Fig. 3: INPP4B promotes localized PI(3,4)P2 conversion to PI(3)P on late endosomes.
From: INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/β-catenin signaling in breast cancer
a MCF-7 cells expressing GFP-INPP4B or GFP-vector were lysed, then subjected to whole cell proteomic analysis by LC-MS/MS. DAVID functional annotation bioinformatics microarray analysis was performed on upregulated proteins in GFP-INPP4B expressing cells versus GFP-vector cells (n = 3 experiments). b MCF-7 cells expressing GFP-INPP4B or GFP-vector were serum-starved overnight then stimulated with EGF (100 ng/mL) for 5 min. Cells were lysed and subjected to immunoprecipitation using GFP-Trap beads. Bound proteins were identified by mass spectrometry using a cut-off of >2-fold change and p < 0.05 (n = 2 experiments). c MCF-7 cells expressing GFP-INPP4B or GFP-vector were lysed and subjected to immunoprecipitation using GFP-Trap beads. Bound fractions and soluble lysates (5% of input) were subjected to immunoblotting to detect endogenous Rab7. d MCF-7 cells expressing GFP-INPP4B were serum starved overnight then stimulated with EGF (100 ng/mL) for 15 min. Cells were fixed and subjected to immuno-electron microscopy analysis using GFP (10 nm) and CD63 (15 nm) antibodies. Representative electron micrographs are shown. Arrows show GFP-INPP4B (white) and CD63 (black) localization. Yellow box indicates area where higher magnification micrograph was captured. e MCF-7 cells expressing HA-Rab7WT, HA-Rab7Q67L, or HA-Rab7T22N were serum-starved overnight then stimulated with EGF (100 ng/mL) for 15 min. Cells were treated with saponin (0.02% w/v) to remove cytoplasmic proteins, then fixed and immunostained using INPP4B, CD63, and HA antibodies. f MCF-7 cells expressing GFP-INPP4B or GFP-vector were fixed and immunostained using purified recombinant GST-2xFYVE, DAPI, and phalloidin. Data represent the number of 2xFYVE+ puncta per cell ± SD (n = 3 experiments, >50 cells per experiment). g MCF-7 cells expressing NT, INPP4B #1, or INPP4B #2 shRNA were fixed and immunostained with PI(3,4)P2 antibodies, DAPI, and phalloidin. h, i MCF-7 cells expressing GFP-INPP4B or GFP-vector were fixed and immunostained using recombinant GST-2xFYVE and EEA1 (h) or CD63 (i) antibodies, and co-stained with DAPI. Data represent the percentage of 2xFYVE+ early endosomes (h) or late endosomes (i) ±SD (n = 3 experiments, >30 cells per experiment). The inset panels at the lower right or under each image are higher power regions of the boxed areas. Scale bar is 200 nm (d), 10 µm (e–i). p values determined by a modified Fisher’s exact test are indicated in a, or by two-tailed unpaired t-test in b, f, i.
