sirna sc29219  (Santa Cruz Biotechnology)

Journal: The Journal of Clinical Investigation

Article Title: GSK3B directs DNA repair choice and determines tumor response to PARP1 inhibition independent of BRCA1

doi: 10.1172/JCI189956

Figure Lengend Snippet: ( A ) Representative images of 53BP1 –/– U2OS cells reconstituted with HA-tagged WT 53BP1 and irradiated with 6 Gy. Cells were costained for BRCA1 and p-T334 phospho-specific antibodies. ( B ) Number of BRCA1 foci per cell stratified by pT334 status at different time points following radiation and ( C ) relative intensity of BRCA1 foci at various time points. ( D ) Representative images of 53BP1 –/– U2OS cells reconstituted with HA-tagged WT 53BP1 and irradiated with 6 Gy. Cells were costained for RAD51 and T334 phospho-specific antibodies. ( E ) Number of RAD51 foci per cell stratified by pT334 status at different time points following radiation and ( F ) relative intensity of RAD51 foci at various time points. Quantification of foci and intensity were calculated for each cell by the Image J software. n = 3. ( G ) Schematic of I-SceI HR reporter assay. Assessment of chromosomal HR repair of DSB as measured by percentage GFP-positive 53BP1 –/– U2OS cells reconstituted with various 53BP1 alleles with or without BRCA1 knockdown. DSBs were induced by adenoviral delivery of I-SceI, and the ability of cells to resolve DSBs by HR was assessed 48 hours later. n = 3. Data were analyzed by 2-way ANOVA followed by Tukey’s multiple comparison. Values are mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Magnification ×65 ( A and D ).

Article Snippet: For BRCA1 knock down, Signal Silence BRCA1 siRNA was purchased from Cell signaling, # 12519.

Techniques: Irradiation, Software, Reporter Assay, Knockdown, Comparison

Journal: The Journal of Clinical Investigation

Article Title: GSK3B directs DNA repair choice and determines tumor response to PARP1 inhibition independent of BRCA1

doi: 10.1172/JCI189956

Figure Lengend Snippet: ( A ) 53BP1 –/– U2OS cells reconstituted with WT or T334A 53BP1 with or without BRCA1 knockdown and in the presence or absence of GSK3B inhibitor following PARPi were assessed by colony formation assay with survival fractions calculated. n = 3. ( B ) Number of RAD51 foci per cell and ( C ) relative intensity of RAD51 foci in BRCA1 -deficient MDA-MB-436 cells with or without BRCA1 reconstituted and with or without GSK3B inhibitor incubated with 15 μM of olaparib. n = 3. ( D ) BRCA1 -deficient MDA-MB-436 cells with or without BRCA1 reconstituted and with or without GSK3B inhibitor were incubated with the indicated concentrations of olaparib, and survival fractions were assessed with colony formation assay. n = 3. ( E ) Mouse 4T1 in vivo tumor study scheme. ( F ) Tumor volumes and ( G ) weights in C57BL6 mice injected with 4T1 cells with or without BRCA1 knock down in the fat pad. Mice were treated with Lithium (30 mg/kg) and/or Olaparib (50 mg/kg). Values are mean ± SEM. Statistical significance was assessed using 1-way ANOVA followed by Tukey’s multiple comparison test and 2-way ANOVA with Bonferroni’s correction. ### P < 0.001; ** P < 0.01; *** P < 0.001; **** P < 0.0001. * indicates comparing same biological group under treatment. # indicates comparing 2 different biological groups under treatment.

Article Snippet: For BRCA1 knock down, Signal Silence BRCA1 siRNA was purchased from Cell signaling, # 12519.

