Journal: iScience

Article Title: HP1β recruits RING1A to ubiquitinate histone H2A for BRCA1-mediated resection of double-stand breaks

doi: 10.1016/j.isci.2025.114582

Figure Lengend Snippet: HP1β interacts with RING1A and enhances BRCA1 recruitment at DSB sites (A) Effect of ATM inhibition on clonogenic survival of irradiated cells. Cells were treated with 10 μM ATM inhibitor (KU-55933) for 1h before IR treatment. (B) p -MDC1 foci formation/dissolution in irradiated H1299 cells is not altered by HP1β depletion. (C) FLAG-RING1A co-immunoprecipitated with HA-HP1β. (D) FLAG-RING1A interaction with FLAG-CD HP1β and FLAG-CSD HP1β. (E) RING1A enrichment after DNA damage at Chr1-A and Chr1-B DSB sites as measured by ChIP-qPCR is decreased by HP1β depletion. (F) AlphaFold 3 model of RING1A and HP1β interaction. (G) AlphaFold 3 model of RING1A, CSD HP1β, and CAF1 complex (CAF1A, CAF1B, RBBP4) interaction. HP1-binding motif PxVxL of CAF1A is located near the CSD HP1β and RING1A-binding interface (close-up view). (H) Impact of RING1A depletion on BRCA1 recruitment at Chr1-A and Chr1-C DSB sites before and after I-Sce1 transfection. (I) Effect of RING1A depletion on IR-induced BRCA1 foci with and without HP1β. (J) Decreased single-strand DNA formation at DSBs in RING1A or HP1β-depleted cells as measured by the ER-AsiSI assay. Experiments were done three times and standard deviation was calculated. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: ATM inhibitor (KU-55933) ATM inhibitor (KU-55933) , MCE Millipore Sigma , Cat# HY-12016 Cat # 1185000.

Techniques: Inhibition, Irradiation, Dissolution, Immunoprecipitation, ChIP-qPCR, Binding Assay, Transfection, Standard Deviation

Journal: iScience

Article Title: HP1β recruits RING1A to ubiquitinate histone H2A for BRCA1-mediated resection of double-stand breaks

doi: 10.1016/j.isci.2025.114582

Figure Lengend Snippet: HP1β modulates H2AK119-ub levels at DSB sites to promote S-phase-specific IR-induced chromosome damage repair (A and B) HP1β depletion decreases H2AK119Ub1 at 1 h and 2-h post IR recovery. Control and HP1β-depleted cells were irradiated by radiation dose of 4Gy. (A) Anti-Ub antibody was used for immunoprecipitation followed by western blot analysis with H2AK119Ub antibody. (B) Quantification of H2AK119Ub1 was performed following normalization with H2A Input western blots. (C) Effect of HP1β depletion on -ub levels at the Chr1-A and Chr1-B sites before and after DSB induction as determined by ChIP-qPCR. (D) Impact of BRCA1 depletion on RING1A enrichment at the DSB. Depletion of BRCA1 has no impact on the enrichment of RING1A at the DSB. (E) Depletion of RING1A but not BRCA1 results in the decreased enrichment of H2AK119-ub at the DSB. (F) H2A-ub reversal of radiosensitivity in HP1β-depleted cells is independent of ATM. Cells with and without HP1β depletion were treated with ATM inhibitor (KU 55933) then treated with increasing IR doses. Effect of H2A-ub expression on cell survival was determined by clonogenic survival. (G) Exogenous H2A-ub partially restores HR in HP1β-depleted cells as determined by the DR-GFP repair assay. (H) Ectopic expression of H2A-ub or FLAG-CSD HP1β reduces S-phase-specific IR-induced residual chromosome damage in HP1β-depleted cells. Three independent experiments were conducted and an SD value between experiments was determined. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: ATM inhibitor (KU-55933) ATM inhibitor (KU-55933) , MCE Millipore Sigma , Cat# HY-12016 Cat # 1185000.

Techniques: Control, Irradiation, Immunoprecipitation, Western Blot, ChIP-qPCR, Expressing

