Figure 5

Assessing autophagy during viral infection and overexpression/knockout approaches. (A), HeLa GL cells were infected with influenza A virus (IAV, left panel, MOI 5), encephalomyocarditis virus (EMCV, middle panel, MOI 10) or measles virus (MeV, right panel, MOI 5). Cells were harvested, saponin treated, fixed, and the eGFP-LC3B fluorescence analyzed by flow cytometry at the indicated time points post infection. Treatment with Chloroquine (1 µM, 4 h) and Rapamycin (1 µM, 4 h) served as controls. Data are shown as mean(n = 3–6) ± SEM. (B), HEK293T GL cells were transiently transfected with an empty vector or a TRIM32-FLAG expressing construct. Cells were saponin treated, fixed, and stained with anti-FLAG antibodies (APC) (left and middle panel). eGFP-LC3B MFI of the transfected cell population was quantified for the TRIM32-FLAG sample and background (= vector) subtracted (right panel). Data are shown as mean(n = 3) ± SEM. (C), Agarose gel depicting the sgRNA amplicon amplified by PCR from genomic DNA isolated from saponin treated Jurkat GL cells that were either mock-infected or transduced with the Human CRISPR Knockout Pooled Library (GeCKO v2) (D) Exemplary distribution of high/low/medium autophagosome content (= eGFP MFI) in Jurkat eGFP-LC3B cells 10 days after transduction with the Human CRISPR Knockout Pooled Library (GeCKO v2) as assessed by FACS sorting (E) Scatter Plot (control population ‘input’ vs low autophagy population ‘low’) of aggregated counts of sgRNAs targeting indicated ATG proteins. Each dot is derived from 6 individual sgRNAs targeting the same gene. The dotted red line indicates an equal abundance of sgRNA counts in both populations. Statistical significance was assessed using unpaired t-test (A) or one-way ANOVA (B). ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Uncropped agarose gel in Supplementary Fig. 5.