arid1a  (Santa Cruz Biotechnology)

Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: In vitro and in vivo synthetic lethality of miR-4653-3p in HCT116 ARID1A −/− cells. A Synthetic lethality of miR-4653-3p in HCT116 ARID1A −/− cells. HCT116 ARID1A +/+ and ARID1A −/− cell lines were transfected with NC or miR-4653-3p mimics for 72 h and the cell viability was shown. Error bars represent s.d. from three independent experiments. ANOVA ** P < 0.01, *** P < 0.001. B-D Preferential induction of apoptosis in HCT116 ARID1A −/− cells by miR-4653-3p. Detection of apoptosis in HCT116 ARID1A +/+ and HCT116 ARID1A −/− cells transfected with NC or miR-4653-3p mimics for 72 h by flow cytometry ( B ) and the percentage of apoptotic cells were quantitated ( C ). Error bars represent s.d. from three independent experiments. Student’s t-test *** P < 0.001. Apoptosis biomarkers (Cleaved PARP and Cleaved caspase 3) were detected by western blot ( D ). E , F Colony formation assay was performed in ARID1A isogenic cell pair to validate the synthetic lethality effects of miR-4653-3p. Cells were transfected with NC or miR-4653-3p mimics for 14 days and colonies were stained with crystal violet. Error bars represent s.d. from three independent experiments. Student’s t-test ** P < 0.01. G Western blot analysis showed overexpression of ARID1A in the two RKO ARID1A OE clones. H Synthetic lethality of miR-4653-3p in RKO ARID1A −/− cells. RKO ARID1A −/− and ARID1A OE cell lines were transfected with miR-4653-3p mimics for 72 h and the cell viability was shown. Error bars represent s.d. from three independent experiments. ANOVA *** P < 0.001. I Preferential induction of apoptosis in RKO ARID1A −/− cells by miR-4653-3p as indicated by apoptosis biomarkers (Cleaved PARP and Cleaved caspase 3). J Schematic illustration of mouse tumor xenograft experiments with the HCT116 ARID1A isogenic cell pair. K , L Tumor growth curves in nude mice bearing HCT116 ARID1A +/+ ( K ) and ARID1A −/− ( L ) xenografts after treatment with NC or miR-4653-3p. M , N Tumor weight of HCT116 ARID1A +/+ ( M ) and ARID1A −/− ( N ) xenografts after treatment with NC or miR-4653-3p. Error bars represent s.d. * P < 0.05 between vehicle and RITA treatment groups ( n = 5). Student’s t-test

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques: In Vitro, In Vivo, Transfection, Flow Cytometry, Western Blot, Colony Assay, Staining, Over Expression, Clone Assay

Journal: Journal of Translational Medicine

Article Title: Targeting miR-4653-3p/SLC25A51/SIRT3 axis to induce synthetic lethality in ARID1A-deficient colorectal cancer via blockade of DNA repair

doi: 10.1186/s12967-026-07760-8

Figure Lengend Snippet: Synthetic lethality of ARID1A and miR-4653-3p is mediated by SLC25A51 and reduced SLC25A51 expression suppressed SIRT3 activity. A Western blot analysis of SLC25A51 expression in HCT116 cells by siRNA silencing. B , C Representative images ( B ) and cell viability ( C ) of HCT116 ARID1A +/+ and ARID1A −/− cell lines after transfected with SLC25A51 siRNA for 72 h. The cell images were taken with Olympus microscope. Scale bars, 100 μm. Cell viability was measured with Image J software. Error bars represent s.d. from three independent experiments. ANOVA * P < 0.05, ** P < 0.01, *** P < 0.001. D HCT116 ARID1A +/+ and ARID1A −/− cell lines were transfected with SLC25A51 siRNA for 72 h, and the expressions of apoptosis biomarkers (Cleaved PARP and Cleaved caspase 3) were detected by western blot. E Western blot analysis of SLC25A51 in HCT116 cells by overexpressing SLC25A51. F Cell viability of HCT116 ARID1A +/+ and ARID1A −/− cell lines with or without miR-4653-3p mimics and SLC25A51 plasmid transfection was shown. Error bars represent s.d. from three independent experiments. Student’s t-test * P < 0.05. G Mitochondrial NAD + levels in HCT116 cells transfected with SLC25A51 siRNA. Error bars represent s.d. from three independent experiments. Student’s t-test ** P < 0.01. H SIRT3 activity in HCT116 cells transfected with SLC25A51 siRNA. Error bars represent s.d. from three independent experiments. Student’s t-test * P < 0.05. I , J HCT116 cells were transfected with or without SLC25A51 siRNA and SIRT3 overexpression plasmids, and the acetylation levels of SOD2 were analyzed by western blot ( I ). Band intensity quantification for acetylation of SOD2 was performed using ImageJ software, normalized to the total levels of SOD2 ( J ). Error bars represent s.d. from three independent experiments. Student’s t-test *** P < 0.001. K Cell viability of HCT116 ARID1A +/+ and ARID1A −/− cell lines after treated with 3-TYP for 72 h was shown. Error bars represent s.d. from three independent experiments. ANOVA *** P < 0.001

Article Snippet: After that, 5% defatted milk was added to the blocked membrane for 2 h. Afterward, primary antibodies ARID1A (Cell Signaling Technology, #12354S, 1:1000 dilution), Cleaved-PARP (Cell Signaling Technology, #5625T, 1:1000 dilution) and Cleaved-caspase 3 antibodies (Cell Signaling Technology, #9661T, 1:1000 dilution), SLC25A51 antibody (Biorbyt, #356775, 1:1000 dilution), Phospho-ATM (Ser1981) (Cell Signaling Technology, #5883T, 1:1000 dilution), Phospho-ATR (Ser428) (Cell Signaling Technology, #2853T, 1:1000 dilution), Phospho-Chk1 (Ser345) (Cell Signaling Technology, #2348T, 1:1000 dilution), Phospho-Chk2 (Thr68) (Cell Signaling Technology, #2197T, 1:1000 dilution), ATM (Abcam, #ab199726, 1:2000 dilution), ATR (Proteintech, #19787-1-AP, 1:1000 dilution), Chk1 (Abcam, # ab40866, 1:10000 dilution), Chk2 (Abcam, #ab109413, 1:50000 dilution), Phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, #9718T, 1:1000 dilution), SIRT3 (Abcam, #ab217319, 1:1000 dilution), SOD2 (Abmart, # T55977 , 1:1000 dilution), SOD2 (acetyl K68) (Abmart, #T510546, 1:1000 dilution), GAPDH (Beyotime, #AF0006, 1:1000 dilution) and alpha Tubulin (Abcam, #ab7291, 1:5000 dilution) were added to incubate membranes overnight at 4 °C, followed by secondary antibody incubation.

Techniques: Expressing, Activity Assay, Western Blot, Transfection, Microscopy, Software, Plasmid Preparation, Over Expression