Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy (2022)
Figure 1

Figure 1: INPP4B promotes PI3Kα-dependent basal autophagic degradation independent of Vps34

A PI(3)P is synthesized directly by the class III PI3K Vps34, or via the sequential actions of PI3Kα, inositol polyphosphate 5-phosphatses and INPP4B in response to growth factor stimulation.

B, C MCF-7 cells expressing GFP-INPP4B or GFP-vector were cultured in growth media or EBSS for the indicated times, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (B). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to growth media-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (C).

D, E MCF-7 cells expressing NT, INPP4B #1 or INPP4B #2 shRNA were cultured in growth media or EBSS for the indicated times, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (D). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to growth media-treated NT shRNA cells which were assigned an arbitrary value of 1 (n=3 experiments) (E).

F, G MCF-7 cells expressing GFP-INPP4B or GFP-vector were cultured in growth media or EBSS for 4 hours, then fixed and immunostained with p62 antibodies, and co-stained with DAPI and phalloidin (F). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (G).

H, I MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (H). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (I) (n=3 experiments).

J, K MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours, then fixed and immunostained with p62 antibodies, and co-stained with DAPI and phalloidin (J). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (K).

L, M MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 2 µM BYL719 or DMSO as a vehicle control for the indicated times. Cells were lysed and immunblotted with LC3B, pAKTS473, AKT(pan), pS6KT389 or S6K antibodies and GAPDH antibodies as a loading control (L). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (M).

N, O MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 1 µM or 10 µM SAR405 or DMSO as a vehicle control for 4 hours. Cells were lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (N). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (O).

Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in F, J. p values determined by one-way ANOVA with Tukey post hoc test in C, G, I, K, M, O, or by one-way ANOVA in E.

Source data are available online for this figure.