Extended Data Fig. 4: Validation of siRNA knock-down of a subset of hits that showed significant changes in the Illusia FRET screen.
From: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells
a & b, Representative images (a; Scale bars, 50 μm) and quantification of reverse transfections in MM231 and TIF cells using an siRNA against GFP; actin staining (SiR-Actin, magenta) and Illusia (Green) (UTC, untransfected control; siNTC, non-targeting control siRNA) (MM231s, n = 220 (UTC), 70 (siNTC), 122 (0 nM), 140 (1 nM), 79 (5 nM), 89 (25 nM), 117 (50 nM) and 98 (100 nM) | TIFs, n = 179 (UTC), 155 (siNTC), 174 (0 nM), 183 (1 nM), 224 (5 nM), 199 (25 nM), 118 (50 nM) and 198 (100 nM) cells pooled from three biological replicates; significance assessed using a one-way ANOVA with a Dunnett’s correction for multiple comparisons; NS, not significant, ***p < 0.001). Data are mean ± SEM. c, Parallel qRT-PCR from RNA samples collected from the RNAi FRET screen cells targeting the top 16 hits (n = 1 experiment, assessed in triplicate). ND, Not detected. d, Representative SE-FRET images of MM231s stably-expressing Illusia showing the normalised FRET (NFRET) from the untransfected controls with or without VO43-, and siRNAs targeting PTPN11 and PTPN12, against the NTC siRNA. Scale bars, 20 µm. e & f, Representative western blots validating siRNA KD efficiency and ITGB1 phosphorylation levels in MM231 (left; n = 5 biological replicates; densitometry for ITGB1(Y783)/ITGB1 is displayed here. Densitometry for Shp2/GAPDH is in Fig. 4e) and TIF (right; n = 6 biological replicates; densitometry for ITGB1(Y783)/ITGB1 is displayed here. Densitometry for Shp2/GAPDH is in Fig. 4f) cells transfected with three different siRNAs (A, B and C) against PTPN11 (e) or PTPN12 (f). Source data and exact p-values are provided in the statistical source data file.
