Fig. 4

NUAK1 regulates Akt signaling under growth factors stimulation. A IB of Akt signaling under NUAK1 inhibition in MDA-MB-231 cells serum-starved overnight followed by 1-h of pretreatment with DMSO or HTH-01-015 (5 µM and 10 µM) before stimulation with EGF for 20 min. B IB of Akt signaling under NUAK1/2 inhibitors in MDA-MB-231 cells serum-starved overnight followed by 1-h of pretreatment with DMSO, HTH-01-015 (10 µM) or WZ4003 (10 µM) before stimulation with EGF for 15 and 30 min. C IB of Akt signaling under NUAK1 inhibition in MDA-MB-231 cells serum-starved overnight followed by 1-h of pretreatment with DMSO or HTH-01-015 (10 µM) before stimulation with EGF for 0, 5, 15, and 30 min. D Quantification of Akt phosphorylation at Ser-473 from C. Each bar represents the mean ± SD, n = 3. Data from 3 independent were analyzed by one-way ANOVA followed by Turkey’s multiple comparison test. E IB of Akt signaling under NUAK1 inhibition in MDA-MB-231 cells serum-starved overnight followed by 1-h of pretreatment with DMSO or HTH-01-015 (10 µM) before stimulation with EGF for 0, 15, 30 and 60 min. F Quantification of Akt phosphorylation at Ser-473 from E. Each bar represents the mean ± SD, n = 3. Data from 3 independent experiments (two for pTSC2) were analyzed by one-way ANOVA followed by Turkey’s multiple comparison test. G IB of Akt Ser-473 phosphorylation using inducible shRNAs for NUAK1. MDA-MB-231 cells stable expressing inducible shRNA vectors for NUAK1 [#1 (left) and #2 (right)] were pretreated with doxycycline or vehicle (used as a negative control) by 4 days. H IB of Akt signaling using inducible shRNA for NUAK1. MDA-MB-231 cells stable expressing inducible shRNA vector for NUAK1 #1 were pretreated with doxycycline or vehicle (used as a negative control) for 3 days followed by serum starvation overnight (with or without doxycycline) before stimulation with EGF. α-tubulin and/or GAPDH were used as loading controls