Fig 2: Axonal SEs transport retrogradely in late endosomes and recycling endosomes. (A) Retrograde SEs show co-localization with late endosomes (Rab7) and recycling endosomes (Rab11). Arrows are co-localized, arrowheads are not. (B) Imaris spot co-localization was used to determine co-localization. (C) Quantification of axonal SE co-localization with endosomal marker (n = 3–4 independent experiments, 15 fields of view per experiment).

Fig 3: Inhibition of LAL leads to accumulation of REs in LAMP1/RAB7-enriched fractions of HepG2 cells. HepG2 cells were incubated with DMEM containing 10 µm Lalistat 2 or a solvent control (DMSO) for 72 h. Then the media were replaced with media containing artificially RP-enriched human TG-rich lipoproteins (final concentration, 10 µm RP) and 10 µm Lalistat 2 or DMSO. After 6 h of incubation, cell lysates were prepared, and endosomal/lysosomal fractions were isolated by density ultracentrifugation. Lipids of cell lysates (A) and endosomal/lysosomal fractions (B, F1 and F2) were extracted into hexane, and retinoids (ROH and RP) were analyzed by HPLC FD. Retinoid levels were normalized to milligram of protein. Data are mean ± S.D. of triplicate determinations and representative of three independent experiments. Statistically significant differences were determined between treatment groups by Student's unpaired t test (two-tailed). ***, p < 0.001. B, inset, enriched fractions (F1–F3) were analyzed by Western blotting using LAMP1/RAB7 and protein disulfide isomerase (PDI) as endosomal/lysosomal and endoplasmic reticulum marker proteins, respectively.

Fig 4: Mevastatin blockade of mevalonate signaling abrogates LBH589-induced autophagy maturation in TNBC cellsPrior to mevastatin plus LBH589 (25 nM) treatment for 24 h, MDA-MB-231 (A) and MDA-MB-468 (B) cells were incubated with or without z-VAD (20 µM) for 2 h. Cell lysates were analyzed by Western blotting for Vps34, Beclin 1, LC3B, p62/SQSTM1, and NBR1, with GAPDH as a loading control. Relative p62/SQSTM1 expression (compared with GAPDH) was analyzed by qPCR. Student's t-test. *P < 0.05; **P < 0.01 (C). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 µM) and/or LBH589 (25 nM) for 24 h. Mevastatin reduced LBH589-dependent expression of Vps34 and Beclin 1 in a dose-dependent manner (D). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 µM) for 24 h and probed for endogenous Vps34 and Beclin 1. Mevastatin abrogated Vps34-Beclin 1 complex formation dose-dependently in the presence of LBH589 (E). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 µM) plus 25 nM LBH589. Immunoblot analyses were performed on immuoprecipitates as indicated. tfLC3 stable MDA-MB-231 cells were analyzed by confocal microscopy (F). MDA-MB-231_tfLC3 cells were treated with mevastatin (2 µM) and/or LBH589 (25 nM) for 24 h. Representative fluorescence images are shown. MDA-MB-231 cells transfected with flag-Rab7 (or pcDNA3.1) were treated with mevastatin (2 µM) and/or LBH589 (25 nM) for 48 h, and cell proliferation was measured. Student's t-test. **P < 0.01; ***P < 0.001 (G). Mevastatin with or without LBH589 (25 nM) reduced prenylated Rab7 in a dose-dependent manner (H). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 µM) and/or LBH589 (25 nM) for 24 hours. Cell lysates were immunoblotted for unprenylated and prenylated Rab7. Prior to treatment with mevastatin (2 µM) and LBH589 (25 nM) for 24 h, MDA-MB-231 cells were incubated with or without mevalonate (250 µM) for 6 h, or with or without compound C (2 µM) for 2 h (I). Cell lysates were immunoblotted for LC3B, p62/SQSTM1, and unprenylated and prenylated Rab7, with GAPDH as a loading control.

Fig 5: The Rab5 detachment occurs in two phases. (A) MDCK cells were stably transfected with invariant chain (MDCK-Ii cells) under the control of an inducible metallothionein promoter. The cells were treated with 25 µM CdCl2 overnight to induce endosomal enlargement and co-transfected with mCh-Rab5. The graph shows the average of mCh-Rab5 detachment on 14 single mCh-Rab5 positive endosomes as fluorescence intensity over time. The figures to the right shows time series of a representative example of mCh-Rab5 detachment from one endosome (white box). (B) MDCK-Ii cells were co-transfected with mCh-Rab5 and EGFP-Rab7a. The graph shows the average of mCh-Rab5 detachment and EGFP-Rab7a recruitment for 14 single mCh-Rab5 positive endosomes as fluorescence intensity over time. White box signifies a representative example of mCh-Rab5 detachment and EGFP-Rab7a recruitment on one endosome (time series in the right panel). (C) MDCK-Ii cells were co-transfected with mCh-Rab5 and EGFP-Rab7aQ67L. The graph shows the average of mCh-Rab5 detachment and EGFP-Rab7aQ67L recruitment for 14 single mCh-Rab5 positive endosomes as fluorescence intensity over time. White box signifies a representative example of mCh-Rab5 detachment and EGFP-Rab7aQ67L recruitment on one endosome (time series in the right panel). (D) This figure (based on A-C) illustrates the detachment pattern of mCh-Rab5 and the recruitment pattern of EGFP-Rab7a/EGFP-Rab7aQ67L as fluorescence intensity over time. Red box (upper left graph) shows the initial rapid detachment phase of single transfected mCh-Rab5, followed by a slower convergence phase (green box). Upper right graph shows a lag phase (blue box) of EGFP-Rab7a recruitment in cells double transfected with mCh-Rab5 and EGFP-Rab7a, whereas the lower left graph shows the lack of the lag phase in cells double transfected with mCh-Rab5 and EGFP-Rab7aQ67L. The bar graph (bottom right) shows the calculated mCh-Rab5 detachment halftime (D1/2) in single- and double-transfected cells. D1/2 was quantified by nonlinear regression (Prism) and averaged over 14 measurements. In single-transfected mCh-Rab5 cells, from A, D1/2=7.005 min±1.195 min s.d., N=14. Double-transfected cells, mCh-Rab5 and EGFP-Rab7a, from B, D1/2=4.216 min±1.659 min s.d., N=14. Double-transfected cells, mCh-Rab5 and EGFP-Rab7aQ67L, from C, D1/2 = 3.865 min±1.958 min s.d., N=14. The analysis shows a significant difference between mCh-Rab5 D1/2 in the control experiment compared to double transfected with EGFP-Rab7a (P<0.0007), or EGFP-Rab7aQ67L (P<0.0012), whereas mCh-Rab5 D1/2 between the double-transected experiments were not significant (ns). Significance was determined using a paired two-tailed t-test. **P=0.0012, ***P=0.0007. Data are mean±s.d.