Fig. 6: Apilimod reactivates secretory autophagy and promotes EV-enclosed virus release in the absence of the EMCV Leader.

a EMCV-L^Zn infected cells were treated with 500 nM apilimod for 16 h prior to infection or 200 nM BafA1 1 h p.i. onwards. EV-containing 100,000×g ultracentrifugation pellets were isolated from supernatants of an equal number of cells at 8 h p.i. and analyzed by western blot for the presence of extracellular released LC3. Depicted is a representative image of n = 2 independent experiments. LC3I PC and LC3II PC are positive control samples for western blot detection of LC3I and LC3II, respectively. b 100 K EVs were labeled with CFSE, purified by density gradient centrifugation, and analyzed by high-resolution flow cytometry. Bars indicate the mean fold increase in EV release relative to untreated controls ±SD from n = 3 (BafA1) or n = 4 (apilimod) independent experiments. **p = 0.0064, ns (left) p = 0.1148 as assessed by a two-tailed one-sample t-test, or two-tailed t-test for ns (right) p = 0.9936. c, d Depicted is the mean fold increase in EV-enclosed infectivity (c) and intracellular virus titers (d) relative to untreated controls ±SD corresponding to the samples in (b) as determined by end-point dilution assay. For c *p = 0.0210, ns p = 0.1464, for d *p = 0.0285, ns p = 0.4226, as assessed by a two-tailed one-sample t-test. Source data are provided as a Source Data file.