Alexa fluor 594 conjugated dextran

For endosomal membrane integrity, H1-HeLa cells were inoculated with equal amounts (~1000 HAV GEs per cell) of gradient-purified HAV or eHAV, or 10 plaque-forming units (PFU) per cell of human rhinovirus-14 (HRV-14) (McKnight and Lemon, 1996 (link)) in supplemented DMEM presence of α-sarcin or Restrictocin A previously reconstituted in sterile ultrapure water and incubated for 6 hr at 37˚C (for HAV) or 33˚C (for HRV-14). Cells were then incubated with supplemented DMEM containing 20 µg.ml⁻¹ puromycin for 20 min, rinsed twice with PBS, and total protein lysates were harvested as described above. Specific puromycin incorporation was validated by pre-treating cells with cycloheximide for 30 min prior to the puromycin pulse (Figure 4—figure supplement 1). For lysosomal membrane integrity analysis, Huh-7.5 cells seeded in 8-well chamber slides were loaded with 100 µg.ml⁻¹ anionic-lysine fixable Alexa Fluor 594-conjugated dextran (10 kDa) (Thermo Fisher Scientific, #D22913) diluted in supplemented DMEM for 16 hr at 37˚C. Cells were rinsed with PBS, inoculated with purified HAV or eHAV (1000 HAV GEs per cell), and fixed as indicated.