Figure 4

25HC restrains active lysosomal function induced by PDCoV infection. A–C LLC-PK1 cells were infected with PDCoV (MOI = 0.1) and treated with 50 μM 25HC for indicated time. A The cells were processed at 12 hpi. LAMP1 was stained red, LD was stained green and nuclei were stained blue. Scale bar, 20 µm. LAMP1 recruitment to LDs was quantified by Fiji-Image J. B The protein expression of LAMP1 and Cathepsin D were analyzed by western blot, GAPDH was used as a loading control. C The immunoblot bands in panel B were quantified by Image J. D NPC1 expression was analyzed by western blot, with GAPDH being a loading control, the relative quantification was conducted by Image J. E LLC-PK1 cells were infected by PDCoV (MOI = 0.1) and treated by 50 μM 25HC, 10 μM U18666A for 12 h. Relative mRNA expression of PDCoV N was measured by qPCR. F LLC-PK1 cells were transfected with four siRNAs targeting NPC1, the relative mRNA expression of NPC1 was analyzed by qPCR. G, H LLC-PK1 cells were transfected with the third siRNA in panel F, followed by infection with PDCoV for 12 h. Relative expression of (G) PDCoV N and (H) IFN-β, ISG15, ISG20 and MX1 were determined by qPCR analysis. I Relative mRNA expression of TFEB was measured by qPCR. J ATGL and TFEB expression were analyzed by western blot, Tubulin was used as a loading control. Immunoblot bands were quantified by Image J. Error bars, mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.