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Fig. 7
CD146 activating YAP signaling activity. A Protein levels of CD146 and Hippo/YAP pathway molecules were determined by Western blotting in GG16 GFP control (pCMV-GFP) and CD146/GFP overexpressing cells under neurospheres (NSP) and differentiated conditions (Diff). CD146 induced YAP expression accompanied by increased phosphorylation of YAP and LATS. B Bar graphs showing relative levels of YAP normalized to β-actin (loading control) together with ratio of pYAP (inactive)/YAP (active). The ratio decreased in CD146 overexpressing cells suggesting increased YAP activity. C The mRNA levels of YAP, CYR61, and CTGF were detected by qRT-PCR, indicating YAP activation by CD146. D Immunofluorescence microscopic images of GG16-GFP or GG16-CD146/GFP cells stained for YAP (red) and nuclei (blue; scale bars = 100 μm); green signal (GFP or CD146-GFP). Nuclear YAP was enhanced by CD146/GFP overexpression. E Western blotting of cytoplasmic and nuclear extracts showing levels of CD146, YAP, and SOX2 in serum-differentiated GG16 GFP control and CD146/GFP cells. CD146 increased YAP levels in both fractions. β-actin used as loading control of cytoplasmic fractions and Lamin B1 for nuclear fractions. SOX2 was included as additional control for purity of extracts. F Western blots detecting CD146, YAP, and Hippo pathway proteins in serum-cultured and neurospheres GSC23 control and CD146-ko cells. CD146 loss reduces YAP expression and increases pLATS and pYAP levels. G Immunofluorescence microscopic images of GSC23 control and GSC23-CD146-ko serum-cultured cells depicting cytoplasmic (inactive) and nuclear (active) YAP (red). Nuclei are counterstained with DAPI (blue). Loss of CD146 reduces nuclear YAP (scale bars, 100 μm). GSC23 control cells showed nuclear YAP, whereas in CD146-ko cells a clear cytoplasmic shift of YAP was detected. H Immunofluorescence of differentiated GSC23 or GSC23 CD146 knockout cells stained for CD146 (red) and nuclei (blue; scale bars, 100 μm). Membranous CD146 is detected in control cells, absent in CD146-ko cells. I qRT-PCR analyses of YAP target genes CYR61 and CTGF showing reduced expression in GSC23-CD146-ko cells. *p < 0.05, **p < 0.01 by Student’s t test