Figure 5

From: Par6G suppresses cell proliferation and is targeted by loss-of-function mutations in multiple cancers

Figure 5

Loss of Par6 activity deregulates Akt pathway. (a) Western blotting analysis shows the impact of PARD6B and PARD6G loss on phospho-PKCz (T410/412) in 2D cultures of MCF10A cells. The cells expressing the indicated shRNAs were grown in 2D culture for 24 h in the presence or absence of essential growth factors insulin and EGF (INS/EGF). Growth factor deprivation was used to reduce background phosphorylation. (b) Silencing of PARD6B or PARD6G does not affect apico-basal polarity in MCF10A acinar structures. The acinar structures expressing the indicated shRNAs were grown in Matrigel for 10 days and immunostained with GM130 golgi marker and α6-integrin to visualize apico-basal polarity. Scale bar 20 μm. (c) Confocal images demonstrating positive phospho-Akt (S473) staining in proliferative day-10 MCF10A acini and negative staining in quiescent day-18 acini. Scale bar 20 μm. (d) Quantification of phospho-Akt (S473) in day-18 PARD6B- and PARD6G-deficient acini with or without Myc activation. The acini containing 1 phospho-Akt-positive cells were scored as phospho-Akt positive. Mean and s.d. were calculated from three independent experiments and P-values by Student’s t-test. (e) Confocal images demonstrating phospho-S6 staining pattern in day-10 and day-18 MCF10A acini. Scale bar 20 μm. (f) Quantification of phospho-S6 in day-18 PARD6B- and PARD6G-deficient acini with or without Myc activation. The acini containing 1 phospho-S6-positive cells were scored as phospho-S6 positive. At least 50 acini were counted in each experiment unless otherwise indicated. Mean and s.d. were calculated from three independent experiments and P-values by Student’s t-test. (g) Western blotting showing the impact of PARD6B and PARD6G loss on Akt phosphorylation at T308 and S473 in growth factor-deprived cells. Cells were grown as in panel (a); in the right panel, cells were treated for 24 h with 4-hydroxytamoxifen (4-OHT) to activate Myc. (h) Loss of PARD6B or PARD6G expression impedes exit from the cell cycle. Control and MCF10A cells with silenced Par6 were deprived from essential growth factors insulin and EGF for 24 h, followed by analysis of Ki-67 positivity. Mean and s.e.m. were calculated from three independent experiments and P-values by Student’s t-test. (i) Western blotting shows the effect of PI3K inhibition (500 nM GDC-0941) and PDK1 inhibition (500 nM BX-795) on PARD6B and PARD6G loss-dependent phosphorylation of Akt at T308. Cells expressing the indicated shRNAs were deprived from growth factors for 24 h as in panel (a) and subsequently treated with inhibitors for 4 h. (j) PDK1 inhibition suppresses the cell cycle effects of PARD6B and PARD6G. MCF10A cells expressing the indicated shRNAs were deprived from growth factors for 24 h and subsequently treated with 500 nM BX-795 for another 24 h. *P<0.5; **P<0.05; ***P<0.005.