Figure S8: RhoA activity is not influenced by RASSF1A depletion whereas constitutive activation of a Rho activity by Narciclasine still impairs RASSF1A-induced invasion. from RASSF1A Suppresses the Invasion and Metastatic Potential of Human Non–Small Cell Lung Cancer Cells by Inhibiting YAP Activation through the GEF-H1/RhoB Pathway

A) GST pull-down assay in HBEC-3 cells. Quantification of active GTP-bound RhoA levels as determined by western blotting. One representative experiment is shown in a top panel of histogram. B & C) HBEC-3 cells were transiently transfected with non-silencing siRNA (siNeg), siRASSF1A-1 in combination or not with B) three plasmids coding for wt-RhoA, RhoA N19 (dominant negative form) & RhoAV14 (constitutively active form) C) Cells were assayed for their invasive capacities with Narciclasine treatment. The invasion capacity was assessed using a BioCoat Matrigel Invasion Chamber. The relative invasion was normalized to that of the cells transfected with siNeg. D) Nuclear intensity of YAP was assessed by immunofluorescence and confocal microscopy (right panel), upon depletion of RASSF1A, transfection of RhoAwt expressing plasmid, co-transfection of siRASSF1A and RhoAwt plasmid, transfection of dominant negative RhoAN19 plasmid alone, or upon RASSF1A depletion, transfection of constitutively active RhoAV14 plasmid alone or upon RASSF1A depletion. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p<0.05, **p<0.01 and ***p<0.001, using an ANOVA test followed by Dunnett's test. E) PP1 protein expression assessed by western-blot upon RASS1A depletion or RASSF1A and 1C depletion. Error bars in histograms indicate the standard error of the mean (SEM) of at least three independent experiments. F) One representative experiment of PP2ACalpha Immunoprecipitation is shown in BEAS-2B cells grew exponentially, followed by PP2ACalpha or RhoB western-blottings, with corresponding specific antibodies. G) Representative images for Ser885-phosphoGEF-H1 staining assessed by immunofluorescence and confocal microscopy, upon RASSF1A, PP2ACalpha, RhoB or GEF-H1 depletion. The cells were stained by Ser885-phosphoGEF-H1 primary antibody (Green) followed by Alexa488 conjugated secondary antibody, along with DAPI for DNA (Blue). H) Quantification of ANKDR1 & CTGF mRNA using actin as an internal control.