Fig. 2

Combination of imatinib and ISRIB specifically inhibits STAT5 signaling in BCR-ABL1-expressing CML cells. A Protein levels of STAT5 and phosphorylated form of STAT5 (pSTAT5) detected by western blot in K562 or LAMA84 CML cells. If indicated (ISRIB alone and imatinib+ISRIB conditions), 250 nM ISRIB was added for 2 hours to protect from ISR. 100 nM thapsigargin was added for 2 hours treatment to all experimental conditions to mimic activation of ISR in vitro. This was followed by treatment with 1 μM imatinib, if indicated (imatinib alone and imatinib+ISRIB conditions). After 16 hours cells were collected for analyses. The ratio of phosphorylated to total STAT5 forms (P/T) calculated based on the densitometry signal is given for each condition. Adequate graphs showing pSTAT5/STAT5 ratios are presented. Statistical analysis: unpaired Student’s t-test with Welch’s correction (*p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0005). Only comparisons with statistical significance are marked. B Flow cytometry analysis of pSTAT5 levels in K562 and LAMA84 cells untreated (control) or treated as indicated above. Data were calculated based on gMFI, fluorescence signal for untreated cells = 1. Statistical analysis: repeated-measures one-way ANOVA with Tukey’s multiple comparisons test (*p ≤ 0.05). Right panel - overlay of the representative histograms presenting fluorescence signals for pSTAT5 estimated in control cells or in cells after treatment. gMFI values are indicated for each condition. Only comparisons with statistical significance are marked. C Protein levels of mTOR and phosphorylated form of mTOR (p-mTOR) detected by western blot in K562 or LAMA84 CML cells untreated (control) or treated with drugs as indicated above. Adequate graphs showing p-mTOR/mTOR ratios in K562 cells and LAMA84 CML cells are presented. Control cells (without drug treatment)=1. Statistical analysis: unpaired Student’s t-test with Welch’s correction (*p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0005). Only comparisons with statistical significance are marked. D Flow cytometry analysis of pS6 levels in K562 and LAMA84 CML cells untreated (control) or treated as indicated. Data are calculated based on gMFI, fluorescence signal for untreated cells = 1. Statistical analysis: repeated-measures one-way ANOVA with Tukey’s multiple comparisons test (*p ≤ 0.05). Only comparisons with statistical significance are marked. E, F, G Protein levels of GSK3β and phosphorylated form of GSK3β (pGSK3b) (E), ERK and phosphorylated form of ERK (pERK) (F) and AKT and phosphorylated form of AKT (pAKT) (G) detected by western blot in K562 or LAMA84 CML cells untreated (control) or treated with drugs as indicated above. Adequate graphs showing phospho/total protein ratios are presented. Statistical analysis: unpaired Student’s t-test with Welch’s correction (*p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0005). Only comparisons with statistical significance are marked. The full-length membranes were adequately cut based on the protein marker size according to target protein sites prior to hybridization with primary antibodies (see Materials and Methods). Cropped blots are presented; original non-cropped membranes are shown in the Supplementary Fig. S9