Journal: Molecular Medicine

Article Title: Loss of XRCC1 promotes cGAS/STING mediated innate immune signaling in gastric cancer

doi: 10.1186/s10020-026-01429-0

Figure Lengend Snippet: ATM inhibitor enhances accumulation of cytosolic DNA in XRCC1 deficient cells. A. Gastric cancer cell line (NCI-N87) were transfected with XRCC1 siRNA (ON TARGET plus siRNA: SMART pool) for 48 hours and XRCC1 knockdown determined by Western blot (β-actin as a loading control). B. ATM inhibitors exacerbate micronuclei formation in XRCC1 deficient cells. XRCC1 proficient and deficient mouse embryonic fibroblast cells (MEFs) treated with ATM inhibitor (Ku55933) for 24 h and nuclear DNA were stained with DAPI and visualized using immunofluorescence assay. C Percent of positive cells with micronuclei in XRCC1 proficient and deficient cells. D Quantification of double stranded DNA from cytosolic fraction and total DNA from total cell extracts using the Cell Fraction Kit (Cat # ab109719, Abcam). E qPCR quantification of cytoplasmic mtDNA from cytosolic fraction normalized with whole extracts using mtDNA specific primers (ND5). F Enrichment of ROS associated genes in XRCC1 proficient and deficient cells. Statistical comparison of the data was analyzed using a Student t-test in GraphPad Prism (** P < 0.01; **** P < 0.0001; ns: represent not significant)

Article Snippet: 4.3.2) in GSEA; C Enrichment of cytosolic DNA sensing genes expression in GC cells were treated with 10 μM ATM inhibitor (ATMi, KU55933) for 24 h. D Volcano graph represent the overall gene expression of inflammatory, cytosolic DNA sensing, and inflammatory cell signaling ; E Heat map of set of genes in volved in interferon response in XRCC1 proficient NCI-N87 (N87-WT) and deficient NCI-N87 (N87-KD)

Techniques: Transfection, Knockdown, Western Blot, Control, Staining, Immunofluorescence, Comparison

Journal: Molecular Medicine

Article Title: Loss of XRCC1 promotes cGAS/STING mediated innate immune signaling in gastric cancer

doi: 10.1186/s10020-026-01429-0

Figure Lengend Snippet: ATM inhibitors enhance cGAS/STING signal activation. A STING/TBK1/IRF-3 signaling pathway activation detected by Western blot of protein extract from siRNA control (WT) and siRNA-XRCC1 knockdown cells treated with and without ATM inhibitor. Anti-IRF3/ p-IRF3 (ser396); TBK1/P-TBK-1 (Ser172) antibodies were obtained from Cell Signaling. Note that fraction of phosphrylated value calculated obtained by dividing the band intensity of physphrylated proteins relative to the total respective unphosphorylated protein band intensity. All band intensity were measured using Bio-Rad Image lab software. B Enrichment of cytosolic DNA sensing genes in WT and XRCC1 knockout GC cells from HallMark data base in enrichment score Gene Set Enrichment Analysis (GSEA ver. 4.3.2) in GSEA; C Enrichment of cytosolic DNA sensing genes expression in GC cells were treated with 10 µM ATM inhibitor (ATMi, KU55933) for 24 h. D Volcano graph represent the overall gene expression of inflammatory, cytosolic DNA sensing, and inflammatory cell signaling ; E Heat map of set of genes in volved in interferon response in XRCC1 proficient NCI-N87 (N87-WT) and deficient NCI-N87 (N87-KD)

Article Snippet: 4.3.2) in GSEA; C Enrichment of cytosolic DNA sensing genes expression in GC cells were treated with 10 μM ATM inhibitor (ATMi, KU55933) for 24 h. D Volcano graph represent the overall gene expression of inflammatory, cytosolic DNA sensing, and inflammatory cell signaling ; E Heat map of set of genes in volved in interferon response in XRCC1 proficient NCI-N87 (N87-WT) and deficient NCI-N87 (N87-KD)

Techniques: Activation Assay, Western Blot, Control, Knockdown, Software, Knock-Out, Expressing, Gene Expression

Journal: Molecular Medicine

Article Title: Loss of XRCC1 promotes cGAS/STING mediated innate immune signaling in gastric cancer

doi: 10.1186/s10020-026-01429-0

Figure Lengend Snippet: Type I interferon response in XRCC1 deficient cells. XRCC1 proficient and deficient NCI-N87 gastric cancer cell line examined for inflammatory response. A Gene expression of inflammatory response change in XRCC1 proficient and deficient NCI-N87 cells from HallMark data base in enrichment score Gene SetEnrichment Analysis (GSEA ver. 4.3.2) in GSEA. B Gene expression of inflammatory response change in XRCC1 proficient and deficient NCI-N87 cells were treated with 10 µM ATM inhibitor (ATMi, KU55933) for 24 h. Preranked mode was performed on data for all contrasts. Each pre-ranked gene list provided consisted of significance-weighted Log2 fold change values for each gene; C mRNA expression of inflammatory cytokines was measured using RT-qPCR. NCI-N87 cells were treated with 10 µM ATM inhibitor (ATMi, KU55933) for 24 h. Statistical comparison of the data was analyzed using a Student t-test in GraphPad Prism (** P < 0.01; *** P < 0.001; **** P < 0.0001)

