Fig. 5
Domatinostat potentiates chemotherapy effect by modulating expression and localization of FOXM1 in CSCs. A. FOXM1 expression in patients with poor (dead; n = 93) and good prognosis (alive; n = 85) in the TCGA PAAD cohort (Wilcox-test W = 2647, p-value< 0.00058). B. FOXM1 expression in sensitive and resistant to the primary therapy in the TCGA PAAD cohort, evaluated as PFS (Wilcox-test W = 2269, p-value< 0.00012). C. FOXM1 expression in good (complete-remission_response patients, n = 43) vs bad (progressive-disease patients, n = 40) responders to chemotherapy, picked in “treatment_outcome_first_course” subset patients (One Way Analysis of variance; p-value< 0.000701). D. Basal protein levels of FOXM1 and stem-cell markers (β-Catenin; Oct-4) in PANC1, PANC28 and ASPC1 spheroids (S) versus differentiated cells (D). β-actin serves as loading protein control. E. Nuclear localization by IF upon domatinostat (0.5 μM) treatment in PANC1 spheroid cells at indicated timing. Bar 50 μM. Magnification 40X. DAPI is for nuclear staining. F. FOXM1-nuclear intensity quantified by Harmony software. G. FOXM1-nuclear spots were quantified by Harmony software. H. WB analysis of nuclear and cytoplasmic FOXM1 in PANC1 spheroids treated with domatinostat (0.5 μM) at the indicated timing. PARP and β-actin serve as nuclear and cytoplasmic loading control, respectively. I. FOXM1 protein expression in PANC1 spheroids, treated with domatinostat (0.5 μM) and domatinostat plus Bortezomib (20 mM) for 6 h. β-actin serves as loading control. L. CAT, GPX2, SOD2, RAD51, XRCC1, BIRC5 and SOX2 mRNA levels in PANC1 spheroids, treated with domatinostat (0.5 μM) for 16 h. M. ChIP-qPCR analysis showing the relative decrease of enrichment of FOXM1 binding to CAT and OCT4 promoters. Data obtained on immunoprecipitated fractions were normalized to input chromatin (IP/Input). The mean of at least two independent experiments with error bars indicating the SD. N. FOXM1, β-Catenin, Oct-4 and γH2AX protein expression in PANC1 spheroids treated for 16 h with domatinostat, GT (IC5096h) or their combination. β-actin serves as loading control. O. OCT4, CAT, SOD2 and GPX2 mRNA levels in PANC1 cells transfected with FOXM1 (OE-FOXM1) or empty vector (EV-FOXM1). P. Mitochondrial ROS amount in OE-FOXM1 and EV-FOXM1 PANC1, treated or untreated with domatinostat (0.5 μM) for 16 h, visualized by mitosox staining. Q. OE-FOXM1 and EV-FOXM1 PANC1 cells were treated for 96 h with domatinostat (1 μM) alone or in combination with GT (respectively, 100 nM and 1.56 nM). Cell growth expressed as percentage of control was assessed by SRB colorimetric assay. The values are the means ±S.D. from at least three independent experiments. Statistically significant results, by 2-way ANOVA test, are reported (***indicates P < 0.0001, **indicates P < 0.005 and *indicates P < 0.05)