phosphorylated histone h2ax γh2ax  (Proteintech)

Journal: Genes & Diseases

Article Title: Targeting fatty acid synthase to overcome PARP inhibitor resistance and to create an artificial synthetic lethality for triple-negative breast cancer

doi: 10.1016/j.gendis.2025.101817

Figure Lengend Snippet: Combination synergy on DNA damage, NHEJ repair, and apoptosis. (A, B) Western blotting and quantification analyses of FASN, PARP1, γ-H2AX, and actin loading control in MDA-MB-231 and MDA-MB-436 cells treated with 5HLS, talazoparib, or the combination. (C, D) The fraction of maximum γ-H2AX induction derived using the Bliss-compatible scaling formula. (E) Host cell reactivation assay of NHEJ activity in MDA-MB-231 and MDA-MB-436 cells treated with 5HLS, talazoparib, or the combination. (F) Comparison between the observed and expected NHEJ activity inhibition by the combination using the Bliss independence model. (G) Caspasae3/7 activity assay of MDA-MB-231 and MDA-MB-436 cells treated with 5HLS, talazoparib, or the combination. (H) The fraction of maximum caspase 3/7 activation derived using the Bliss-compatible scaling formula. (I) Annexin V staining as an indicator of apoptosis in MDA-MB-231 and MDA-MB-436 cells following treatments with 5HLS, talazoparib, or the combination. (J) The fraction of maximum apoptosis induction derived using the Bliss-compatible scaling formula. n = 3; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: Antibodies against PARP1 (#66520) and phosphorylated histone H2AX (γH2AX) (#613402) were from Proteintech (Rosemont, Illinois, USA) and BioLegend (San Diego, California, USA), respectively.

Techniques: Western Blot, Control, Derivative Assay, Host-Cell Reactivation, Activity Assay, Comparison, Inhibition, Activation Assay, Staining

Journal: Genes & Diseases

Article Title: Targeting fatty acid synthase to overcome PARP inhibitor resistance and to create an artificial synthetic lethality for triple-negative breast cancer

doi: 10.1016/j.gendis.2025.101817

Figure Lengend Snippet: In - vivo effect of 5HLS and talazoparib combination on tumor growth. (A) Tumor volume and body weight of mice treated by vehicle (Veh), 5HLS, talazoparib (Tal), or combination (Comb) of 5HLS and talazoparib. (B, C) Gross anatomy (B) and wet weight (C) of dissected xenograft tumors at the end of the study. (D) Synergy analysis. Tumor growth inhibition (TGI) was derived using the wet weight of tumors. The expected combination inhibition (C (E) ) was calculated from that of 5HLS and talazoparib alone using the Bliss independence model (see Materials and Methods ). C (O) represents the observed combination inhibition. (E) Immunohistochemical analyses of FASN, PARP, and γH2AX. Scale bar, 50 μm. (F) Western blotting analyses of FASN, BRCA1, PARP1, cleaved PARP1 (cPARP1), γH2AX, and actin loading control in xenograft tumors from mice treated with vehicle, 5HLS, talazoparib, and the combination of 5HLS and talazoparib. Each lane represents mixed samples of five tumors in equal proportion within the treatment group. n = 5; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Antibodies against PARP1 (#66520) and phosphorylated histone H2AX (γH2AX) (#613402) were from Proteintech (Rosemont, Illinois, USA) and BioLegend (San Diego, California, USA), respectively.