Techniques: Knockdown, Colony Assay, Incubation, In Vivo, Injection, Comparison

Journal: The Journal of Clinical Investigation

Article Title: GSK3B directs DNA repair choice and determines tumor response to PARP1 inhibition independent of BRCA1

doi: 10.1172/JCI189956

Figure Lengend Snippet: ( A ) Representative images of 53BP1 –/– U2OS cells reconstituted with HA-tagged WT 53BP1 and irradiated with 6 Gy. Cells were costained for BRCA1 and p-T334 phospho-specific antibodies. ( B ) Number of BRCA1 foci per cell stratified by pT334 status at different time points following radiation and ( C ) relative intensity of BRCA1 foci at various time points. ( D ) Representative images of 53BP1 –/– U2OS cells reconstituted with HA-tagged WT 53BP1 and irradiated with 6 Gy. Cells were costained for RAD51 and T334 phospho-specific antibodies. ( E ) Number of RAD51 foci per cell stratified by pT334 status at different time points following radiation and ( F ) relative intensity of RAD51 foci at various time points. Quantification of foci and intensity were calculated for each cell by the Image J software. n = 3. ( G ) Schematic of I-SceI HR reporter assay. Assessment of chromosomal HR repair of DSB as measured by percentage GFP-positive 53BP1 –/– U2OS cells reconstituted with various 53BP1 alleles with or without BRCA1 knockdown. DSBs were induced by adenoviral delivery of I-SceI, and the ability of cells to resolve DSBs by HR was assessed 48 hours later. n = 3. Data were analyzed by 2-way ANOVA followed by Tukey’s multiple comparison. Values are mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Magnification ×65 ( A and D ).

Article Snippet: For BRCA1 knock down, Signal Silence BRCA1 siRNA was purchased from Cell signaling, # 12519.

Techniques: Irradiation, Software, Reporter Assay, Knockdown, Comparison

Journal: The Journal of Clinical Investigation

Article Title: GSK3B directs DNA repair choice and determines tumor response to PARP1 inhibition independent of BRCA1

doi: 10.1172/JCI189956

Figure Lengend Snippet: ( A ) 53BP1 –/– U2OS cells reconstituted with WT or T334A 53BP1 with or without BRCA1 knockdown and in the presence or absence of GSK3B inhibitor following PARPi were assessed by colony formation assay with survival fractions calculated. n = 3. ( B ) Number of RAD51 foci per cell and ( C ) relative intensity of RAD51 foci in BRCA1 -deficient MDA-MB-436 cells with or without BRCA1 reconstituted and with or without GSK3B inhibitor incubated with 15 μM of olaparib. n = 3. ( D ) BRCA1 -deficient MDA-MB-436 cells with or without BRCA1 reconstituted and with or without GSK3B inhibitor were incubated with the indicated concentrations of olaparib, and survival fractions were assessed with colony formation assay. n = 3. ( E ) Mouse 4T1 in vivo tumor study scheme. ( F ) Tumor volumes and ( G ) weights in C57BL6 mice injected with 4T1 cells with or without BRCA1 knock down in the fat pad. Mice were treated with Lithium (30 mg/kg) and/or Olaparib (50 mg/kg). Values are mean ± SEM. Statistical significance was assessed using 1-way ANOVA followed by Tukey’s multiple comparison test and 2-way ANOVA with Bonferroni’s correction. ### P < 0.001; ** P < 0.01; *** P < 0.001; **** P < 0.0001. * indicates comparing same biological group under treatment. # indicates comparing 2 different biological groups under treatment.

Article Snippet: For BRCA1 knock down, Signal Silence BRCA1 siRNA was purchased from Cell signaling, # 12519.

Techniques: Knockdown, Colony Assay, Incubation, In Vivo, Injection, Comparison

Journal: Redox Biology

Article Title: Targeting GPX4-mediated ferroptosis protection sensitizes BRCA1 -deficient cancer cells to PARP inhibitors

