phosphorylated h2ax  (Cell Signaling Technology Inc)

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: Acta Pharmaceutica Sinica. B

Article Title: USP51/GRP78/ABCB1 axis confers chemoresistance through decreasing doxorubicin accumulation in triple-negative breast cancer cells

doi: 10.1016/j.apsb.2025.03.004

Figure Lengend Snippet: Ectopic ubiquitin-specific protease 51 (USP51) plays a pivotal role in triple-negative breast cancer (TNBC) chemoresistance. (A) Western blotting analysis of polyubiquitinated protein expression in Cal51 and Cal51 R cells. (B–D) RNA sequencing (B), q-PCR (C) and Western blotting (D) of MIU-containing novel DUB family member 1 (MINDY1), OTU domain-containing ubiquitin aldehyde-binding protein 2 (OTUB2) and USP51 expression in Cal51 and Cal51 R cells. (E) Western blotting of MINDY1 expression in MINDY1-knockdown Cal51 R cells. (F) Western blotting of OTUB2 expression in OTUB2-knockdown Cal51 R cells. (G) Western blotting of USP51 expression in USP51-knockdown Cal51 R cells. (H) Cell Counting Kit-8 (CCK-8) assay of Cal51 R cells with knockdown of MINDY1, OTUB2 or USP51. (I) Western blotting analysis of polyubiquitinated protein expression in the USP51-knockdown Cal51 R cells. (J–N) Analyses of colony formation (J), cell apoptosis (K, L) and γ H2AX expression (M, N) in USP51-knockdown Cal51 R cells treated with doxorubicin (DOX) (1 μmol/L). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; the data in C, J, K and M were assessed via unpaired Student's t -test. The data are presented as the mean ± SD in (C, H, J, K, M). The data are representative of four (H) or three (C, J, K, M) independent experiments.

Article Snippet: The cells were subjected to 24-h treatment with various drugs before fixation in 4% paraformaldehyde, followed by overnight incubation with a phosphorylated histone H2AX ( γ H2AX) antibody (Cell Signaling Technology, MA, USA) at 4 °C.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, RNA Sequencing, Binding Assay, Knockdown, Cell Counting, CCK-8 Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP51/GRP78/ABCB1 axis confers chemoresistance through decreasing doxorubicin accumulation in triple-negative breast cancer cells

doi: 10.1016/j.apsb.2025.03.004

Figure Lengend Snippet: USP51 promotes TNBC chemoresistance via its DUB activity. (A) Western blotting of USP51 expression in USP51-expressing Cal51 cells. (B–G) Analyses of cell viability (B), colony formation (C), cell apoptosis (D, E) and γ H2AX expression (F, G) in USP51-expressing Cal51 cells treated with DOX (1 μmol/L). (H) Representative image of tumors dissected from xenograft model mice treated with DOX (2 mg/kg). (I, J) Quantification of the volume (I) and weight (J) of tumors derived from the respective xenograft tumors. (K) Immunohistochemical staining of Ki67, cleaved caspase-3 and γ H2AX in the respective xenograft tumors. ∗∗ P < 0.01, ∗∗∗ P < 0.001; ns represents no significant difference; the data in C, D, F and I–K were assessed via unpaired Student's t -test. The data are presented as the mean ± SD in (B–D, F, I–K). The dots represent individual samples in (I–K). The data are representative of four (B), three (C, D, F) or five (I–K) independent experiments.

Article Snippet: The cells were subjected to 24-h treatment with various drugs before fixation in 4% paraformaldehyde, followed by overnight incubation with a phosphorylated histone H2AX ( γ H2AX) antibody (Cell Signaling Technology, MA, USA) at 4 °C.

Techniques: Activity Assay, Western Blot, Expressing, Derivative Assay, Immunohistochemical staining, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP51/GRP78/ABCB1 axis confers chemoresistance through decreasing doxorubicin accumulation in triple-negative breast cancer cells

doi: 10.1016/j.apsb.2025.03.004

Figure Lengend Snippet: USP51 promotes TNBC chemoresistance by deubiquitinating GRP78. (A) Western blotting of GRP78 and USP51 expression in GRP78-expressing shUSP51/Cal51 R cells. (B–G) Analyses of cell viability (B), colony formation (C), cell apoptosis (D, E) and γ H2AX expression (F, G) in GRP78-expressing shUSP51/Cal51 R cells treated with DOX (1 μmol/L). (H) Representative image of tumors dissected from xenograft model mice treated with DOX (2 mg/kg). (I, J) Quantification of the volume (I) and weight (J) of tumors derived from the respective xenograft tumors. (K) Immunohistochemical staining of Ki67, cleaved caspase-3 and γ H2AX in the respective xenograft tumors. ∗∗∗ P < 0.001; ns represents no significant difference; the data in C, D, F and I–K were assessed via unpaired Student's t -test. The data are presented as the mean ± SD in (B–D, F, I–K). The dots represent individual samples in (I–K). The data are representative of four (B), three (C, D, F) or five (I–K) independent experiments.

