Loss of EHD2 expression impairs caveolae mechanosensing and gene transcription in breast cancer cells. (A) Immunoblot (left) and quantification (right) of EHD2, Cav1, and cavin1 protein levels normalized to CHC in Hs578T and MDA-MB-436 cells. (B and C) Representative TIRF images of changes in cell surface Cav1 spot numbers (left) and quantification (right) under resting (Iso) and hypo-osmotic (Hypo) conditions in Hs578T (B) or MDA-MB-436 (C) cells. Cells are delineated by dashes. Scale bar = 10 µm. (D) Quantification of changes in cell surface Cav1 spot numbers in MDA-MB-436 cells transfected or not (CTRL) with EHD2-EGFP under Iso and Hypo conditions. (E) Quantification of Cav1, Cav2, cavin1, and cavin2 mRNA levels in MDA-MB-436 cells transfected or not (CTRL) with EHD2 or Cav1 and in MDA-MB-436 cells depleted for Cav1 (siCav1) and transfected or not by EHD2 under hypo-osmotic conditions. (F) Quantification of Cav1, Cav2, cavin1, and cavin2 mRNA levels in Hs578T cells transfected with control siRNA (CTRL), siEHD2, or siCav1 after 30 min of cyclic stretch. For all panels, n ≥ 3 independent experiments; mRNA levels are compared with resting conditions (dotted line); *, P < 0.05; **, P < 0.01; ***, P < 0.001; two tailed t test; data are mean ± SEM; numbers of cells are indicated on histogram bars.