doi: 10.1016/j.redox.2024.103350

Figure Lengend Snippet: BRCA1 promotes ferroptosis susceptibility in ovarian cancers. (A) Western blot analysis of BRCA1 protein expression in UWB1.289 and UWB1.289+BRCA1(UWB-BRCA1) cells. (B) Volcano plot of quantitative proteomics displays the differentially expressed proteins between UWB1.289 and UWB-BRCA1 cells. (C) Gene Ontology (GO) pathway enrichment analysis of significantly changed proteins. (D) Indicated cells were treated with increasing concentrations of RSL3 for 24 h in presence or absence of 100 μM DFO (desferoxamine), dead cells were measured by PI staining. Data were presented as mean ± standard deviation (SD), n = 3. (E) Western blot analysis of BRCA1 expression in A2780 cells transfected with control (Ctrl) or BRCA1 siRNA. (F–G) Control (Ctrl) or BRCA1 siRNA transfected A2780 cells were treated with indicated dosages of RSL3 for 24 h, followed by analysis of cell viability (F) and PI staining (G). (H–J) UWB1.289/UWB-BRCA1 cells (H), Ctrl/BRCA1 siRNA transfected A2780 cells (I), BRCA1 small-guide RNA (sgRNA) mediated - A2780 knockout (KO) cells (J) were treated with or without 0.5 μM RSL3 for 24 h, lipid peroxidation was detected by BODIY 581/591 C11 labeling. The representative flow cytometry profile (left) and quantification of lipid peroxidation (right) was shown. The depletion of BRCA1 in A2780 BRCA1 KO cells were confirmed by Western blot (upper right, J). Data were presented as mean ± SD from three replicates. (K) Immunohistochemistry (IHC) analysis of 4-HNE(4-Hydroxynonenal) in wild-type (WT) or BRCA1 -mutant ovarian cancer tissues. The presentative staining (left) or quantification of staining score (right) was shown. Data were presented as mean ± standard deviation (SD), n = 9. **p < 0.01 and ***p < 0.001 by 2-tailed t -test.

Article Snippet: Human BRCA1 siRNA (target sequence: #1, 5′-CCA CAC GAT TTG ACG GAA A-3’; #2, 5′-CTA CTC ATG TTG TTA TGA A-3′) were obtained from Ribobio (Guangzhou, China), in vivo cholesterol-modified GPX4 siRNA (target sequence: 5′-GGA GTA ACG AAG AGA TCA A-3′) were purchased from Ribobio (Guangzhou, China).

Techniques: Western Blot, Expressing, Quantitative Proteomics, Staining, Standard Deviation, Transfection, Control, Knock-Out, Labeling, Flow Cytometry, Immunohistochemistry, Mutagenesis

Journal: Redox Biology

Article Title: Targeting GPX4-mediated ferroptosis protection sensitizes BRCA1 -deficient cancer cells to PARP inhibitors

doi: 10.1016/j.redox.2024.103350

Figure Lengend Snippet: BRCA1 negatively regulates GPX4 protein stability. (A) Western blot analysis of indicated protein levels in UWB1.289, UWB1.289 re-expressing BRCA1(UWB-BRCA1), control (ctrl) or BRCA1 siRNA transfected-A2780 cells. (B–C) Western blot analysis of GPX4 protein levels in Ctrl/ BRCA1 -KO A2780 cells (B), SKOV3 and OVCAR-3 cells transfected with ctrl or BRCA1 siRNA(C). (D–E) Relative mRNA levels of BRCA1 and GPX4 were quantified in UWB1.289/UWB-BRCA1 cells (D), or BRCA1-silenced A2780, SKOV3 and OVCAR-3 cells (E). (F) UWB1.289 and UWB-BRCA1 cells were treated with 100 μg/ml cycloheximide (CHX) for 0–8 h, followed by Western blot analysis of indicated proteins. (G) A2780 cells were transfected with ctrl or BRCA1 siRNA, 48 h after transfection, cells were treated with 100 μg/ml cycloheximide (CHX) for 0–8 h, then indicated proteins were assessed by Western blot. (H–I) IHC(H) and Western blot (I) analysis of GPX4 expression in WT or BRCA1 -mutant ovarian cancer tissues. (J–K) UWB1.289, UWB-BRCA1 or UWB-BRCA1 cells stably expressing flag-GPX4 were treated with or without 0.5 μM RSL3 for 24 h, followed by analysis of lipid peroxidation using BODIY 581/591 C11 labeling (J) and cell death using PI staining (K). The GPX4 expressions in above cells were confirmed by Western blot. Data were presented as mean ± standard deviation (SD). *p < 0.05, **p < 0.01 and ***p < 0.001 by two-way ANOVA test. n.s. stands for no significance.