Article Snippet: The cells were subjected to 24-h treatment with various drugs before fixation in 4% paraformaldehyde, followed by overnight incubation with a phosphorylated histone H2AX ( γ H2AX) antibody (Cell Signaling Technology, MA, USA) at 4 °C.

Techniques: Western Blot, Expressing, Derivative Assay, Immunohistochemical staining, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP51/GRP78/ABCB1 axis confers chemoresistance through decreasing doxorubicin accumulation in triple-negative breast cancer cells

doi: 10.1016/j.apsb.2025.03.004

Figure Lengend Snippet: GRP78 promotes TNBC chemoresistance by regulating ATP binding cassette subfamily B member 1 (ABCB1)-mediated DOX efflux. (A) Fluorescence detection of DOX in Cal51 and Cal51 R cells. (B) Western blotting of GRP78 and fluorescence detection of DOX in GRP78-treated Cal51 R cells treated with DOX (1 μmol/L) in the presence or absence of Encequidar (100 nmol/L). (C) Co-IP assay of GRP78 and ABCB1 in Cal51 R cells. (D) Immunofluorescence staining of GRP78 and ABCB1 in ABCB1-Myc/Cal51 R . (E) Analysis of ABCB1 nanodiscs via electron microscopy. (F) Western blotting of ABCB1 expression in nanodiscs derived from Ctrl/HeLa and ABCB1/HeLa cells. (G) ATPase activity assay of ABCB1 nanodiscs with purified GST or GST-GRP78 proteins. (H–M) Cell viability (H), colony formation (I), cell apoptosis (J, K) and γ H2AX foci formation (L, M) were examined in shGRP78/Cal51 R cells treated with DOX alone or in combination with Encequidar (100 nmol/L). ∗∗∗ P < 0.001; ns represents no significant difference; the data in A, B, I, J and M were assessed via unpaired Student's t -test. The data are presented as the mean ± SD in (A, B, G–J, M). The data are representative of three (A, B, G, I, J, M) or four (H) independent experiments.

Article Snippet: The cells were subjected to 24-h treatment with various drugs before fixation in 4% paraformaldehyde, followed by overnight incubation with a phosphorylated histone H2AX ( γ H2AX) antibody (Cell Signaling Technology, MA, USA) at 4 °C.

Techniques: Binding Assay, Fluorescence, Western Blot, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Electron Microscopy, Expressing, Derivative Assay, Activity Assay, Purification

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP51/GRP78/ABCB1 axis confers chemoresistance through decreasing doxorubicin accumulation in triple-negative breast cancer cells

doi: 10.1016/j.apsb.2025.03.004

Figure Lengend Snippet: WCY-4-1 treatment confers DOX sensitivity in TNBC. (A–F) Cell viability (A), colony formation (B), cell apoptosis (C, D) and γ H2AX expression (E, F) were assessed in shUSP51/Cal51 R cells treated with DOX alone or in combination with WCY-4-1 (80 nmol/L). (G) Representative images of tumors dissected from xenograft model mice treated with DOX (2 mg/kg) and/or WCY-4-1 (2 mg/kg). (H, I) Quantification of the volume (H) and weight (I) of tumors derived from the respective xenograft model groups. (J) Immunohistochemical staining of Ki67, cleaved caspase-3 and γ H2AX in the respective xenograft tumors. ∗∗ P < 0.01, ∗∗∗ P < 0.001; ns represents no significant difference; the data in B, C, E and H–J were assessed via unpaired Student's t -test. The data are presented as the mean ± SD in (A–C, E, H–J). The dots represent individual samples in (H–J). The data are representative of four (A), three (B, C, E) or five (H–J) independent experiments.

Article Snippet: The cells were subjected to 24-h treatment with various drugs before fixation in 4% paraformaldehyde, followed by overnight incubation with a phosphorylated histone H2AX ( γ H2AX) antibody (Cell Signaling Technology, MA, USA) at 4 °C.

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Staining