Fig. 4

Knockdown of Akt1 promoted EGFR activation by dephosphorylating and inactivating PIKfyve. (a) knockdown of Akt1 decreased PI5P production in MCF-7 and MDA-MB-231 cells, PI: Phosphoinositides, ***p < 0.001, one-way ANOVA, n = 5 per group. (b) Knockdown of Akt1 induced an increased EGFR co-localization with the early endosomes marker EEA.1 in MCF-7 and MDA-MB-231 cells. (c) Co-immunoprecipitation assay was performed to determine the phosphorylation status of PIKfyve by Akt1. Note that Flag-PIKfyve WT but not a S318A mutant PIKfyve binds to Akt1 in MCF-7 and MDA-MB-231 cells, n = 5 per group. (d) Expression of a phosphorylation-mimic PIKfyve S318D mutant in breast cancer cells could induce a lower expression of EGFR and β-catenin protein than did WT PIKfyve in MCF-7 and MDA-MB-231 cells, n = 3 per group. (e) Western blot analysis showed that inhibition of PIKfyve with YM201636 enhanced the phosphorylation and total protein expression of EGFR in MCF-7 and MDA-MB-231 cells, n = 5 per group. (f) Western blot analysis showed that inhibition of PIKfyve with YM201636 could upregulate the expression of β-catenin total and nuclear protein in MCF-7 and MDA-MB-231 cells, n = 5 per group. (g) Immunofluorescence staining showed PIKfyve inhibitor YM201636 could induce β-catenin nuclear accumulation in MCF-7 and MDA-MB-231 cells. (h-i) Transwell assay with Matrigel demonstrated that PIKfyve inhibitor YM201636 enhanced the ability of invasion in MCF-7 and MDA-MB-231 cells. H: representative of images of breast cancer cells; I: analysis of the invade cells. **p < 0.01, ***p < 0.001, two-tailed unpaired t-test, n = 5 per group