Article Snippet: Human BRCA1 siRNA (target sequence: #1, 5′-CCA CAC GAT TTG ACG GAA A-3’; #2, 5′-CTA CTC ATG TTG TTA TGA A-3′) were obtained from Ribobio (Guangzhou, China), in vivo cholesterol-modified GPX4 siRNA (target sequence: 5′-GGA GTA ACG AAG AGA TCA A-3′) were purchased from Ribobio (Guangzhou, China).

Techniques: Western Blot, Expressing, Control, Transfection, Mutagenesis, Stable Transfection, Labeling, Staining, Standard Deviation

Journal: Redox Biology

Article Title: Targeting GPX4-mediated ferroptosis protection sensitizes BRCA1 -deficient cancer cells to PARP inhibitors

doi: 10.1016/j.redox.2024.103350

Figure Lengend Snippet: BRCA1 interacts with GPX4 via RING domain. (A) Co-immunoprecipitation (co-IP) analysis of BRCA1/GPX4 interaction from lysates derived from A2780 cells. (B–C) A2780 cells were co-transfected with indicated plasmids, followed by IP analysis of BRCA1/GPX4 association using anti-flag affinity resin. (D) Proximity ligation assay (PLA) for the interaction between BRCA1 and GPX4 in A2780 cells. (E) The Coomassie blue staining of purified His-GPX4 proteins. (F) 100 ng of recombinant(r) his-GPX4 was incubated with or without purified flag-BRCA1/BARD1 protein in EBC buffer (50 mm Tris-HCl pH 7.6–8.0, 120 mm NaCl, 0.5 % NP-40, 1 mm EDTA, 1 mm β-mercaptoethanol, 50 mm NaF, and 1 mmNa 3 VO 4 ) for 20 min, followed by IP with anti-flag affinity resin. (G) Schematic diagram of BRCA1 protein and various myc tagged BRCA1 deletion mutants. (H) A2780 cells were co-transfected with Flag-GPX4 along with indicated myc tagged BRCA1 truncations, followed by IP with anti-flag affinity resin and Western blot with indicated antibodies. (I–J) Nuclear(N) and cytoplasmic (C) Fractions were isolated from UWB1.289/UWB-BRCA1 cells (I), parental or BRCA1 -KO A2780 (J) cells, and subjected to Western blot analysis with indicated antibodies. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Human BRCA1 siRNA (target sequence: #1, 5′-CCA CAC GAT TTG ACG GAA A-3’; #2, 5′-CTA CTC ATG TTG TTA TGA A-3′) were obtained from Ribobio (Guangzhou, China), in vivo cholesterol-modified GPX4 siRNA (target sequence: 5′-GGA GTA ACG AAG AGA TCA A-3′) were purchased from Ribobio (Guangzhou, China).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Derivative Assay, Transfection, Proximity Ligation Assay, Staining, Purification, Recombinant, Incubation, Western Blot, Isolation

Journal: Redox Biology

Article Title: Targeting GPX4-mediated ferroptosis protection sensitizes BRCA1 -deficient cancer cells to PARP inhibitors