Techniques: In Vivo, Inhibition, Derivative Assay, Immunohistochemical staining, Western Blot, Control

Journal: bioRxiv

Article Title: The BRCA1- RAD51 Axis Regulates SCAI/REV3 Dependent Replication Fork Maintenance

doi: 10.1101/2025.11.25.689574

Figure Lengend Snippet: A. WT RPE1 cells transfected with the indicated siRNAs for 60 h, then treated with 2 mM hydroxyurea (HU) for 18 h, harvested, and processed for western blotting with the specified antibodies. ND, no drug. B. Western blots (WB) from WT and SCAI-null U2OS cell lines treated with 1 mM HU for 18 h. C. WT and ΔSCAI U2OS cells were transfected with either backbone plasmids (Vector) or plasmids expressing SCAI that is resistant to CRISPR/Cas9 editing. After 30 h, cells were treated with 1.5 mM HU for another 18 h before immunoblotting. D. WT RPE1 cells were transfected with the indicated siRNAs, then treated with vehicle (DMSO), 1 µM, or 2 µM aphidicolin (APH) for 18 h before being processed for western blotting with the specified antibodies. E-F . Comet assay of hTERT-RPE1 ( E ) and U2OS ( F ) cells treated with 2 mM HU for 40 h. Indicated siRNAs were transfected two days before HU treatment. Red line represents median, three independent experiments, representative experiment shown. Kruskal-Wallis followed by Dunn’s multiple comparison test. G. WT RPE1 cells were transfected with the indicated siRNAs, then treated with 1 mM HU for 18 h and assayed for the indicated proteins by WB. γH2AX values are normalized to vinculin loading control. H - J . Representative microscopy images showing chromatin bound RPA and γH2AX, following transient knockdown of SCAI or REV3 and 16 h treatment with 1.5 mM HU in hTERT RPE1 wildtype cells (e) scale bar indicates 25 micron. Quantification of the integrated fluorescent intensity of RPA (f) and γH2AX (g) of cells in e, red bar indicates mean and SEM of about 2000 nuclei. ****p< 0.0001, Tukey’s test.

Article Snippet: The antibodies used in this work include the following: Anti-vinculin 1:1000 (Sigma-Aldrich, V9131-.2ML), vinculin (7F9) 1:200 (Santa Cruz Biotechnology, sc-73614), SCAI 1:500 (Abcam, ab124688), Chk1 (2G1D5) 1:1000 (Cell Signaling, 2360S), P-Chk1 (S345) (133D3) Rabbit mAb 1:1000 (Cell Signaling, 2348S), RPA 32 kDa subunit (9H8) 1:200 (Santa Cruz Biotechnology, sc-56770, only used for western blots), RPA32 1:200 (Genetex, GTX70258, only used for IF), Rabbit x-Phospho RPA32 (S4/S8) 1:1000 (Bethyl, A300-245A), H2AX 1:1000 (Bethyl, A300-082A), Anti-phospho-Histone H2A.X (Ser139) 1:1000 (EMD Millipore Corp., 05-636), REV7 1:200 (Santa Cruz Biotechnology, sc-135977), Anti-Exonuclease 1 (EXO1) 1:1000 (Bethyl, A302-640A), ORC2 1:1000 (Abcam, ab68348), BRCA1 1:1000 (EMD Millipore Corp., 07-434), 53BP1 1:1000 (Bethyl, A300-272A), RIF1 1:1000 (Bethyl, A300-569A), CtIP 1:1000 (Bethyl, A300-488A), SmarcAL1 (A-2) 1:200 (Santa Cruz Biotechnology, sc-376377), ZRANB3 1:1000 (Bethyl, A303-033A), FBH1 1:1000 (Abcam, ab58881), RAD51 1:1000 (Abcam, ab63801), MUS81 1:200 (Santa Cruz Biotechnology, sc-53382), SLX4 1:1000 (Bethyl, A302-270A), H3 1:1000 (Cell Signaling, 9715), MRE11 1:1000 (Abcam, ab214), Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP 1:2500 (Invitrogen, 31430), Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP 1:2500 (Invitrogen, 31460), Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 1:300 (Invitrogen, A32731), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 1:300 (Invitrogen, A32742), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 1:300 (Invitrogen, A32723).

