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. 2022 Aug 25;14:933979. doi: 10.3389/fnagi.2022.933979

FIGURE 2.

FIGURE 2

Autophagy cargo receptors, p62 and optineurin, inhibit small EV secretion through the ubiquitin association domain. (A) Schematic of p62 full length and p62ΔC proteins with their respective domains. (B) Representative images of DiI-stained small EVs secreted from NIH3T3 cells expressing GFP, GFP-p62, or GFP-p62ΔC. (C) Quantification of secreted small EVs for (B). Data are presented as means ± SEM. n = 3 independent experiments, 1-way ANOVA, followed by Dunnett’s post-hoc, #P < 0.0001. (D) Representative immunoblots of GFP-p62, GFP-p62ΔC, GFP, and β-Actin protein levels in lysates from cells for (B). (E) Representative images of DiI-stained small EVs secreted from NIH3T3 cells expressing GFP, GFP-OPTN, or GFP-OPTN E478G. (F) Quantification of secreted small EVs for (E). Data are presented as means ± SEM. n = 3 independent experiments, 1-way ANOVA, followed by Dunnett’s post-hoc, #P < 0.0001. n.s. = not significant. (G) Representative immunoblots for GFP, GFP-OPTN, and β-Actin protein levels in cell lysates for (E). (H) Representative images of DiI-stained small EVs secreted from NIH3T3 cells transfected with control siRNA or p62 siRNA. (I) Quantification of secreted small EVs for (H). Data are presented as means ± SEM. n = 4 independent experiments, t-test, #P < 0.0001. (J) Representative immunoblots for p62 and β-Actin protein levels in cell lysates for (H).