doi: 10.1016/j.redox.2024.103350

Figure Lengend Snippet: BRCA1/BARD1 catalyzes K6-linked polyubiquitination of GPX4 on lysine 47 and 58. (A) Flag-GPX4 was co-transfected with or without HA-ubiquitin (Ub) into UWB1.289/UWB-BRCA1 cells, and then treated with 5 μM MG132 for 6 h before harvesting for analysis of flag-GPX4 ubiquitination. (B) A2780 cells were co-transfected with indicated plasmids, IP was performed using anti-flag affinity resin after MG132 treatment for 6 h and ubiquitination of flag-GPX4 was analyzed by Western blot. (C) A2780 cells with or without BRCA1 knockdown were co-transfected with indicated plasmids, ubiquitination of flag-GPX4 was analyzed by IP with anti-flag affinity resin after MG132 treatment. (D–E) Purified His-GPX4 was incubated in presence of Ub/E1/E2, purified flag-BARD1, and either WT or I26A mutant flag-BRCA1 1-303 protein, then his-GPX4 ubiquitination was analyzed. (F–G) WT, K11R, K47R or K58R flag-GPX4 was co-transfected with WT (F) or K6-specfic(G) HA-ubiquitin, along with or without myc-BRCA1/BARD1 into A2780 cells, then flag-GPX4 ubiquitination was analyzed after MG132 treatment. (H) WT or K47RK58R (2 KR) flag-GPX4 was co-transfected with HA-Ub and myc-BRCA1/BARD1 complex, followed by analysis of flag-GPX4 ubiquitination after MG132 treatment.

Article Snippet: Human BRCA1 siRNA (target sequence: #1, 5′-CCA CAC GAT TTG ACG GAA A-3’; #2, 5′-CTA CTC ATG TTG TTA TGA A-3′) were obtained from Ribobio (Guangzhou, China), in vivo cholesterol-modified GPX4 siRNA (target sequence: 5′-GGA GTA ACG AAG AGA TCA A-3′) were purchased from Ribobio (Guangzhou, China).

Techniques: Transfection, Ubiquitin Proteomics, Western Blot, Knockdown, Purification, Incubation, Mutagenesis

Journal: Redox Biology

Article Title: Targeting GPX4-mediated ferroptosis protection sensitizes BRCA1 -deficient cancer cells to PARP inhibitors

doi: 10.1016/j.redox.2024.103350

Figure Lengend Snippet: GPX4 promotes growth in BRCA1 -deficient ovarian cancer. (A) IHC analysis of BRCA1 and GPX4 protein expression in ovarian cancer tissue. The representative IHC staining (left) and correlation analysis of GPX4 immunoscore and BRCA1 immunoscore (right) were shown. N = 96. (B) BRCA1 -KO A2780 cells were transfected with or without cholesterol-modified in vivo GPX4 siRNA, followed by Western blot analysis of indicated proteins. (C–D) Tumor growth curve(C) and tumor weights (D) of indicated A2780 xenografts treated with control (ctrl) or GPX4 siRNA. (E) IHC analysis of GPX4 and 4-HNE levels in indicated xenograft tumors. The representative IHC staining (left) and their quantification (right) were shown. Data were presented as mean ± standard deviation (SD), n = 6. **p < 0.01by two-way ANOVA test.

Article Snippet: Human BRCA1 siRNA (target sequence: #1, 5′-CCA CAC GAT TTG ACG GAA A-3’; #2, 5′-CTA CTC ATG TTG TTA TGA A-3′) were obtained from Ribobio (Guangzhou, China), in vivo cholesterol-modified GPX4 siRNA (target sequence: 5′-GGA GTA ACG AAG AGA TCA A-3′) were purchased from Ribobio (Guangzhou, China).

Techniques: Expressing, Immunohistochemistry, Transfection, Modification, In Vivo, Western Blot, Control, Standard Deviation

Journal: Redox Biology

Article Title: Targeting GPX4-mediated ferroptosis protection sensitizes BRCA1 -deficient cancer cells to PARP inhibitors

