Additional file 5 of The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

Additional file 5: Figure S5 shows the level of re-expression of PPM1F WT and PPM1F DA mutant in PPM1F-deficient A172 cells and the resulting effect on cellular levels of pT788/pT789 integrin β1. Furthermore, the re-introduction of PPM1F into PPM1F-deficient A172 cells does not alter cell growth. Figure S5: Related to Fig. 7 and Fig. 9. (A) WCLs from A172 wildtype cells, control cells (sgRNA against Cerulean), PPM1F KO cells (sgRNA against Cerulean and PPM1F) and PPM1F KO cells re-expressing mKate2, re-expressing active PPM1F-mKate2, or re-expressing the inactive PPM1FD360A-mKate2 were analyzed by Western blotting with α-human PPM1F or α-integrin β1 antibodies. α-Tubulin antibody was used as loading control. (B) Serum-starvedA172 cell lines as in (A) were seeded onto 2 µg/ml FNIII9-11 for 45 min and WCLs were subjected to Western blotting with indicated antibodies to analyze T788/T789 phosphorylation of integrin β1. α-Tubulin antibody was used as loading control. (C) Cell growth is not altered by PPM1F re-expression. 5 × 103 A172 wildtype, PPM1F KO and PPM1F KO cells re-expressing wildtype PPM1F were seeded into 96-well plates in triplicate and examined for cell proliferation after one, two, three, and four days by paraformaldehyde fixation, crystal violet staining, dye elution with 10% acetic acid and absorbance measurement at 590 nm in a microplate reader. Shown are mean ± SEM values from one representative experiment repeated twice.