Fig. 5 | Nature Communications

Fig. 5

From: Identification of a novel anoikis signalling pathway using the fungal virulence factor gliotoxin

Fig. 5

FAK and p190RhoGAP are inactivated by GT. a Western blot analysis of total extracts of BEAS-2B cells either untreated (NT) or treated with 1 µM GT for the indicated time periods showing that the activating phosphorylation of FAK at Y397 (pFAK) was lost within 1 h. b Paxillin (pPax) was dephosphorylated and degraded and JNK was phosphorylated/activated (pJNK) with the same kinetics as FAK inactivation. c Concomitantly, p190RhoGAP was also dephosphorylated at Y1105. d Treating BEAS-2B cells with 10 µM of the specific FAK inhibitor FAK14 caused rapid cell detachment within 6 h. Scale bar = 100 µm. e, f Cell survival of BEAS-2B cells measured as relative metabolic activity using the XTT assay gradually decreased with increasing doses of FAK14 (0–10 µM) (e). Concomitantly, 50 µM FAK14 triggered cell death of BEAS-2B cells (as measured by annexin V-FITC staining) with similar time kinetics as GT (f). g Western blot analysis of total extracts of BEAS-2B cells either non-treated (NT) or treated with 50 µM of FAK14 (g) or 10 µM of FAK14 (h) for 30 min to 4 h. The inhibitor caused FAK and p190RhoGAP dephosphorylation and MKK4, JNK and Bim phosphorylations. Tubulin was used as loading control in ac, g and h. i FAK is active in adherent epithelial cells causing the activation of p190RhoGAP by its phosphorylation at Y1105. This inactivates RhoA. In response to GT, FAK and p190RhoGAP get inactivated, therefore allowing RhoA activation. Graphs in e and f show the means of at least three independent experiments ± s.e.m.; p-values: *0.05–0.01, **0.01–0.001, ***<0.001, one-way ANOVA, post hoc: Bonferroni compared to NT

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