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. 2012 Oct 29;199(3):425–435. doi: 10.1083/jcb.201207089

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© 2012 Joshi et al.

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Figure 5. — Transcriptional regulation of Cav1 and cavin-1 through endogenous Cav1 in metastatic MDA-231 breast cancer cells. (A, top) Metastatic MDA-231 breast cancer cells treated with jasplakinolide (Jasp.), H₂O₂/vanadate (H₂O₂/van), or LatA or transfected with control (Ctrl si) or Cav1 siRNAs were then transiently transfected with control pRL, Cav1-luciferase (Luc), or cavin-1–luciferase constructs for 48 h and subjected to luciferase assay. (bottom) Altered promoter activity caused by jasplakinolide, H₂O₂/vanadate, or LatA treatment is Cav1 dependent as it is not observed in Cav1 siRNA–transfected cells. (B) CHIP assays for Egr1 binding to Cav1 and cavin-1 promoters in MDA-231 cells were performed after H₂O₂/vanadate, jasplakinolide, or LatA treatment on control (Ctrl) or Cav1 siRNA–transfected cells. Negative control IgG immunoprecipitation is shown. (C) MDA-231 cells were not treated (NT) or treated with PP2, Y27632, R0, or staurosporine (Stauro; left) or with PMA (right) and then Western blotted for pCav1, Cav1, cavin-1, and β-actin (**, P < 0.001). (D) MDA-231 cells treated with jasplakinolide or mechanically stretched (left) or treated with jasplakinolide and either PP2, Y27632, R0, or staurosporine (right) were Western blotted for Cav1, cavin-1, and β-actin. (E) MDA-231 cells transiently transfected with Cav1-luciferase or cavin-1–luciferase for 48 h were subjected to luciferase assay after treatment with PP2, Y27632, R0, staurosporine (Stauro), or PMA. (F) MDA-231 cells were cotransfected with Cav1-luciferase or cavin-1–luciferase and either control (siCtl) or Egr1 (siEgr1) siRNA and treated with jasplakinolide and either PP2, Y27632, R0, or staurosporine before luciferase assay (*, P < 0.05). Error bars show means ± SEM.