Journal: bioRxiv
Article Title: Luminescence-based screening for extracellular vesicle release modulators reveals a role for PI4KIIIβ in exosome biogenesis upon lysosome inhibition
doi: 10.1101/2023.02.23.529257
Figure Lengend Snippet: Kinase inhibitor screening reveals PI4KIIIβ as modulator of bafilomycin-induced EV secretion. a) Correlation plot depicting normalized HA-NL-CD63 secretion for individual kinase inhibitors from the kinase inhibitor screen performed under basal and bafilomycin-induced conditions. Grey area corresponds to the mean ± 2 times the standard deviation of the DMSO controls under bafilomycin-induced conditions. Kinase inhibitors in the green area were considered selective inhibitors of bafilomycin-induced HA-NL-CD63 secretion. b) Effect of PI4KIIIβ inhibition (PI4KIII Beta inhibitor 3, 1 μM) on bafilomycin- induced HA-NL-CD63 secretion. Data represents the mean ± S.E.M of four independent experiments. **P<0.01 using a one- sample, two-tailed T-test. c) Dose-inhibition curve of the PI4KIII Beta inhibitor 3 on bafilomycin-induced HA-NL-CD63 secretion. Data is a representative of three independent experiments. The IC 50 value of 6.4 ±1.5 nM (mean + S.E.M) was determined based on three independent experiments. d) Confirmation of PI4KIIIβ knockout at the protein level in HA-NL- CD63-CRISPR HEK293 cells. e) Effect of PI4KIIIβ KO on HA-NL-CD63 secretion under basal and bafilomycin-induced conditions. Data represents the mean ± S.E.M of three independent experiments. **p <0.01 using a two-sample, two-tailed Student’s T test. f) Western blot against EV marker proteins on cell lysates and isolated small EVs from control and bafilomycin-induced HEK293 cells in the presence or absence of PI4KIIIβ inhibitor (PI4KIII Beta inhibitor 3, 1 μM). Representative of two independent experiments. g) Effect of PI4KIIIβ inhibition (PI4KIII Beta inhibitor 3, 1 μM) on sEV secretion as determined by Nanoparticle Tracking Analysis. Representative of four independent experiments. h) Quantification of (g). Combined data of four independent experiments. Data is color-coded by biological replicate, where dots represent each biological replicate, and the horizontal bars represent mean ± SEM. **, P < 0.01 using a Student’s two-tailed two-sample t-test paired by biological replicate. i) Effect of PI4KIIα (PI-273, 2 μM) and PI4KIIIα (10 nM, GSK-A1) inhibition on HA-NL-CD63 secretion under basal and bafilomycin-induced conditions. Data represents the mean ± S.E.M of three (PI4KIIα) or four (PI4KIIα) independent experiments.
Article Snippet: U18666A (cat. no. U3633), Ionomycin (cat. no. IO634) and GSK-A1 (cat. no. SML2453) were obtained from Merck.
Techniques: Standard Deviation, Inhibition, Two Tailed Test, Knock-Out, CRISPR, Western Blot, Marker, Isolation
