Fig 2: Inhibiting Hsc70/Hsp70 blocks pexophagy.a Quantifying LC3B accumulation on ROS-stressed peroxisomes (as in Fig. 1h–j). Depletion of cellular Hsc70 and Hsp70 delayed LC3B appearance on ROS-stressed peroxisomes (x-axis represents time after 559 nm illumination; siControl, n = 8 cells; siHsp70 + siHsc70, n = 18 cells). *P = 4.25E−09, **P = 8.32E−09, ***P = 3.22E−07 (one-tailed t test). b Hsp70/Hsc70 inhibition on LC3B accumulation. Treating cells with PES-Cl delayed LC3B appearance on ROS-stressed peroxisomes (Mock, n = 10 cells; PES-Cl, n = 14 cells). *P = 0.0011, **P = 0.0004, and ***P = 3.89E−05 (one-tailed t test). c Inhibiting Hsc70/Hsp70 with PES-Cl delayed EGFP-p62 accumulation on ROS-stressed peroxisomes (Mock, n = 9 cells; PES-Cl, n = 10 cells). **P = 0.0018 and ***P = 0.0001 (one-tailed t test). d PES-Cl treatment delayed EGFP-Ub accumulation on ROS-stressed peroxisomes (Mock, n = 11 cells; PES-Cl, n = 15 cells). *P = 0.0227, **P = 0.0046, and #P = 0.0071 (one-tailed t test). All error bars in a–d represent mean ± SEM. e Same experimental setup as Fig. 2a, b, but with PES-Cl treatment. Peroxisomes within the white circular region in a NIH3T3 cell expressing PMP34-EGFP-TagBFP2 and diKillerRed-PTS1 were illuminated with 559 nm light (top panels). The EGFP and TagBFP2 signals of 559 nm illuminated peroxisomes were monitored 7 h after illumination (middle panels). The white arrows indicate that EGFP fluorescence persisted on ROS-stressed peroxisomes (bottom panels; magnified view of the white square region in middle panels). f The percentage of peroxisomes that entered lysosomes (TagBFP2+ EGFP−) 7 h following 559 nm illumination (Mock, n = 13 cells; PES-Cl, n = 17 cells; siHsc70 + siHsp70, n = 14 cells. ***P = 3.67E−09, ###P = 1.56E−08, one-tailed t test). All error bars represent the mean + SEM. All scale bars: 5 μm. Source data are provided as a Source data file.

Fig 3: Association of HSP expression with polyp size: (a) fold change in HSPA1 expression; (b) HSPA1 expression in polyps; (c) HSPA1 expression in polyp-adjacent (normal) tissue; (d) fold change in HSP90AA1 expression; (e) HSP90AA1 expression in polyps; (f) HSP90AA1 expression in polyp-adjacent (normal) tissue. Data presented on logarithmic scale as geometric means (medians for HSPA1 in polyp and adjacent mucosa) (red close squares) with 95% confidence interval (whiskers) and analyzed with one-way ANOVA (Kruskal-Wallis H-test). Groups differing significantly (p < 0.05) in a post-hoc analysis (Conover test) were indicated by symbols of the same type (* or ^). P/N, polyp-to-normal ratio; NRQ, normalized relative quantity.

Fig 4: Association of HSP expression with polyp grade of dysplasia: (a) fold change in HSPA1 expression; (b) HSPA1 expression in polyps; (c) HSPA1 expression in polyp-adjacent (normal) tissue; (d) fold change in HSP90AA1 expression; (e) HSP90AA1 expression in polyps; (f) HSP90AA1 expression in polyp-adjacent (normal) tissue. Data presented on logarithmic scale as geometric means (red close squares) with 95% confidence interval (whiskers) and analyzed with t-test for independent samples. P/N, polyp-to-normal ratio; NRQ, normalized relative quantity; LGD, low grade dysplasia; HGD, high grade dysplasia; A, adenocarcinoma in polyp.

Fig 5: JESS automated western blotting analysis, transmission electron microscopy (TEM) analysis and profiling heatmaps. (A) Confirmation that ultracentrifugation pellet contains exosomes. The JESS automated western blot analysis shows the detection of specific exosomal markers CD9, CD63, Hsp70, and CD81 in both plasma- and salivary-exosomal samples except for the Hsp 70 from CP salivary-exosomal representative sample showed faint band. [PD = plasma diseased sample/CP plasma-exosomal representative sample; PH = plasma healthy/healthy plasma-exosomal representative sample; SD = saliva diseased/healthy salivary-exosomal representative sample; and SH = saliva healthy/healthy salivary-exosomal representative sample]. (B) TEM image of a single exosome originated from healthy plasma-exosomal representative sample. (C) TEM image of a single exosome originated from healthy salivary-exosomal representative sample. (D) Heatmap illustrating all 33 significantly down-regulated miRNAs in CP as compared to healthy plasma-exosomal samples. (E) Heatmaps illustrating all significantly up-regulated miRNAs in CP as compared to healthy salivary-exosomal samples. (F) Heatmaps illustrating top ten significantly down-regulated miRNAs in CP as compared to healthy salivary-exosomal samples. Color shading reflects expression level [color version = with red indicating low expression and green indicating high expression; black-and white version = with darker black indicating low expression and white or greyer color indicating high expression]. (G) Venn diagram shows the distribution of total miRNAs that are down-regulated only in plasma- (DP) and in salivary- (DS) exosomal, up-regulated in only salivary-exosomal (US), and down-regulated in both plasma- and salivary-exosomal (DS-O-DP) CP samples as compared to their respective exosomal healthy samples.