Fig. 6: INPP4B promotes Wnt/β-catenin activation.
From: INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/β-catenin signaling in breast cancer
a Differentially expressed (>2-fold) Wnt pathway genes identified from nanoString RNA profile analysis of GFP-INPP4B versus GFP-vector expressing MCF-7 cells using the nCounter PanCancer Pathways codeset. Wnt/β-catenin target genes are indicated. b RNA was extracted from MCF-7 cells expressing GFP-INPP4B or GFP-vector, and two-step quantitative RT-PCR was performed using primers for AXIN2, LEF1, DKK1, MYCN, MYC, or CCND1. Expression was normalized to GAPDH. Expression was determined using the ΔΔCt method and expressed relative to GFP-vector control cells (±SD), which were assigned an arbitrary value of 1 (n = 3 experiments). c–e MCF-7 cells expressing GFP-INPP4B or GFP-vector were stimulated with 50% Wnt3a-conditioned media ± 10% R-spondin-conditioned media or 50% L-cell control media. c RNA was extracted and two-step quantitative RT-PCR was performed using primers for AXIN2 and expression was normalized to GAPDH. Expression was determined using the ΔΔCt method and expressed relative to L-cell control media-treated GFP-vector control cells (±SD) which were assigned an arbitrary value of 1 (n = 3 experiments). d Cells were lysed and subjected to immunoblotting with non-phospho β-cateninS33/S37/T41 (active-β-catenin), β-catenin or GSK3β antibodies, and GAPDH antibodies were used as loading control. e Data represent the mean GSK3β levels relative to GAPDH ± SD (n = 3 experiments). f, g MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and NT or Hrs shRNA, were lysed and subjected to immunoblotting with GSK3β antibodies or GAPDH antibodies as a loading control (f). Data represent the mean GSK3β levels relative to GAPDH ± SD (n = 4 experiments) (g). h MCF-7 cells expressing GFP-INPP4B or GFP-vector were permeabilized with 6.5 μg/mL digitonin, then treated with 1 µg/mL proteinase K in the presence or absence of 0.1% Triton X-100. Cells were lysed and immunoblotted with GSK3β antibodies, and GAPDH antibodies were used as loading control. Yellow box indicates GSK3β protein protected from proteinase K treatment. i MCF-7 cells co-expressing RFP-GSK3 and GFP-INPP4B or GFP-vector were incubated with 50 nM LysoTracker Deep Red for 1 h, then fixed and stained with DAPI. The inset panels at the lower right of each image are higher power regions of the boxed areas. j, k MCF-7 cells expressing GFP-INPP4B or GFP-vector were suspended in 0.3% soft agar in the presence of 2 µM IWP-2, or DMSO as a vehicle control, and cultured for 4 weeks to allow anchorage-independent colony growth (j). Data represent the relative colony size ± SD (n = 3 experiments in triplicate, 50 colonies/experiment) (k). Scale bar is 10 µm (i), 1 mm (j). p values determined by two-tailed unpaired t-test are indicated in b, by one-way ANOVA in c, e, g, or by one-way ANOVA with Tukey post hoc test in k.