doi: 10.1016/j.redox.2024.103350

Figure Lengend Snippet: Inhibition of GPX4 potentiates therapeutic efficacy of PARP inhibitor in BRCA1 -deficient ovarian cancers. (A) UWB1.289 cells were treated increasing concentrations of Olaparib (Ola) for 24 h, and the lipid peroxidation was analyzed by BODIY 581/591 C11. The representative flow cytometry profiles (left) and quantification of lipid peroxidation (right) were shown. (B) Western blot analysis of indicated proteins in UWB1.289 cells stably expressing control (ctrl) or GPX4 shRNA. (C–D) Ctrl or GPX4 shRNA transfected UWB.1289 cell were treated with or without 100 μM Olaparib (Ola) for 48 h, and lipid peroxidation (C) and cell death (D) were analyzed by BODIY 581/591 C11 labeling and PI staining respectively. (E) UWB1.289 cells expressing ctrl or GPX4 shRNA were grown in presence or absence of 2 μM Olaparib for 7–10 days, followed by colony formation analysis. (F) Western blot analysis of GPX4 expression in control or BRCA1 -KO A2780 cells expressing ctrl or GPX4 shRNA. (G) BRCA1 -KO A2780 cells with or without GPX4 knockdown were grown in presence or absence of 2 μM Olaparib for 7–10 days, followed by colony formation analysis. (H–I) BRCA1 -KO A2780 cells expressing ctrl or GPX4 shRNA were treated with or without 100 μM Olaparib (Ola) for 48 h, and lipid peroxidation (H) and cell death (I) were analyzed by BODIY 581/591 C11 labeling and PI staining respectively. Data represents the mean ± SD from at least three independent replicates, *p < 0.01, and ***p < 0.001 by two-way ANOVA test.

Article Snippet: Human BRCA1 siRNA (target sequence: #1, 5′-CCA CAC GAT TTG ACG GAA A-3’; #2, 5′-CTA CTC ATG TTG TTA TGA A-3′) were obtained from Ribobio (Guangzhou, China), in vivo cholesterol-modified GPX4 siRNA (target sequence: 5′-GGA GTA ACG AAG AGA TCA A-3′) were purchased from Ribobio (Guangzhou, China).

Techniques: Inhibition, Drug discovery, Flow Cytometry, Western Blot, Stable Transfection, Expressing, Control, shRNA, Transfection, Labeling, Staining, Knockdown

Journal: Redox Biology

Article Title: Targeting GPX4-mediated ferroptosis protection sensitizes BRCA1 -deficient cancer cells to PARP inhibitors

doi: 10.1016/j.redox.2024.103350

Figure Lengend Snippet: Combination of PARP and GPX4 inhibitors synergistically inhibits BRCA1 -deficient cancers. (A) UWB1.289 or HCC1937 cells were combined treated with serial dosages of Olaparib and ML210 for three days, followed by Bliss synergy analysis. (B) Colony formation analysis of UWB1.289 or HCC1937 cells treated with 0.5 μM Olaparib, 0.5 μm ML210 or their combination. (C–D) UWB.1289 cells were treated with 100 μM Olaparib, 2 μM ML210, or their combination for 48 h, and lipid peroxidation (C) and PI staining (D) assays were performed. (E–F) BRCA1 -mutant ovarian cancer organoids (PDO) were treated with serial concentrations of olaparib, ML210, or their combination for 5 days, then the organoid viability was determined. The representative organoid after treatment (E) The ZIP synergy score (F) were shown. (G) BRCA1-mutant ovarian cancer organoids were treated with Olaparib (50 μM), ML210 (5 μM), ferrostatin-1(Fer-1) (10 μM) or their combination as indicated for 5 days, and organoid viability was measured. (H–I) BRCA1 -KO A2780 xenograft bearing-mice were administrated with PBS (ctrl), olaparib (100 mg/kg), ML210 (30 mg/kg) or their combination, tumor volumes were measured and tumor growth curve (H) and tumor wights (I) were shown. (J) IHC analysis of 4-HNE in indicating drug-treated xenograft tumor tissues. The representative IHC staining (left) and quantification of IHC staining (right) were shown. Data were presented as means ± SD, n = 6. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA test.

Article Snippet: Human BRCA1 siRNA (target sequence: #1, 5′-CCA CAC GAT TTG ACG GAA A-3’; #2, 5′-CTA CTC ATG TTG TTA TGA A-3′) were obtained from Ribobio (Guangzhou, China), in vivo cholesterol-modified GPX4 siRNA (target sequence: 5′-GGA GTA ACG AAG AGA TCA A-3′) were purchased from Ribobio (Guangzhou, China).

Techniques: Staining, Mutagenesis, Immunohistochemistry