Fig 2: Atg3 overexpression in SKM-1 cells induces autophagy.(A-C) At 96 h post-transfection, cells of each group were subjected to Western blotting for LC3 and p62 after Baf A1 treatment for 2 h.(D-F) The expression of proteins involved AKT, pAKT, mTOR and p-mTOR were analyzed at 96 h post-transfection. GAPDH served as a loading control. Protein levels were normalized to GAPDH expression. (G and H) The formation of LC3 puncta was assayed by confocal microscopy, and the percentage of cells with punctate staining was calculated. Representative results from triplicate experiments are shown as the mean±SD (n = 3). *p<0.05, **p<0.01, ***p<0.001.

Fig 3: The effects of exogenous Aß 25-35 treatment and endogenous overexpression of APPswe on autophagic flux. (A) Fluorescence microscopy images of control SH-SY5Y, APPWT, APPswe and SH-SY5Y +Aß 25-35 (30 µM) cells infected with mRFP-GFP-LC3 adenovirus for 24 h and then treated with 100 nM rapamycin for 10 min (scale bar, 10 µm). (B) mRFP and GFP puncta were quantified to determine the number of APs and ALs per cell. For each group, 30 cells from 5 visual fields were randomly selected for counting (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Western blot showing the expression of ATG5, ATG12, ATG16L, BECN1, ATG14, LC3, p62 and Lamp1 in APPWT and APPswe cells. (D and E) Relative protein gray density analysis. (F) Western blot analysis showing the expression of ATG5, ATG12, ATG16L, BECN1, ATG14, LC3, p62 and Lamp1 in SH-SY5Y and SH-SY5Y +Aß 25-35 (30 µM) cells. (G and H) Protein band relative gray density analysis. (I) Different concentrations of Aß 25-35 (0, 20, 40, and 60 µM) were used to treat SH-SY5Y cells for 24 h and the expression of LC3, p62, BECN1, ATG14 and Lamp1 were assessed by Western blot. (J and K) Relative protein gray density analysis. (* P < 0.05, ** P < 0.01, *** P < 0.001; “ns” denotes that there was no significant difference, n = 3).

Fig 4: ZOL increases autophagic activity instead of inhibition of lysosomal degradation. Human umbilical vein endothelial cells were pretreated with vehicle or 1 µM CQ for 1 h, then exposed to 100 µM ZOL for 48 h. The protein levels of (A) LC3B-I and -II, and (B) SQSTM1 were determined by western blot and densitometric analysis was performed. Data are presented as the mean ± standard error, n=6. **P<0.01 vs. V alone; ##P<0.01 vs. ZOL + V group; ZOL, Zoledronate; V, vehicle; CQ, chloroquine; LC3B, microtubule-associated proteins 1A/1B light chain 3B; SQSTM1, sequestome 1.

Fig 5: (See previous page). Starvation induces autophagy and myofibroblast differentiation. (A) Upper panel: Western blot showing LC3B-I and -II protein levels in WI-38 fibroblasts exposed to serum-free (starvation) medium (SS). Lower panel: Densitometric analysis of LC3B-II relative to LC3B-I normalized to time 0 (representative of 4 independent experiments, *P < 0.05 t = 0 vs 1 h). (B) Western blot showing LC3B-I and -II protein levels in WI-38 fibroblasts at baseline, starved for 4 h or 1 d and exposed to DMSO (V) or bafilomycin A1 (20 nM; Baf). Representative of 3 independent experiments. (C) Evaluation of LC3B puncta by confocal microscopy in WI-38 fibroblasts infected with a baculovirus vector expressing GFP-LC3B and exposed to normal conditions (medium with 10% FBS; N), serum-free medium with DMSO (V) or serum-free medium with bafilomycin A1 (20 nM) for 1 d. Representative of 3 independent experiments. (D) Upper panel: Western blot showing SQSTM1 and tubulin (TUBA) protein levels in starved WI-38 fibroblasts. Lower panel: Densitometric analysis of SQSTM1 protein levels relative to tubulin. Data are presented as mean ± s.e.m. (representative of 4 independent experiments, *p = 0.02 4 h vs 2 d and 4 h vs 4 d). (E) Upper panel: Western blot showing ACTA2 protein levels in WI-38 fibroblasts exposed to SS medium or grown under normal conditions (N). Tubulin was used as a loading control. Lower panel: Densitometric analysis of ACTA2 protein levels relative to tubulin normalized to time 0 (representative of 4 independent experiments; *p = 0.0170 SS vs N at 4 d). (F) Evaluation of the myofibroblast markers ACTA2 (red) and stress fiber (green) by immunofluorescence microscopy in cells grown in normal medium (N) or maintained without serum (SS) for 4 d. Representative of 3 independent experiments. (G) Real-time qPCR evaluation of mRNA levels of COL1A1 and COL3A1 after 4 d in N or SS. GAPDH was used as the reference gene (***P < 0.001 N vs SS, representative of 2 independent experiments performed in triplicate).