Extended Data Fig. 3: Impaired autophagy induction by acid-mediated bypass of endocytic viral entry, confirmation of indicated gene knockdown in HeLa cells, and requirement for SNX5 in autophagy induced by diverse viruses.
From: Sorting nexin 5 mediates virus-induced autophagy and immunity
a, b, Representative fluorescent micrographs (a) and quantitation (b) of GFP–LC3 puncta in HeLa-GFP–LC3 cells that were either mock-infected or infected with SIN or HSV-1ΔBBD (MOI = 50 and 25, respectively; 4.5 h) in the presence (pH 5.4) or absence (pH 7.4) of an acidic pulse (that induces viral entry at the plasma membrane) after viral attachment. Arrows in a denote representative GFP–LC3 puncta that would be scored as positive in b. Scale bars, 20 μm. Bars in b represent mean ± s.d. of triplicate samples (100-150 cells analysed per sample). c, d, Viral entry efficiency in HeLa-GFP–LC3 cells treated similarly as in a and b. Bars in c and d represent mean ± s.d. of SIN minus-strand RNA levels (c) and HSV-1 immediate early gene ICP27 mRNA levels (d) of triplicate samples at indicated time points, respectively. e, Representative fluorescent micrographs of GFP–LC3 puncta in HeLa-GFP–LC3 cells that were treated with indicated siRNAs (72 h) and then either mock-infected or infected with indicated virus for 4.5 h (MOI = 5 for HSV-1ΔBBD; MOI = 10 for SIN, Zika virus, WNV, CHIKV and IAV; and MOI = 20 for poliovirus and CVB3). Scale bars, 20 μm. Arrows in e denote representative GFP–LC3 puncta that would be scored as positive in Fig. 1b. f–i, Confirmation of gene knockdown in indicated siRNA-treated HeLa-GFP–LC3 cells (72 h) by western blot analyses of indicated proteins (f–h) or quantitative real-time PCR of SNX32 (i) for the experiment shown in Fig. 1b. Bars in i represent mean ± s.d. of triplicate samples. j, Western blot detection of SNX5 and actin in wild-type HeLa-GFP–LC3 cells (WT), HeLa SNX5KO/GFP–LC3 cells (KO), and two clones of reconstituted HeLa SNX5KO/GFP–LC3/SNX5 cells used in the experiment shown in k. In b, one-way ANOVA with Dunnett’s test for multiple comparisons was used to compare means of SIN or HSV-1ΔBBD infection versus mock infection. In b–d, unpaired two-tailed t-tests were used to compare means of pH 7.4 versus pH 5.4 conditions. For e–h, j, similar results were observed in three independent experiments. In i, an unpaired two-tailed t-test was used to compare means of NC versus SNX32 knockdown. k, GFP–LC3 puncta in reconstituted HeLa SNX5KO/GFP–LC3 cells mock-infected or infected with SIN or HSV-1ΔBBD (MOI = 10 and 5, respectively; 4.5 h). Bars represent mean ± s.d. of three independent replicates (100-150 cells analysed per sample). P values, one-way ANOVA with Dunnett’s test for multiple comparisons. For gel source data, see Supplementary Fig. 1.
