human brca1 sirna  (Cell Signaling Technology Inc)

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

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Two-color single molecule fluorescence coincidence of BRCA1-GFP with ( A ) mCherry control, ( B ) mCherry-cavin1, ( C ) mCherry-cavin2, ( D ) mCherry-cavin3, and ( E ) mCherry-CAV1 coexpressed in MCF7 cells. The green curve represents BRCA1-GFP-only events, the red curve represents mCherry-only events, and the yellow curve represents BRCA1-GFP + Cherry events. ( F ) Distribution of burst brightness measured for BRCA1-GFP (blue) and GFP control (green). ( G ) Schematic representation of domain organization of full-length wildtype (WT) BRCA1 and the truncated (Tr) 1–300 BRCA1 constructs. NES: nuclear export signal; BRCT domain: BRCA1 C terminus domain; N: N terminus. ( H–J ). Two-color single-molecule fluorescence coincidence of 1–300 BRCA1 with ( H ) cavin1, ( I ) cavin2, and ( J ) cavin3 expressed in Leishmania cell-free lysates. More than 1000 events were collected in all cases.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Fluorescence, Construct

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A–E ) Immunofluorescence microscopy in combination with PLA for protein-protein interactions (red dots) within single cells of stably expressing ( A ) MCF7-GFP, ( B ) MCF7-cavin1-GFP, ( C ) MCF7-cavin2-GFP, ( D ) MCF7-cavin3-GFP, and ( E ) MCF7-CAV1-GFP using monoclonal GFP and polyclonal BRCA1 antibodies. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. ( F ). Number of red dots/PLA signals in 40–50 cells for each MCF7-GFP-expressing cell line was quantified from three independent experiments using a nested ANOVA. Each biological replicate is color-coded, and the mean ± SEM is presented as a black bar. **p<0.05, **p<0.01.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Immunofluorescence, Microscopy, Stable Transfection, Expressing

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Representative image of MCF7 cells stably expressing GFP alone, cavin1-GFP, and cavin3-GFP fixed and stained with a BRCA1 antibody. ( B ) Percentage of MCF7 cells showing strictly nuclear, nuclear-cytoplasmic, or cytoplasmic localization of BRCA1 was counted for 50 cells from 4 to 5 independent experiments as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. Each biological replicate was color-coded. NS: not significant, *p<0.05, **p<0.01. ( C ) Lysates from stably expressing MCF7 cells western blotted for GFP, BRCA1, and Tubulin as a load control. ( D ) MCF7-GFP and MCF7-cavin3-GFP cells, untreated (-) or treated with MG-132 for 6 hr. Lysates were western blotted with GFP, BRCA1, and Tubulin antibodies as a loading control. ( E ) A431 cells treated with control siRNAs (Con) or two siRNAs specific to cavin3. Lysates were western blotted using cavin3, BRCA1, CAV1 antibodies, and Tubulin as the loading control. ( F ) A431 cells treated with control siRNAs or two siRNAs specific to BRCA1. Lysates were western blotted using cavin3, BRCA1, CAV1 antibodies, and Tubulin as the loading control. ( G ) A431 cells treated with control (Con) or siRNAs specific to cavin3, untreated or treated with MG132 for 6 hr. Lysates were western blotted using cavin3, BRCA1, and Tubulin as a loading control. Quantitation of all blots in is provided in . Figure 4—source data 1. Raw western data for MCF7 cells with molecular weight markers for . ( A ) Western blot analysis of anti-rabbit BRCA1, ( B ) anti-mouse Tubulin, and ( C ) anti-mouse GFP antibodies in (1) GFP lysates, (2) cavin1-GFP lysates, and (3) cavin3-GFP lysates. Figure 4—source data 2. Raw western data for MCF7 cells with molecular weight markers for . ( A ) Western blot analysis of anti-mouse GFP, ( B ) anti-rabbit BRCA1, and ( C ) anti-mouse Tubulin antibodies in (1) MCF7/GFP untreated, (2) MCF7/GFP + MG132-treated lysates, and (3) MCF7/cavin3-GFP untreated lysates. Figure 4—source data 3. Raw western data for A431 cells with molecular weight markers for . ( A ) Western blot analysis of anti-rabbit cavin3, ( B ) anti-rabbit CAV1, ( C ) anti-mouse Tubulin, and ( D ) anti-rabbit BRCA1 antibodies in (1) A431 cells treated with control siRNA oligos, (2) A431 cells treated with cavin3-specific siRNA oligo 1, and (3) A431 cells treated with cavin3-specific siRNA oligo 2. Figure 4—source data 4. Raw western data for A431 cells with molecular weight markers for . ( A ) Western blot analysis of anti-rabbit BRCA1, ( B ) anti-mouse Tubulin, ( C ) anti-rabbit cavin3, and ( D ) anti-rabbit CAV1 antibodies in A431 cells treated with (1) control siRNA oligos, (2) A431 cells treated with BRCA1-specific siRNA oligo 1, and (3) A431 cells treated with BRCA1-specific siRNA oligo 2. Figure 4—source data 5. Raw western data for A431 cells with molecular weight markers for . ( A ) Western blot analysis of anti-rabbit BRCA1, ( B ) anti-rabbit cavin3, and ( C ) anti-mouse Tubulin antibodies in (1) A431 cells treated with control siRNA (control KD) oligos no treatment, (2) A431 cells treated with control siRNA oligos (control KD) and MG132 for 6 hr, (3) cavin3-specific siRNA (cavin3 KD) oligo 1 no treatment, (4) cavin3-specific siRNA (cavin3 KD) oligo 1 and MG132 for 6 hr, (5) cavin3-specific siRNA (cavin3 KD) oligo 2 no treatment, and (6) cavin3-specific siRNA (cavin3 KD) oligo 2 and MG132 for 6 hr.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Stable Transfection, Expressing, Staining, Western Blot, Quantitation Assay, Molecular Weight

