Fig. 8: Loss of AFDN or SCRIB disrupts ERK and AKT activation kinetics and cell motility in a growth factor-dependent manner.

Activation of the MAPK and PI3K pathways following EGF stimulation of MCF7 parental (a), AFDN KO (b) or SCRIB KO (c) cells. pERK or pAKT Western blots measure pathway activity 2.5, 5, 15, 30 or 45 minutes after addition of EGF. Total ERK and AKT Western blots confirmed expression and serve as loading controls. Quantification of MAPK (d; ERK activation) and PI3K (e; AKT activation) activity following addition of EGF over time. Center is the mean and error bars SD as derived from n = 3 independent replicates. f Effect of AFDN or SCRIB KO on cell proliferation compared to parental MCF7 cells over a 72-hour time course from n = 4 independent replicates. Center represents mean and error bars SD. **P < 0.005 (P = 0.003), ns = not significant (P = 0.298) as measured by two-way ANOVA. g KO of SCRIB or AFDN results in motility defects in response to EGF. Wound closure (%) was measured at 24, 48, 72 or 96 hours for MCF7 (P = 0.0001 within group), AFDN KO (P = 0.0145), SCRIB KO (P = 0.0001), and AFDN/SCRIB KO (P = 0.0001) cells. 100 ng/ml EGF was supplemented to the media at time 0 and every 24-hour interval thereafter. n values denote independent replicates. Line represents the median, box the IQR and whiskers min/max. ***P < 0.001, *P < 0.01 as measured by RM one-way ANOVA (within group) or two-way ANOVA (between group). P = < 0.0001 for all between group comparisons. h Phase contrast images of MCF7, SCRIB KO and AFDN KO cells during wound closure. 100 ng/ml EGF was supplemented to the media every 24 hours. Inset are enlarged images of cells at the wound edge. Scale bar represents 100 µm. i AFDN and SCRIB KO cells do not polarize towards the wound. Cells were stained for GM130 (Golgi), which typically orients towards the wound at the leading edge (see parental MCF7 cells; top). Cells were fixed 24 hours after wounding and EGF stimulation. Arrows indicate direction of motility. Scale bars represent 20 µm. All source data are provided in the Source Data files.