n = 1; t = 1). Samples are from same experiment as in panel (G). (J) Cell apoptosis assay using 10 4 NALM6-RFP + (expressing red fluorescent protein; RFP) as target cells. The apoptosis reagent IncuCyte Annexin V Green was added (final dilution 1:200) into the target cells 1 h after seeding, followed by immediate addition of the indicated effector cells in different ratios (K = 10 3 cells). Effector cells were primary NK cells from donor NK2 (15 days after isolation from blood and kept in culture), KOLF2-derived NK cells and NK derived from iPSC line ROSA26_3 CAR-GFP (cl. A4). As positive control, absolute ethanol was added to 3 replicate wells to a final concentration 10%, 2 h after initial target cell seeding. NK Basal Medium was used for all cell suspensions in the assay. Each treatment consists of 3 technical replica wells. The graph shows the average ratio of NALM6 RFP + AnnexinV + /NALM6 RFP + and SD of 3 technical replicas (t = 3). The assay was done as one biological replica (n = 1). Cells were imaged by IncuCyte system at 20× magnification by non-adherent cell-by-cell format. See also Figure S6 .

Article Snippet: IncuCyte Annexin-V Green reagent for apoptosis , Sartorius , Cat#4642.

Techniques: Clone Assay, Staining, Immunostaining, Flow Cytometry, Apoptosis Assay, Expressing, Isolation, Derivative Assay, Positive Control, Concentration Assay

Journal: iScience

Article Title: CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells

doi: 10.1016/j.isci.2023.108287

Figure Lengend Snippet:

Article Snippet: IncuCyte Annexin-V Green reagent for apoptosis , Sartorius , Cat#4642.

Techniques: Control, Virus, Selection, Recombinant, Knock-Out, Clone Assay, Plasmid Preparation, Modification, Staining, Reverse Transcription, Generated, Software, Flow Cytometry