Article Snippet: 4.3.2) in GSEA; C Enrichment of cytosolic DNA sensing genes expression in GC cells were treated with 10 μM ATM inhibitor (ATMi, KU55933) for 24 h. D Volcano graph represent the overall gene expression of inflammatory, cytosolic DNA sensing, and inflammatory cell signaling ; E Heat map of set of genes in volved in interferon response in XRCC1 proficient NCI-N87 (N87-WT) and deficient NCI-N87 (N87-KD)

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Comparison

Journal: Molecular Medicine

Article Title: Loss of XRCC1 promotes cGAS/STING mediated innate immune signaling in gastric cancer

doi: 10.1186/s10020-026-01429-0

Figure Lengend Snippet: Inhibition of ATM enhances the transcriptional expression of PD-L1 in XRCC1 deficient gastric tissues and GC cells. A Western blot analysis of PD-L1 expression in gastric tissue from Cre-XRCC1 flox/flox versus Cre+XRCC1 Δ/Δ mice. B Quantitative RT-PCR analysis of PD-L1 mRNA expression in gastric tissue from Cre-XRCC1 flox/flox versus Cre+XRCC1 Δ/Δ mice. C Western blot analysis of PD-L1 expression in NCI-N87 GC cells. Note that the intensity of band measured from each lane and normalized to loading control and fold chage calculated relative to control. All band intensity were measured using Bio-Rad Chemdoc Image analysis software. D XRCC1 proficient NCI-N87(siRNA-control) and deficient (NCI-N87, siRNA-XRCC1) cells treated with ATM inhibitor for 24 h and mRNA expression of PD-L1 measured using RT-qPCR. Statistical comparison of the data was analyzed using a ANOVA-Two way in GraphPad Prism (** P < 0.01; P ***<0.001; P ****<0.0001; ns: not significant)

Article Snippet: 4.3.2) in GSEA; C Enrichment of cytosolic DNA sensing genes expression in GC cells were treated with 10 μM ATM inhibitor (ATMi, KU55933) for 24 h. D Volcano graph represent the overall gene expression of inflammatory, cytosolic DNA sensing, and inflammatory cell signaling ; E Heat map of set of genes in volved in interferon response in XRCC1 proficient NCI-N87 (N87-WT) and deficient NCI-N87 (N87-KD)

Techniques: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, Control, Software, Comparison

Journal: iScience

Article Title: SKA2 promotes gastric cancer progression by regulating glutathione metabolism

doi: 10.1016/j.isci.2026.115202

Figure Lengend Snippet: Knocking down SKA2-induced cell-cycle arrest and apoptosis through the SKA2/ROS/ATM axis in GC cell lines (A) Western blotting analysis of γ-H2AX (Ser139), ATM, p -ATM (Ser1981), p -Chk2 (Thr68), and SKA2 expression in SNU638 and NUGC3 SKA2-knockdown cell lines. (B) Western blotting analysis of SKA2 overexpression in SNU638 and NUGC3 SKA2-knockdown cell lines using antibodies against γ-H2AX (Ser139), ATM, p -ATM (Ser1981), p -Chk2 (Thr68), and SKA2. (C) Western blotting analysis of KU-55933 treatment in SNU638 and NUGC3 SKA2-knockdown cell lines using antibodies against cyclin D1, cyclin A2, cyclin B1, p -Chk2 (Thr68), PARP, Cleaved-Caspase3, JNK, p -JNK (Thr183/Tyr185), ATM, p -ATM (Ser1981), and SKA2. (D) Western blotting analysis of BML-277 treatment in SNU638 and NUGC3 SKA2-knockdown cell lines using antibodies against cyclin D1, cyclin A2, cyclin B1, p -Chk2 (Thr68), and SKA2. (E) Western blotting analysis of P38, p-P38 (Thr180/Tyr182), ERK, p -ERK1/2 (Thr202/Tyr204), JNK, p -JNK (Thr183/Tyr185), and SKA2 expression in SNU638 and NUGC3 SKA2-knockdown cell lines. (F) Western blotting analysis of the rescue effect of SKA2 overexpression on MAPK pathway markers (ERK, p -ERK1/2, JNK, and p -JNK) in SNU638 and NUGC3 SKA2-knockdown cell lines. (G) Western blotting analysis of JNK-IN-8 treatment in SNU638 SKA2-knockdown cell lines using antibodies against PARP, cleaved-caspase3, JNK, p -JNK (Thr183/Tyr185), and SKA2. α-Tubulin was used as the internal control for all blots. Representative blotting images are shown from 3 independent experiments.

Article Snippet: KU-55933 , sangon biotech , Cat# A423600.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Control