Fig. 5: NEDD9 promotes ST-mediated FAK-AKT activation and helps ST evade lysosomal degradation.

A Western blot analysis of Nedd9^wt/wt and Nedd9^-/- mBMDMs infected with ST and (B) densitometric analysis of FAK protein level normalized to GAPDH, n = 3, 2-way ANOVA: p (Nedd9^-/- UI vs. Nedd9^-/- ST 4 h) = 0.0430, p (Nedd9^wt/wt ST 4 h vs. Nedd9^-/- ST 4 h) = 0.0166 and (C) phosphorylated FAK protein level normalized to GAPDH 1 and 4 h post ST infection, n = 3, 2-way ANOVA: p (Nedd9^wt/wt ST 1 h vs. Nedd9^-/- ST 1 h) = 0.0219, p (Nedd9^wt/wt ST 4 h vs. Nedd9^-/- ST 4 h) = 0.0090. D Infection of Nedd9^wt/wt and Nedd9^-/- mBMDMs with ST (1 h, 4 h, 24 h post infection, uninfected) and quantification of pFAK and NEDD9 colocalization, n = 5, biological replicates, unpaired t-test: p = 0.0028. E Infection of Nedd9^wt/wt and Nedd9^-/- mBMDMs with ST and Western blot analysis followed by densitometric analysis, n = 3, of (F) phosphorylated AKT Thr308 normalized to GAPDH, 2-way ANOVA: p (Nedd9^wt/wt UI vs. ST 4 h) = 0.0079, p (Nedd9^wt/wt ST 4 h vs. Nedd9^-/- ST 4 h) = 0.0199 and (G) phosphorylated AKT Ser473 normalized to GAPDH at indicated time points, n = 3, 2-way ANOVA: p (Nedd9^wt/wt UI vs. ST 4 h) < 0.0001, p (Nedd9^wt/wt ST 4 h vs. Nedd9^-/- ST 4 h) = 0.0003. Densitometric analysis of Western blots showing (H) phosphorylated mTOR Ser2448, n = 3, 2-way ANOVA: p (Nedd9^wt/wt UI vs. ST 1 h) = 0.0401, p (Nedd9^wt/wt UI vs. ST 4 h) < 0.0001, p (Nedd9^-/- UI vs. ST 4 h) = 0.0036, p (Nedd9^wt/wt ST 4 h vs. Nedd9^-/- ST 4 h) = 0.0029 and (I) phosphorylated NDRG1 normalized to GAPDH in Nedd9^wt/wt and Nedd9^-/- mBMDMs 1 and 4 h post ST infection, 2-way ANOVA: p (Nedd9^wt/wt UI vs. ST 1 h) = 0.0067, p (Nedd9^wt/wt UI vs. ST 4 h) = 0.0112, p (Nedd9^wt/wt ST 1 h vs. Nedd9^-/- ST 1 h) = 0.0038, p (Nedd9^wt/wt ST 4 h vs. Nedd9^-/- ST 4 h) = 0.0040. J Bacterial burden (CFU/mL) of ST infected Nedd9^-/- mBMDMs untreated and treated for 4 h with 1 µM AKT activator SC-79 in vitro, n = 4, unpaired t-test: p = 0.0418. Densitometric analysis of Western blots showing (K) LC3-I to LC3-II conversion normalized to ß-Actin in Nedd9^wt/wt and Nedd9^-/- mBMDMs 1 h and 4 h post ST infection, n = 3, 2-way ANOVA: p (Nedd9^-/- UI vs. Nedd9^-/- ST 4 h) = 0.0203, p (Nedd9^wt/wt ST 1 h vs. Nedd9^-/- ST 1 h) = 0.0128, p (Nedd9^wt/wt ST 4 h vs. Nedd9^-/- ST 4 h) = 0.0006 and (L) p62, n = 3, 2-way ANOVA: p (Nedd9^wt/wt UI vs. ST 4 h) < 0.0001, p (Nedd9^-/- UI vs. ST 4 h) = 0.0012, p (Nedd9^wt/wt ST 1 h vs. Nedd9^-/- ST 1 h) = 0.0179, p (Nedd9^wt/wt ST 4 h vs. Nedd9^-/- ST 4 h) < 0.0001. p > 0.05 equals not significant (ns), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.