FIG 1.

AJUBA LIM proteins regulate YAP activity in proliferating epithelial cells but not growth-arrested epithelial cells. (A) MCF10A cells transfected with control (CTL) scrambled RNAi (SCR) or AJUBA plus LIMD1 (LD1) RNAi were plated at low or high density for 24 h, and BrdU uptake was measured. +ve, positive. (B) Quantification of results in panel A. At least 100 cells were scored under each condition. (C) MCF10A cells were transfected with the indicated RNAi. Control cells were transfected with a scrambled RNAi. After 24 h, the cells were split and cultured at either LD or HD for another 24 h. The cells were lysed, and Western blotting with the indicated antibodies was performed. The pS127YAP/total YAP ratio is shown below each lane. The pYAP/YAP level ratio in control cells at LD was arbitrarily set as 1. (D) The same cells as in panel B were stained with YAP antibody or phalloidin (F-actin), and an immunofluorescence assay was performed. Nuclei were identified with DAPI stain. (E) Quantification of results in panel D. Relative nuclear YAP levels are presented. The amount of nuclear YAP in control LD cells was arbitrarily set as 1. At least 100 cells were scored under each condition. Blue bars, control cells; red bars, AJUBA plus LD1 KD cells. (F) MCF10A cells stably expressing a GTIIC-luciferase TEAD reporter cassette were transfected with the same set of RNAi as in panel C and then plated at LD or HD. The bioluminescence per group was determined, and the results are reported as relative luciferase activity. CTL cells at LD were arbitrarily set to 1. **, P < 0.01; *, P < 0.05; ns, no significant difference. Each experiment was performed at least 3 times, and a representative example is shown. The data are presented as means ± standard deviations (SD). Scale bars, 100 μm (A) and 50 μm (D). Blue bars, control cells; orange bars, AJUBA KD cells; green bars, LD1 KD cells; red bars, AJUBA plus LD1 KD cells.