FIG 3.

In mammalian cells, AJUBA LIM proteins do not influence mechanical signal regulation of YAP. (A) MCF10A cells were transfected with CTL (scrambled) or AJUBA plus LIMD1 (LD1) RNAi and then split and cultured at either LD or HD on soft (80- to 120-Pa) or stiff (120-kPa) fibronectin-coated polyacrylamide hydrogels for 24 h. Immunofluorescence assays with the indicated antibodies or stains were then performed. (B) Quantification of YAP nuclear/cytoplasmic immunofluorescence from cells in panel A. KD refers to AJUBA and LIMD1 RNAi-depleted cells. At least 50 cells in multiple fields were scored. Blue bars, control cells; red bars, AJUBA plus LD1 KD cells. (C) Quantitative PCR (Q-PCR) for YAP and the CTGF gene (a YAP-regulated gene) in cells from panel A at intermediate density. KD refers to AJUBA and LIMD1 RNAi-depleted cells. Blue bars, YAP mRNA levels; red bars, CTGF mRNA levels. (D) Western blot of cell lysates from cells in panel A with the indicated antibodies. (E) Representative images of CAFs plated on micropatterned coverslips containing fibronectin-coated pillars of different sizes and stained for DAPI, actin, and YAP-TAZ. (F) Quantification of YAP-TAZ nuclear/cytoplasmic ratios from cells in panel D. All the quantified experiments were performed 2 or 3 times, and a representative example is shown. Blue bars, control cells; red bars, AJUBA plus LD1 KD cells. **, P < 0.01; *, P < 0.05; ns, no significant difference. The data are presented as means ± SD. Scale bars, 25 μm (A) and 20 μm (D).