Figure 3: 2-O-Bn-InsP₅ inhibits PLCγ1-PDK1 complex formation and PLCγ1 tyrosine phosphorylation in MDA-MB-231 cells.

(A) Localisation of PLCγ1 and PDK1 visualised by high definition confocal scan in MDA-MB-231 overexpressing PLCγ1 and PDK1. Cells were left untreated or treated with 2-O-Bn-InsP₅ 50 μM and then stimulated with EGF for 3 min. Arrows indicate plasma membrane localisation. Bar: 20 μm. (B) Percentage of FRET in MDA-MB-231 cells co-expressing PLCγ1 and PDK1 at the indicated time of EGF stimulation. Columns are the means ± SEM of percentage of FRET measured at protrusion level in proximity of the plasma membrane. (C) Endogenous PLCγ1 and PDK1 were co-immunoprecipitated from MDA-MB-231 untreated or treated with 2-O-Bn-InsP₅ 50 μM and stimulated with EGF 50 ng/ml for the indicated time points. PLCγ1 was immunoprecipitated using a specific anti-PLCγ1 antibody and association of the two enzymes was assessed by immunoblotting using anti-PLCγ1 and anti-PDK1 antibodies. Parallel co-immunoprecipitation using mouse IgG was performed as control. (D) Percentage of MDA-MB-231 cells displaying plasma membrane rearrangement presenting both PLCγ1 and PDK1 staining at the plasma membrane. Cells expressing exogenous PLCγ1 and PDK1 were stimulated with EGF for the indicated times in the presence or absence of 2-O-Bn-InsP₅ 50 μM. Graph shows the means ± SEM from n = 3 independent experiments. (E,F) Representative Western blot (E) and corresponding densitometry analysis (F) of EGF-induced PLCγ1 tyrosine phosphorylation in MDA-MB-231 untreated or treated with 2-O-Bn-InsP₅ 50 μM or GSK2334470 1 μM. In E, GAPDH was used as loading control. Data in F are means ± SEM from n = 4 independent experiments. P value: *<0.05; ^#≤0.01.