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) LDH release of MCF7-GFP, cavin3-GFP, and cavin1-GFP cells subjected to UV treatment and a 6 hr chase. LDH release is expressed as a percentage to control GFP cells from six independent experiments presented as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. ( B ) Annexin V-positive cells after UV treatment and a 6 hr recovery time in MCF7 cells presented as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. ( C ) 7-AAD-positive cells after UV treatment and a 24 hr recovery time in MCF7 cells presented as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. ( D ) A431 cells and ( E ) MDA-MB231 cells were transfected with GFP, cavin3-GFP, or BRCA1-GFP. Results are the relative percentage of LDH release to GFP as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from at least three independent experiments. ( F ) A431 cells and ( G ) MDA-MB231 cells were treated with control, cavin3, or BRCA1 specific siRNAs. Cavin3-depleted A431 and MDA-MB231 cells were transfected with BRCA1-GFP for 24 hr. BRCA1-depleted A431 and MDA-MB231 cells were transfected with cavin3-GFP for 24 hr. All cells were UV treated, and LDH release was measured and calculated relative to control siRNA UV-treated cells. The results represent independent experiments as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. Each biological replicate is color-coded. NS: not significant; *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Transfection

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Immunofluorescence microscopy in combination with PLA to detect and visualize protein-protein interactions (red dots) within single cells of stably expressing MCF7-GFP and ( B ) MCF7-cavin3-GFP cells using monoclonal BRCA1 (MS 110) and polyclonal GFP antibodies. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. ( C ) Number of red dots/PLA signals in 40–50 cells for each MCF7-GFP-expressing cell line quantified from three independent experiments using a nested ANOVA. Each biological replicate is color-coded with the mean ± SEM presented as a black bar. **p<0.01.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Immunofluorescence, Microscopy, Stable Transfection, Expressing

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Fluorescence microscopy analysis of PLA signals generated in MCF7 cells transfected with cavin3-GFP using mouse GFP and rabbit BRCA1 antibodies from , ( B ) GFP antibody alone, ( C ) BRCA1 antibody alone, ( D ) the absence of PLA probes, or ( E ) primary antibody controls. Representative images are from at least two independent experiments as shown.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Fluorescence, Microscopy, Generated, Transfection

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Equal numbers of A431 cells treated with control siRNA or cavin3-specific siRNAs were subjected to UV treatment and a recovery time of 6 hr. ( B ) Equal numbers of A431 cells treated with control siRNAs or BRCA1-specific siRNAs were subjected to UV treatment (2 min) and a recovery time of 6 hr. ( C ) Equal numbers of MDA-MB231 cells treated with control siRNAs or cavin3-specific siRNAs were subjected to UV treatment and a recovery time of 6 hr. ( D ) Equal numbers of MDA-MB231 cells treated with control siRNAs or BRCA1-specific siRNAs were subjected to UV treatment and a recovery time of 6 hr. LDH release was measured from the cell supernatant and was calculated relative to control siRNA UV-treated cells as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. ( E ) MCF7 cells depleted of BRCA1 (-cavin3, -BRCA1), depleted of BRCA1 and transfected with cavin3-GFP (-BRCA1, +cavin3), left untreated (-cavin3, +BRCA1), or transfected with cavin3-GFP (+BRCA1, +cavin3). All cells were subjected to UV treatment and a 6 hr recovery time. LDH release was measured from the cell supernatant and was calculated relative to control MCF7 cells lacking both BRCA1 and cavin3 as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. Each biological replicate is color-coded. NS: not significant; *p<0.05, ***p<0.001, ****p<0.0001.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Transfection

