Fig. 4

p110α^H1047R expression leads to centrosome overduplication through RhoA and ROCK pathway activation. a Assessment of RhoA activity in Pik3ca ^WT;Flpe-ER ^T2 and Pik3ca ^H1047R+neo;Flpe-ER ^T2 MEFs, 3 days after treatment with 4-OHT. The experiment was performed three times using three independent Pik3ca ^WT;Flpe-ER ^T2 and Pik3ca ^H1047R+neo;Flpe-ER ^T2 MEFs. A representative experiment is shown. b Cyclin E protein levels in Pik3ca ^WT;Flpe-ER ^T2 and Pik3ca ^H1047R+neo;Flpe-ER ^T2 MEFs, 3 days after treatment with 4-OHT. Cell extracts from three independent MEF lines per genotype are shown. c Activation of signalling pathways involved in centrosome duplication by p110α^H1047R expression, and effect of p110α inhibition (by 24 h of treatment with 3 µM A66). Cell extracts from two independent MEF lines per genotype are shown. d Effect of inhibition of CDK2 (by 2 h of treatment with 1 µM NU6140) or Akt (by 2 h of treatment with MK2206) on signalling pathways involved in centrosome duplication. e Impact of inhibition of p110α (by 3 µM A66), Akt (by 1 µM Akti X) or ROCK (by 10 µM Y27632 or 0.5 µM H1152) on p110α^H1047R-induced centrosome amplification in primary MEFs. A total of 100 cells were scored per condition using two independent p110α^H1047R MEF lines; values = mean ± SD. f Impact of inhibition of Akt (by 1 µM Akti X) or ROCK (by 10 µM Y27632) for 15 days on centrosome numbers in p110α^H1047R Nutu cells. Centrosomes were revealed by γ-tubulin staining. In total, 200 cells were scored per cell line and per condition