Fig. 4: An RNAi FRET screen to identify regulatory PTPs for ITGB1 NPxY sites.
From: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells
a, A waterfall plot from an RNAi sensitized emission (SE)-FRET screen of MM231 cells stably expressing Illusia to assess changes after KD of 96 out of the 108 PTPs in the human genome, using three siRNAs/target (A, B and C) (n = 3; one-way analysis of variance (ANOVA) with a Tukey correction for multiple comparisons after normalizing replicates into robust z-scores). PTPs that significantly (P < 0.01) increased phosphorylation upon KD are indicated with red text and circles (15 hits), while those that significantly (P < 0.01) decreased phosphorylation upon KD are indicated with blue text and circles (15 top hits). The data are the mean ± s.e.m. b,c, Representative FLIM–FRET images (left) and quantification (right) of MM231 and TIF cells with stable Illusia expression and transfected with siRNAs (A, B or C) against either PTPN11 (Shp2) (b) or PTPN12 (PTP-PEST) (c) (MM231 PTPN11 silencing, n = 100 (siNTC), 98 (siRNA A), 100 (siRNA B) and 100 (siRNA C); PTPN12 silencing, n = 99 (siNTC), 99 (siRNA A), 98 (siRNA B) and 97 (siRNA C) cells pooled from three biological replicates; TIF PTPN11 silencing, n = 75 (siNTC), 76 (siRNA A), 76 (siRNA B) and 78 (siRNA C); PTPN12 silencing, n = 74 (siNTC), 74 (siRNA A), 77 (siRNA B) and 74 (siRNA C) cells pooled from four biological replicates; one-way ANOVA with a Dunnett correction for multiple comparisons). n.s., not significant. Scale bars, 20 μm. The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas on the boxplots highlight the IQR of the control conditions. siNTC, non-targeting control siRNA. d,e, Densitometry from western blots of PTPN11 (representative western blot) (d) (Extended Data Fig. 4e) and PTPN12 (representative western blot) (e) (Extended Data Fig. 4f) siRNA KD in MM231 (n = 5 biological replicates) and TIF (n = 5 (Shp2) or 6 (PTP-PEST) biological replicates) cells. The data are the mean ± s.e.m. *P < 0.05, ***P < 0.001.
