Fig 2: Endolysosomal abnormalities in oocytes from Tsg101 d/d mice are age‐dependent. (A) GV oocytes from Tsg101 f/f and Tsg101 d/d mice at the indicated ages were stained with anti‐EEA1 antibody. The graph shows oocytes with an abnormal EEA1 pattern. The numbers of oocytes are shown within bars, and the numbers of mice are given at the end of each bar. (B) GV oocytes were stained with anti‐Rab7a (late endosome marker) and anti‐Lamp‐1 (lysosome‐specific marker) antibodies. Scale bar = 20 μm. (C) Pattern of EEA‐1 localization in oocytes during maturation.

Fig 3: pY374-PKCδ stabilizes association of RAB5 and RAB7 with EGFR-containing endosomes. (a–f) EGFR-containing endosomes (Alexa Fluor 488) and the indicated RABs (Alexa Fluor 594) in control pCas9Vec (clone a) or PTPN14-KO (two clones, c1 and o) BT-549 cells were fluorescently tagged as described in Fig. 6. Representative confocal micrographs of EGFR-containing endosomes costained with RAB5 (a), RAB7 (c), or RAB11 (e) 60 min after inducing endocytosis with 20 ng/ml EGF (arrowheads indicate colocalization), and quantified data of colocalization of EGFR-containing endosomes with RAB5 (b), RAB7 (d), or RAB11 (f) from confocal micrographs (mean ± SEM; n > 30 cells; ns, not significant; **, P < 0.01; ***, P < 0.001 using one-way ANOVA with Bonferroni multiple comparisons post-test).

Fig 4: Analysis of autophagy in HeLa cells expressing CMT2B-causing RAB7 mutant proteins. HeLa cells transfected for 48 h with empty vector or plasmids encoding HA-tagged RAB7WT, RAB7Q67L, RAB7T22N and CMT2B-causing RAB7 mutants (RAB7L129F, RAB7K157N, RAB7N161T, RAB7V162M), were incubated in full medium (FM) or starvation medium (ST) for 30 min, or in the presence of 100 nM bafilomycin A1 (BAF) for 3 h. (A) The colocalization rate between RAB7 and LC3B was analyzed for the each different RAB7 isoform and for each condition (FM, ST, BAF). (B) The number of LC3B-positive dots per cell was evaluated for each sample. (C) The autophagic flux was calculated as the ratio of LC3B dots between BAF and FM of the same sample and normalized on empty vector. Quantification of results was performed by unbiased intensitometric analysis of fluorescence using the Quantitation Module of Volocity software.69 Statistical analysis one-way ANOVA test was performed between the same conditions (FM, ST and BAF) selecting RAB7WT as the referring sample. All statistical comparisons are from the sample indicated with asterisks and RAB7WT. Means ± SEM for each value are shown in the graphs (n = 3). * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Fig 5: Stx17 overexpression recovered the Meth-induced autophagosome-late endosome/lysosome fusion deficiency. (a) Co-localization of LC3 and Stx17. (b) Co-localization of LC3 and Rab5. (c) pHBAd-EF1-Stx17-3 flag (MOI = 40, 36 h) or empty adenovirus vector (Ad) was overexpressed in primary hippocampal neurons. (d) Co-localization of LC3 and Rab7 after the Stx17 overexpression. (e) Co-localization of LAMP1 and Rab7 after the Stx17 overexpression. (f) Co-localization of LC3 and LAMP1 after the Stx17 overexpression. Scale bar: 5 μm. (g) Co-localization of LC3-Rab7, LAMP1-Rab7, and LC3-LAMP1 was quantified and expressed as Pearson coefficient value (∗∗p < 0.01 and ∗∗∗p < 0.001 compared with the control group and ###p < 0.001 compared to the Meth group).