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. 2018 Apr 9;9:679. doi: 10.3389/fimmu.2018.00679

Figure 5.

Figure 5

Wnt5A-induced bacterial clearance is mediated through autophagy. (A–D) Effect of 3-methyladenine (3-MA, 1 mM) and wortmanin (WM, 100 nM), respectively, on rWnt5A-mediated killing of Pseudomonas aeruginosa (PA) and Streptococcus pneumoniae (SP) (n = 3). Percent killing was calculated for 3 h (T3) killing time-point. DMSO (DM) and PBS were used as vehicle control. (E) LC3BII accumulation assessed at different killing time-points (T0–T4) for rWnt5A and PBS pretreated RAW 264.7 cells infected separately with PA and SP by western blotting (n = 3). T0 represents bacterial killing in 0 time (post 1 h of infection). (F) Influence of WM, 3-MA, Rac1 inhibitor, and Dvl inhibitor (Dvli) on rWnt5A-induced LC3BII accumulation at 3 h (T3) postinfection with PA (n = 3). (G,H) LC3-punctae in Wnt5A vs. PBS sets as assessed by confocal microscopy (G) and punctae dot calculation (H) (n = 3). (I) Presence of Wnt5A in L5A conditioned medium (L5A: L cells expressing Wnt5A) but not in L conditioned medium (used as control) (n = 3). (J,K) Difference in bacterial load between L5A Phg (phagosome isolated from L5A treated cells 2 h postinfection: T2) and L Phg (corresponding control) (n = 3). (L) Higher levels of LC3BII, ATG5-ATG12, Rac1, p62, LAMP-1, and p-Dvl-2 (phosphorylated Disheveld-2) in L5A Phg compared to L Phg. Both sets contain similar levels of precursor and mature forms of cathepsin D (CathD). Data represented as mean ± SEM; *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.