Fig. 8

NUAK1 regulates FOXO3a subcellular localization, the expression of p21^CIP1, p27^KIP1, FoxM1, and cancer cell survival. A Representative confocal images of FLAG-tagged FOXO3a under NUAK1 inhibition. MDA-MB-231 cells expressing FLAG-tagged FOXO3a were serum-starved overnight followed by 1-h of pretreatment with DMSO or HTH-01-015 (10 µM) before 60 min of stimulation with EGF. Red, FLAG FOXO3a; Green, Phalloidin (F-actin); Blue, nuclei. B Quantification in % of FLAG-tagged FOXO3a expression from MDA-MB-231 IF experiments from A. N = nuclear fraction. C = Cytoplasmic fraction. Each bar represents the mean ± SD, Student t test. C–E p21 and p27 expression under NUAK1 inhibition. MDA-MB-231 cells were serum-starved overnight followed by 1-h of pretreatment with DMSO or HTH-01-015 (10 µM) before stimulation with EGF for 4 and 6 h for mRNA levels (C, D) (each bar represents the mean ± SD. Student t test, n = 3), and 0, 4, 6 and 8 h for protein levels (E). F IB of p21 and p27 expression under NUAK1 overexpression. MDA-MB-231 cells stable for FLAG-tagged NUAK1 inducible expression were pretreated with doxycycline or vehicle (used as a negative control) by 12 h followed by serum-starved overnight (with or without doxycycline) before stimulation with EGF for 0, 4 and 6 h. G, H IB of p21 and p27 expression under NUAK1 inhibition in U87 (G) and SW480 (H) cells serum-starved overnight followed by 1-h of pretreatment with DMSO or HTH-01-015 (10 µM) before stimulation with EGF for 0, 4 and 6 h. All IB are representative of at least three independent experiments. GAPDH and/or α-tubulin were used as loading controls. I FoxM1 mRNA levels under NUAK1 inhibition. MDA-MB-231 cells were serum-starved overnight followed by 1-h of pretreatment with DMSO or HTH-01-015 (10 µM) before stimulation with EGF for 4 h. Each bar represents the mean ± SD, Student t test, n = 3. J Quantification of the crystal violet staining to evaluate NUAK1 effect on cell number under complete medium. MDA-MB-231 cells were treated with DMSO or HTH-01-015 (10 µM) for 24 h. Each bar represents the mean ± SD, Student t test, n = 3. K, L Quantification of the crystal violet staining to evaluate NUAK1 effect on cell number under EGF- or insulin-stimulation. MDA-MB-231 cells were serum-starved overnight followed by 1-h pretreatment with DMSO or HTH-01-015 (5 µM or 10 µM) before stimulation with EGF (K) or Insulin (L) for 24 h. Each bar represents the mean ± SD, one-way ANOVA, n = 3. M NUAK1 effect on senescence. MDA-MB-231 cells were treated with DMSO, Palbociclib (1 µM) (used as a positive control) or HTH-01-015 (5 µM) for 4 days (n = 3). N, O NUAK1 effect on cell death in MDA-MB-231 (N) and SW480 (O) cells under normal growth conditions or EGF stimulation using Incucyte. Each bar represents the mean ± SD, Student t test, n = 5 or one‐way ANOVA, n = 5. P NUAK1 effect on cell viability in spheroids from MDA-MB-231, U87, and DLD-1 cells. Spheroids were pretreated with DMSO or HTH-01-015 (5 µM or 10 µM) before stimulation with EGF for 96 h. Each bar represents the mean ± SD, one‐way ANOVA, n = 6