Figure 11.

CSFV release requires the formation of autolysosomes. (A) Detection of the cell viability with the treatment of CQ by CCK-8 test. (B-F) CSFV infected PK-15 cells were treated with CQ for 4 h at 20 hpi, then the cell samples were analyzed by western blot with anti-E2, anti-LC3B, anti-SQSTM1, and anti-ACTB/β-actin (loading control) antibodies(B), the virus titers of extracellular and intracellular were measured by endpoint dilution titrations by using immunofluorescence (C-D). (E) The ratios of CSFV titers in extracellular and intracellular were calculated to evaluate viral release. (F) CSFV infected plaques formed by intracellular cell lysates, extracellular culture supernatants (with or without antibody), and isolated autophagic EVs were detected by immunofluorescence. Scale bar: 200 μm. Results are shown as the mean ± SD (n = 3). ns, not significant; **, P < 0.01. (G) Detection of the autolysosomes in CSFV infected cells by using mCherry-GFP-LC3B. Transfected cells treated with DMSO, rapamycin, 3-MA, and CQ were used as control. Red arrows point to autolysosomes. (H) Detection of the autophagosome-lysosome fusion in CSFV-infected cells by confocal microscope. Autophagosomes were detected by anti-LC3B antibody (in green), lysosomes were detected by anti-LAMP2 antibody (in red). Cells treated with rapamycin and CQ used as control. (I) Detection of the CSFV in lysosomes. CSFV was detected by anti-E2 antibody (in green), lysosomes were detected by anti-LAMP2 antibody (in red). Cells treated with CQ used as control. Cell nuclei were stained with DAPI (in blue). Scale bar: 10 μm.