Fig. 3

Rho activation by MLN stimulates TJ expression. A Effect of MLN on TJ transcription measured by CLDN4 promoter luciferase reporter and RT–qPCR of CLDN4 and OCLN transcripts in LNCaP cells. B Drug effect on the stimulation of Cldn4 expression by MLN (100 nM in control). Inhibitors of PI3K/AKT kinase are indicated in dark red, those of Roc/Cdc42 are indicated in red, and those of Rho signaling are indicated in blue. C Rho signaling is required for the stimulation of Cldn4 expression by MLN. Conditions: 100 nM MLN, charcoal-stripped serum (CSM), 20 nM staurosporine (Stau), 5 µM MBQ167 (MBQ), 10 µM Y27632, 5–10–20 µg/ml C3E, 10 µM lysophosphatidic acid (LPA), 50 µM calpeptin (CAL), and 1 nM CNFy. D Effect of the drugs (see above) on individual Rho isoforms. The active GTP-bound isoforms are hash-tagged. E The effect of 100 nM MLN on LNCaP cell morphology and VCaP spheroid assembly was reversed by 10 µM Y27632 and 20 µg/ml C3E. Scale bars = 50 µm (LNCaP) and 200 µm (VCaP). F Effect of knocking down Rho isoforms and specific mechanoresponsive transcriptional regulators on the stimulation of Cldn4 expression by 100 nM MLN. G Interaction of ZO1 and YBX3 proteins revealed by western blotting after immunoprecipitation. On the right: Effect of 100 nM MLN on the levels of nuclear ZO1 and YBX3 analyzed by immunofluorescence (histogram) and cell fractionation (western blot). Statistical significance: ***-p < 0.001