Fig 2: Immortalized human mesenchymal stem cells show little autophagosome turnover unless stimulated. Immortalized human mesenchymal stem cells grown under normal culture conditions were tagged with a tandem green fluorescent protein-red fluorescent protein (GFP-RFP) light chain 3 (LC3) via viral transfection (see Materials and methods). ihMSCs were then stimulated with bafilomycin to halt autophagy for 1 hour or with rapamycin to induce autophagy for 15 minutes and 1 hour. The representative of experiments repeated at least three times is shown.

Fig 3: Characterization of sEV secreted from control- or DDA-treated B16F10 cells. Analysis of sEV isolated from the culture media of B16F10 cells treated with the solvent vehicle (C-sEV) or DDA (DDA-sEV) for 24 h. (a) Representative flow cytometry analysis of sEV tetraspanin content (n = 3). (b) Representative immunoblot analysis of markers of sEV (Alix, Tsg101, Flotilin (Flo), LC3 II, Annexin A1 (AnxA1) and the melanocytic marker tyrosinase (Tyr) (n = 3) with densitometry values showing changes in protein expression relative to C-sEV and normalized to Alix. (c) Representative flow cytometry analysis of sEV calreticulin content (n = 5). (d) GC/MS analysis of cholesterol content in sEV (n = 3). (e) Representative flow cytometry analysis of BMP quantification in sEV (n = 5). (f) Immunocapture of CD63-positive sEV with an anti-CD63 antibody fixed to beads and analysis of the presence of BMP in CD63-positive sEV by flow cytometry analysis (n = 4). Data is expressed as percentage of positive beads and are the mean ± SEM of four independent experiments (*P < 0.05, t test, two-tailed). (g) Quantification of sEV production from B16F10 cells, treated with increasing concentrations of DDA, monitored after sEV labelling with the lipid membrane probe bodipy-ceramide as described in the schematic diagram, (n = 3). (h) sEV production recovered from 106 cells was measured by measuring protein content (n = 5). (i) Mice (15/group) were implanted with B16F10 tumour cells in the right flank and treated two times, at day 0 and day 7, in the contralateral flank with PBS or with 2 µg of C-sEV or DDA-sEV. Mean tumour volumes (± SEM) are shown, two-way ANOVA, **P < 0.01, ****P < 0.0001. ns: not significant. Data are representative of three independent experiments. The plots in (c) and (e) show the median fluorescence intensity (MFI) of DDA- sEV relative to C-sEV. Data in c, d, e, g, h are the means ± SEM of 3–5 independent experiments (*P < 0.05 and **P < 0.01, t test, two-tailed; ns: not significant)

Fig 4: RP loss increases phospo-S6 kinase signaling.(A) Western blot analysis showing the expression of phosphorylated S6 kinase in GFP-LC3 HEK cells transfected with siScr or siRPS19 and either untreated or treated with 100 nM rapamycin overnight. (B) Western blot analysis of phosphorylated S6 kinase expression in CD34+ cord blood cells infected with a control shRNA (shScr) or an shRNA against RPS19 (shRPS19). (C) Western blot analysis of phosphorylated S6 kinase expression in LCLs derived from a normal control or DBA patients. (D) Western blot analysis of phosphorylated S6 kinase expression in 2 dpf zebrafish embryos.

Fig 5: S6 kinase phosphorylation and autophagy is induced by reactive oxygen species (ROS).(A) Confocal microscopy analysis of GFP-LC3 HEK cells transfected with siScr or siRPS19 and either untreated or treated with 10 mM Trolox overnight. Size bars = 10 µM. (B) Quantification of the average number of GFP-LC3 puncta per cell in (A). (C) Western blot analysis of phosphorylated S6 kinase expression in GFP-LC3 HEK transfected with siScr or siRPS19 and either untreated or treated with 10 mM Trolox overnight. (D) Western blot analysis of phosphorylated S6 kinase and phosphorylated AKT substrate expression in 2 dpf zebrafish embryos untreated or treated with 10 mM Trolox overnight. (E) Survival rates of embryos treated overnight with 10 mM Trolox. The results are the compilation of three independent experiments with N = 25 embryos representing each mutation. The statistics shown are compared to wild type. (F) Analysis of p53 stabilization in 2 dpf wild type, rpS7-deficient embryos, p53M214K/M214K mutant embryos, or double mutants (rpS7;p53) by western blotting. (G) Analysis of p53 stabilization by western blotting of zebrafish mutants treated overnight with 100 µM Trolox.