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: Two-color single-molecule fluorescence coincidence of BRCA1-GFP with mCherry tagged ( A ) cavin1, ( B ) cavin2, ( C ) cavin3, ( D ) CAV1, and ( E ) mCherry control expressed in MDA-MB231 cells. The green curve represents BRCA1-GFP-only events, the red curve represents mCherry-only events, and the yellow curve represents BRCA1-GFP + Cherry events. More than 1000 events were collected.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Fluorescence

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Immunofluorescence microscopy in combination with proximity ligation assay (PLA) visualization of endogenous protein-protein interactions (red dots) within MDA-MB231 cells in untreated (Unt.) cells, ( B ) UV treatment (2 min) and a 30 min chase time, ( C ) 200 μM H 2 O 2 for 30 min, and ( D ) hypo-osmotic treatment (90% H 2 O in Dulbecco’s modified Eagle’s medium [DMEM]) for 10 min. ( E ) Number of red dots/PLA signals in 40–50 cells for cavin-BRCA1 was quantified from three independent experiments and is presented as mean ± SEM using a nested ANOVA. Each biological replicate is color-coded with the mean presented as a black bar. ( F ) Number of red dots/PLA signals in 40–50 cells for cavin3-cavin1 was quantified from three independent experiments and is presented as mean ± SEM using a nested ANOVA. Each biological replicate is color-coded with the mean presented as a black bar. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Immunofluorescence, Microscopy, Proximity Ligation Assay, Modification

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) A431 cells were treated with isotonic (ISO) or hypo-osmotic (HYPO) medium, and proximity ligation assay (PLA) was performed using cavin3 and BRCA1, ( B ) cavin3 and PCNA, ( C ) cavin3 and flotillin1, and ( D ) cavin3 and Aurora kinase antibodies as controls for PLA. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. ( E ) Total number of PLA signals in the cytosol and the nucleus of cells as defined by DAPI staining in 50 cells for each pair of antibodies quantified from three independent experiments using a nested ANOVA with the mean ± SEM represented by the black bar, *p<0.05, **p<0.01.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Proximity Ligation Assay, Staining

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Immunofluorescence microscopy in combination with proximity ligation assay (PLA) visualization of endogenous protein-protein interactions (red dots) within A431 cells in ( A ) untreated (Unt.) cells, ( B ) UV treated and a chase time of 30 min, ( C ) 200 μM H 2 O 2 (H 2 O 2 ) for 30 min, and ( D ) hypo-osmotic treatment (HYPO) for 10 min. Top panel: BRCA1 and cavin3; bottom panel: cavin1 and cavin3. ( E ) PLA signals/cell for cavin3-BRCA1 association in 50 cells/biological replicate with three independent experiments. ( F ) PLA time-course analysis after UV treatment and a chase time up to 360 min in 50 cells/biological replicate with three independent experiments. ( G ) PLA signals/cell for cavin1-cavin3 association in 50 cells/biological replicate with three independent experiments. All data was quantified from three independent experiments using a nested ANOVA. Each biological replicate is color-coded with the mean ± SEM presented as a black bar. NS: not significant; *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Immunofluorescence, Microscopy, Proximity Ligation Assay

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: Representative immunofluorescence images of control knockdown, cavin3 knockdown, and BRCA1 knockdown cells for cavin3 (green), BRCA1 (red), and DAPI (nuclei). Images are representative of three independent experiments.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Immunofluorescence

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: A431 cells treated with control (Con) or siRNAs specific to cavin3 or BRCA1 (oligo 1 and 2). Cells were left untreated or subjected to UV treatment. Cells were subject to immunofluorescence microscopy in combination with PLA using monoclonal mouse BRCA1 and polyclonal rabbit cavin3 antibodies. The number of PLA signals in 40–50 cells was quantified from three independent experiments using a nested ANOVA. Each biological replicate is color-coded with the mean ± SEM presented as a black bar. *p<0.05, **p<0.001.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Immunofluorescence, Microscopy

