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. 2021 Jan 18;28(6):1865–1879. doi: 10.1038/s41418-020-00713-8

Fig. 3. Akt activation does not promote ciliogenesis in RPE1 cells.

Fig. 3

Activated Chk2 did not contribute to ETO-induced ciliogenesis. A Chk2 was activated in ETO-treated RPE1 cells. Extracts of cells treated with ETO at 100 µM for 24 h were analyzed by immunoblotting with antibodies against phosphorylated Chk2 (p-Chk2), Chk2, phosphorylated Chk1 (p-Chk1), Chk1, and tubulin (Tub.). Depletion of Chk2 did not inhibit ETO-induced ciliogenesis. B Chk2 was depleted efficiently. Extracts of RPE1 cells transfected with siRNA against Chk2 was analyzed by immunoblotting with antibodies against Chk2 and tubulin (Tub.). C Quantitative results of frequency of ciliated RPE1 cells treated with 100 µM ETO for 24 h in control or Chk2-deficient cells. Activation of Akt did not induce ciliogenesis. D Akt was activated by ETO treatment. Extracts of cells treated with 100 µM ETO for 24 h were analyzed by immunoblotting with antibodies against phosphorylated Akt (p-Akt), Akt, and tubulin (Tub.). E inhibition of Akt led to robust cell death upon ETO treatment. Quantitative results of relative cell numbers after treatment with 100 µM ETO for 24 h in the presence or absence of Akt inhibitor IV (Akti, 5 µM). F Inactivation of Akt diminished ETO-induced ciliogenesis. Quantitative results of the frequency of ciliated cells after treatment with 100 µM ETO for 24 h in the presence or absence of Akt inhibitor IV (Akti, 5 µM). The results are presented as the mean ± SD of three independent experiments; more than 100 cells were counted in each individual group. G Akt was depleted efficiently. Extracts of RPE1 cells transfected with siRNA against Akt was analyzed by immunoblotting with antibodies against Akt and tubulin (Tub.). H Depletion of Akt had modest effect on cell death upon ETO treatment. Quantitative results of relative cell numbers after treatment with 100 µM ETO for 24 h in the control or Akt-deficient cells. C Quantitative results of frequency of ciliated RPE1 cells treated with 100 µM ETO for 24 h in control or Akt-deficient cells. **P < 0.01; ***P < 0.001; n.s. no significance.