Skip to main content
. 2021 Jun 18;10:e61407. doi: 10.7554/eLife.61407

Figure 4. Cavin3 regulates BRCA1 protein expression and localization.

(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 Figure 4 is provided in Figure 4—figure supplement 1A–E.

Figure 4—source data 1. Raw western data for MCF7 cells with molecular weight markers for Figure 4C.
(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 Figure 4D.
(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 Figure 4E.
(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 Figure 4F.
(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 Figure 4G.
(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.

Figure 4.

Figure 4—figure supplement 1. Reciprocal regulation of BRCA1 and cavin3 protein levels.

Figure 4—figure supplement 1.

(A) Relative protein expression of BRCA1 in MCF7 cells expressing GFP (white), cavin1-GFP (blue), and cavin3-GFP (orange) using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (B) BRCA1 gene expression was analyzed using the TaqMan Gene Expression assay as described in Materials and methods in MCF7 cells. Results are expressed as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. NS: not significant. (C). Relative protein expression of BRCA1 in MCF7 cells expressing GFP or cavin3-GFP untreated (-) or treated with MG132 (+) using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (D) Relative protein levels of cavin3 and BRCA1 in cells treated with control (Con) siRNAs or siRNAs specific to cavin3 (oligo1 and oligo2) in A431 cells using a one-way ANOVA and Bonferroni’s multiple comparisons test from 3 to 4 independent experiments. (E). Representative western blots of MCF7, MDA-MB231, A431, and HeLa cells were western blotted for protein expression of cavin3, CAV1, BRCA1, and Tubulin as the loading control. (F) Relative protein levels of cavin3 and BRCA1 in cells treated with control (Con) siRNAs or siRNAs specific to BRCA1 (oligo1 and oligo2) in A431 cells using a one-way ANOVA and Bonferroni’s multiple comparisons test from 3 to 4 independent experiments. For each experiment, each biological replicate was color-coded. NS: not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 4—figure supplement 1—source data 1. Raw western data for HeLa WT and cavin3 KO cells with molecular weight markers for Figure 4—figure supplement 1E.
(A) Western blot analysis of anti-rabbit cavin3, (B) anti-mouse Tubulin, (C) anti-rabbit CAV1, and (D) anti-rabbit BRCA1 antibodies in (1) MCF7, (2) MDA-MB231, (3) A431, and (4) HeLa cells.
Figure 4—figure supplement 2. Validation of loss of cavin3 and BRCA1 in MDA-MB231 cells.

Figure 4—figure supplement 2.

(A) Representative western blot analysis of cavin3, BRCA1, CAV1, and Tubulin as the loading control in MDA-MB231 cells following treatment with two siRNAs targeting cavin3 oligo 1 and 2 (or control). (B) Relative protein expression of cavin3 and BRCA1 in MDA-MB 231 cells transfected with cavin3 oligo 1 and cavin3 oligo 2 compared to control-treated cells from three independent experiments using a one-way ANOVA with Bonferroni’s multiple comparisons tests. Each biological replicate is color-coded. (C). Western blot analysis of BRCA1, cavin3, and Tubulin as the loading control in MDA-MB 231 cells following treatment with two siRNAs targeting BRCA1 oligo 1 and 2 (or control). (D) Relative protein expression of cavin3 and BRCA1 in MDA-MB 231 cells transfected with cavin3 oligo 1 and cavin3 oligo 2 compared to control-treated cells from three independent experiments using a one-way ANOVA with Bonferroni's multiple comparisons test. Each biological replicate is color-coded. **p<0.01, ***p<0.001.
Figure 4—figure supplement 2—source data 1. Raw western data for MDA-MB231 cells with molecular weight markers for Figure 4—figure supplement 2A.
(A) Western blot analysis of anti-rabbit BRCA1, (B) anti-rabbit cavin3, and (C) anti-mouse Tubulin in (1) MDA-MB231 treated with control siRNAs, (2) MDA-MB231 cells treated with cavin3-specific siRNA oligo 1, and (3) MDA-MB231 cells treated with cavin3-specific siRNA oligo 2.
Figure 4—figure supplement 2—source data 2. Raw western data for MDA-MB231 cells with molecular weight markers for Figure 4—figure supplement 2B.
(A) Western blot analysis of anti-rabbit CAV1, (B) anti-rabbit cavin3, (C) anti-rabbit cavin1, (D) anti-mouse Tubulin, (E) anti-rabbit BRCA1, and (F) anti-mouse BRCA1 in (1) MDA-MB231 cells treated with control siRNAs, (2) MDA-MB231 cells treated with BRCA1-specific siRNA oligo 1, (3) MDA-MB231 cells treated with BRCA1-specific siRNA oligo, and (4) MDA-MB231 cells treated with cavin3-specific oligo 1.
Figure 4—figure supplement 3. Reciprocal loss of BRCA1 and cavin3 in A431 cells.

Figure 4—figure supplement 3.

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.