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. 2017 Feb 6;27(3):371–385. doi: 10.1016/j.cub.2016.12.038

Figure 3.

Figure 3

VAPB or PTPIP51 Overexpression Inhibits Basal Autophagy and Autophagic Flux

(A and B) Representative images of HEK293 cells co-transfected with EGFP-LC3 and either control empty vector (CTRL), Myc-VAPB, or HA-PTPIP51 and treated with either vehicle (A) or bafilomycin A1 (BafA1) (B) as indicated. Cells were immunostained for VAPB and PTPIP51 via their epitope tags and LC3 visualized via the EGFP tag. Transfection of VAPB or PTPIP51 decreases the numbers of EGFP-LC3 structures in both vehicle- and bafilomycin A1-treated cells. The scale bars represent 10 μm. The bar charts show numbers of EGFP-LC3 dots per cell in the different experiments. Data were analyzed by one-way ANOVA and Tukey’s post hoc test. n = 100–130 cells per condition from three independent experiments. Error bars are SEM; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001.

(C) VAPB or PTPIP51 overexpression inhibits autophagic flux. HeLa cells were transfected with either control empty vector (CTRL), Myc-VAPB, or HA-PTPIP51 and treated with either vehicle (−) or bafilomycin A1 (BafA1+) as indicated. Samples were then probed on immunoblots for LC3 and α-tubulin as a loading control. Protein molecular mass markers are indicated in kD. Both LC3-I and LC3-II isoforms are shown; arrow indicates LC3-II isoform. Also shown are immunoblots for PTPIP51 and VAPB, which were detected via their epitope tags. VAPB and PTPIP51 expression decreases the levels of LC3-II in both vehicle- and bafilomycin-A1-treated cells. The bar chart shows relative LC3-II levels following quantification of signals from immunoblots. LC3-II levels were normalized to α-tubulin signals. Data were analyzed by one-way ANOVA and Tukey’s post hoc test; n = 3. Error bars are SEM; ∗p ≤ 0.05; ∗∗p ≤ 0.01.

(D) VAPB and PTPIP51 expression increases EGFP-HD74Q aggregation in HEK293T cells. Cells were co-transfected with EGFP-HD74Q and either control empty vector (CTRL), Myc-VAPB, or HA-PTPIP5 and immunostained for VAPB and PTPIP51 via their epitope tags. Cells were analyzed 48 hr post-transfection. Arrows indicate cells containing EGFP-HDQ74 aggregates; blue represents DAPI staining of nuclei. The scale bar represents 10 μm. The bar chart shows percentage of EGFP-HD74Q-transfected cells displaying aggregates. Data were obtained from 320-580 EGFP-HD74Q-transfected cells per condition from five independent experiments. Data were analyzed by one-way ANOVA and Tukey’s post hoc test. Error bars are SEM; ∗p ≤ 0.05; ∗∗∗p ≤ 0.001.