Fig. 6: RSV-induced cholesterol accumulation in lysosomes blocks autophagy flux by inhibiting autolysosome degradation.
HEK293T cells transiently expressing mRFP-GFP-LC3B, HEp-2, 16HBE, or HBECs cells were either mock-infected or infected with RSV (MOI = 1) in the presence or absence of U18666A (10 μM), CQ (5 μM), RAPA (200 nM), MβCD (100 μM), or BafA1 (200 nM) for 24 h. a The fluorescence intensity of mRFP-GFP-LC3B in infected HEK293T cells was measured using a confocal microscope with Airyscan. Scale bar: 10 μm. Data are one representative of three independent experiments. b–g The fluorescence intensity of LysoSensor in infected cells was measured using a confocal microscope with Airyscan (HEp-2 (b, c); 16HBE (d, e); HBECs (f, g)). Scale bar: 10 μm. Data (n = 20 micrographs) are one representative of three independent experiments. h, i The effect of RSV on lysosomal activity in infected HEp-2 cells was detected using flow cytometry (n = 3 independent experiments). j–o The number of autolysosomes in infected cells was examined using TEM (HEp-2 (j, k); 16HBE (l, m); HBECs (n, o)). The red arrow indicates the autolysosome. Scale bar: 2 μm or 500 nm. Data (n = 20 cells) are one representative of three independent experiments. Image parameters: scaling-per pixel (a: 0.025 × 0.025 μm2; b, d, f: 0.038 × 0.038 μm2); image size-pixels (a, b, d, f: 2048 × 2048); image size-scaled (a: 50.71 × 50.71 μm2; b, d, f: 78.01 × 78.01 μm2); objective (a, b, d, f: plan-apochromat 63×/1.40 oil DIC M27); scan zoom (a: 2.0; b, d, f: 1.3). Data are shown as the mean ± SD, statistical analysis using two-sided Student’s t-test (k, m, o) or one-way ANOVA (c, e, g, i) (####P < 0.0001 compared to the blank control group; ****P < 0.0001 compared to the viral control group).