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Z-score for HeLa WT and cavin3 KO cells (replicates Rep. 1–3) showing upregulated proteins (red) and downregulated proteins (blue). ( B ) Volcano plot showing proteins (red dots) identified by Gene Ontology Biological Process (GOBP) involved in DNA repair. ( C ) Volcano plot showing DNA repair proteins upregulated in cavin3 KO cells. ( D ) Volcano plot showing proteins of the BRCA1 A-complex, BRCA1, BRCC3, MDC1, and UBE4A downregulated in cavin3 HeLa KO cells and upregulation of 53BP1 with a heatmap analysis of the expression of each of these proteins in replicate (Rep. 1–3) HeLa WT and cavin3 KO cells.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Expressing

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Representative western blot analysis of WT and cavin3 KO cells UV time course for cavin3, BRCA1, CAV1, Rad51, and Tubulin. ( B ) Protein components of the BRCA1 A-complex. Blue-colored circles: proteins downregulated in the label-free quantitative (LFQ) proteomics; yellow-colored circles: proteins not detected in the LFQ proteomics of cavin3 KO cells. ( C ) Representative western blot analysis of cavin3, BRCA1, pH2AX, UIM1C/Rap80, BARD1, Rad51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV, PKM, PGK1, and Actin in WT and cavin3 KO HeLa cells untreated (-) or UV treated (UV) followed by a 4 hr chase. Quantitation of protein levels from three independent experiments is presented in . ( D ) Representative immunofluorescence images of BRCA1 foci after UV treatment in WT HeLa cells. ( E ) Percentage of cells with more than five BRCA1 foci, Rap80 foci, and γH2AX foci in WT and cavin3 KO cells following UV treatment and a 30 min chase. The results are presented as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. ( F ) WT and cavin3 KO cells untreated or treated with the PARP inhibitor (AZD2461, PARPi) 5 nM for 6 days were subjected to comet assays. The results are presented as the mean ± SEM using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. Each biological replicate is color-coded. Extent Tail Moment was calculated as described in Materials and methods. NS: not significant; **p<0.01, ***p<0.001; ****p<0.0001. Figure 8—source data 1. Raw western data for HeLa WT and cavin3 KO cells time course after UV treatment with molecular weight markers for . Western blot analysis of ( A ) anti-rabbit cavin3, ( B ) anti-rabbit CAV1, ( C ) anti-rabbit BRCA1, ( D ) anti-rabbit RAD51, and ( E ) anti-mouse Tubulin antibodies in (1) WT control, (2) WT UV 30 min chase, (3) WT UV 60 min chase, (4) WT UV 120 min chase, (5) WT UV 240 min chase, (6) cavin3 KO control, and (7) cavin3 KO UV 30 min chase, cavin3 KO UV 60 min chase, cavin3 KO UV 120 min chase, and cavin3 KO 240 min chase. Figure 8—source data 2. Raw western data for HeLa WT and cavin3 KO cells untreated or UV treatment for 4 hr with molecular weight markers for . ( A ) Western blot analysis of anti-rabbit cavin3, ( B ) anti-rabbit BRCA1, ( C ) anti-rabbit Rad51 and γH2AX, ( D ) anti-rabbit BRCC36, ( E ) anti-sheep Merit40, ( F ) anti-rabbit BRCA2, ( G ) anti-rabbit CAV1, ( H ) anti-rabbit PKM, ( I ) anti-rabbit PGK1, and ( J ) anti-actin antibodies in (1) WT untreated cells, (2) WT + UV treatment and a 4 hr chase, (3) cavin3 KO cells, and (4) cavin3 KO + UV treatment and a chase 4 hr chase time.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Western Blot, Quantitation Assay, Immunofluorescence, Molecular Weight

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: Densitometry analysis was performed of the protein levels of cavin3, BRCA1, P139 γH2AX, RAP80, BARD1, RAD51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV1, PKM, PGK1, and Actin in in WT and cavin3 KO cells subjected to UV treatment and a 4 hr chase from 2 to 3 independent experiments presented as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. NS: not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques:

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet:

Article Snippet: Human cavin3 Stealth siRNAs (set of three – HSS174185, 150811, 150809) and Human BRCA1 Stealth siRNAs (set of three – HSS101089, 186096, 186097) were purchased from Life Technologies Australia Pty Ltd. Two siRNA oligonucleotides to cavin3 or BRCA1 were found to reduce protein levels (oligo 1 and oligo 2) and were transfected into cells at 24 hr and 48 hr after plating using Lipofectamine 2000 reagent (Invitrogen) with a ratio of 6 μl Lipofectamine to 150 pmol siRNA.

Techniques: Sequencing, In Situ, CRISPR, Protease Inhibitor, Software