Techniques: Transfection, Western Blot, Plasmid Preparation, Expressing, CRISPR, Single Cell Gel Electrophoresis, Comparison, Control, Microscopy, Knockdown

Journal: bioRxiv

Article Title: The BRCA1- RAD51 Axis Regulates SCAI/REV3 Dependent Replication Fork Maintenance

doi: 10.1101/2025.11.25.689574

Figure Lengend Snippet: A. WT and BRCA1 null RPE1 cells were transfected with the indicated siRNAs, then treated with 1 mM HU for 18 h prior to blotting for the indicated proteins. B. WT and BRCA1-null RPE1 cells were transfected with the indicated siRNAs. Cells were then treated with 1 mM HU for 18 h before WCLs (whole cell lysates) were prepared and WB were performed against the indicated proteins. C. IF images showing RPA foci in WT RPE1 cells following treatment with the indicated siRNAs. Cells were treated with 1.5 mM HU for 18 h prior to IF. Scale bars indicate 5 microns. D. Quantification of ( C ). ****P < 0.0001, unpaired Welch’s t test (two-tailed). E. IF analyses showing representative γH2AX foci in WT and BRCA1-null RPE1 cells post-treatment with indicated siRNAs. Cells were treated with 1.5 mM HU for 18 h prior to IF analyses. Scale bars indicate 5 microns. F . Quantification of ( E ). *P = 0.0337, unpaired Welch’s t test (two-tailed). G . Comet assay of hTERT-RPE1 cells treated with 2 mM HU for 40 h. Indicated siRNAs were transfected two days before HU treatment. Red line represents median, two independent experiments, representative experiment shown. Kruskal-Wallis followed by Dunn’s multiple comparison test. ns – not significant (P ≥ 0.05). H. WT and BRCA1-null RPE1 cells were transfected with the indicated siRNAs, then treated with 1 mM HU for 18 h and fractioned prior to blotting for the indicated proteins. CE – cytoplasmic extract. SCAI values are normalized to ORC2 loading control.

Article Snippet: The antibodies used in this work include the following: Anti-vinculin 1:1000 (Sigma-Aldrich, V9131-.2ML), vinculin (7F9) 1:200 (Santa Cruz Biotechnology, sc-73614), SCAI 1:500 (Abcam, ab124688), Chk1 (2G1D5) 1:1000 (Cell Signaling, 2360S), P-Chk1 (S345) (133D3) Rabbit mAb 1:1000 (Cell Signaling, 2348S), RPA 32 kDa subunit (9H8) 1:200 (Santa Cruz Biotechnology, sc-56770, only used for western blots), RPA32 1:200 (Genetex, GTX70258, only used for IF), Rabbit x-Phospho RPA32 (S4/S8) 1:1000 (Bethyl, A300-245A), H2AX 1:1000 (Bethyl, A300-082A), Anti-phospho-Histone H2A.X (Ser139) 1:1000 (EMD Millipore Corp., 05-636), REV7 1:200 (Santa Cruz Biotechnology, sc-135977), Anti-Exonuclease 1 (EXO1) 1:1000 (Bethyl, A302-640A), ORC2 1:1000 (Abcam, ab68348), BRCA1 1:1000 (EMD Millipore Corp., 07-434), 53BP1 1:1000 (Bethyl, A300-272A), RIF1 1:1000 (Bethyl, A300-569A), CtIP 1:1000 (Bethyl, A300-488A), SmarcAL1 (A-2) 1:200 (Santa Cruz Biotechnology, sc-376377), ZRANB3 1:1000 (Bethyl, A303-033A), FBH1 1:1000 (Abcam, ab58881), RAD51 1:1000 (Abcam, ab63801), MUS81 1:200 (Santa Cruz Biotechnology, sc-53382), SLX4 1:1000 (Bethyl, A302-270A), H3 1:1000 (Cell Signaling, 9715), MRE11 1:1000 (Abcam, ab214), Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP 1:2500 (Invitrogen, 31430), Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP 1:2500 (Invitrogen, 31460), Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 1:300 (Invitrogen, A32731), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 1:300 (Invitrogen, A32742), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 1:300 (Invitrogen, A32723).

Techniques: Transfection, Two Tailed Test, Single Cell Gel Electrophoresis, Comparison, Control