Fig 2: PLK1 inhibition induces a sustained change in cell fate.A Schematic of the drug wash-out experiment. SUM149PT cells were treated for 3 days with rigosertib or DMSO. Subsequently, the drug was washed out and cells were cultured for eight more days without the drug and then harvested for downstream experiments. B Immunoblot showing levels of ERa and ERK2 (loading control) in SUM149PT cells treated with the indicated concentrations of rigosertib as depicted in Fig. 4A. C Bar graphs representing average mRNA expression of ESR1 and downstream targets in SUM149PT cells treated with 100 nM rigosertib or DMSO as depicted in Fig. 4A. n = 2 experimental replicates with 2 technical replicates each. Unpaired Student’s t-test. Data are means ± SD. D Representative flow-cytometry dot plots of EdU/Hoechst cell cycle staining of SUM149PT cells treated with 100 nM rigosertib or DMSO as depicted in Fig. 4A. E Bar graph depicting the proportion of cells in different cell cycle states based on EdU/Hoechst cell cycle staining shown in Fig. 4D. n = 3 experimental replicates. Ordinary two-way ANOVA with multiple comparisons. Data are means ± SD. F Schematic of the experimental setup for in vitro treated SUM149PT cells grown as mouse xenografts in NSG mice. Cells were treated for 3 days with 1 µM rigosertib or DMSO prior to injection. G Kinetics of primary tumour growth of SUM149PT cells treated in vitro with 1 µM rigosertib (n = 5 mice) or DMSO (n = 5 mice) as depicted in Fig. 4F. Mann–Whitney U-test. Data are means ± SD. H Left panel: Pie charts depicting quantification of tumour incidence upon in vitro treatment of SUM149PT cells as in Fig. 4F. Right panel: Table summarizing the frequency of tumour initiating cells (TICs) and respective statistical analysis. Chi-square test.

Fig 3: Developmental expression and function of P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in early and mid-gestation primary human fetal brain endothelial cells (hfBECs). (A) Representative images of western blots (B) densitometric analysis of total P-gp and (C) BCRP protein comparing early and mid-gestation hfBECs. ERK2 is used as loading control. (D) Relative ABCB1 (encoding P-gp) and (E) ABCG2 (encoding BCRP) mRNA expression in early and mid-gestation hfBECs. Data are normalized by the geometric mean of TOP1 and YWHAZ (reference genes), (F) calcein-AM (P-gp substrate) and (G) Ce6 (BCRP substrate) accumulation in early and mid-gestation hfBECs. Data are expressed as mean ± SD. n = 6/gp. Statistical differences were conducted using an unpaired t-test. * p = 0.05.

Fig 4: Proliferation and apoptosis analysis of the small intestine.A: Quantification of the total number of cells per crypt. A minimum of 50 crypts were quantitated per animal of each genotype and mean values are shown + SEM. Genotypes were: GSK3a+/+;GSK3ß+/+ (n = 3), KI/KI = GSK3aS21A/S21A;GSK3ßS9A/S9A (n = 3). No statistically significant difference was observed using the Student’s t-test. B: Immunohistochemical staining of WT/WT and KI/KI small intestinal crypts using antibodies for BrdU or phospho-Histone H3. Scale bars, 20µm. C: Quantification of BrdU-positive cells per crypt within the WT/WT and KI/KI small intestine. Staining in 50 crypts of three mice of each genotype was quantified. No statistically significant difference was observed using the Student’s t-test. D: The position of BrdU-positive cells within the WT/WT and KI/KI crypts was quantified. Position 0 represents the base of the crypt. A minimum of 50 crypts were quantified in three mice of each genotype. No statistically significant difference was observed using the Student’s t-test. E: Immunohistochemical staining of WT/WT and KI/KI villi using an antibody for cleaved caspase 3. Scale bars, 50µm. F: Western blot analysis of small intestinal protein samples from WT/WT or KI/KI mice using antibodies for phosphorylated GSK3a/ß, cleaved PARP or cleaved caspase-3. Immunoblotting for ERK2 and GAPDH was used to determine equal protein loading.

Fig 5: (A) Pharmacological inhibition of the AKT/PKB and ERK pathways reduces eIF4E phosphorylation and translation of the target mRNA of the eIF4F complex in cervical cancer cell lines. (A) Steady-state levels of the phosphorylated forms of eIF4E (Ser-209), ERK (Tyr-204), and AKT (Ser-473) were evaluated in C33A (HPV negative), CasKi (HPV16), and HeLa (HPV18) cell lines by immunoblot assays. Caski and HeLa cells were treated with the indicated concentrations of PD184352 (B), Palomid-529, and Ribavirin (C) over 48 h, while control cells (CT) were incubated with DMSO. Cell lysates were resolved by SDS/PAGE and immunoblotted with antibodies against the phosphorylated form of eIF4E (Ser-209), ERK (Tyr-204), AKT (Ser-473), 4EBP1 (Thr-70), and total levels of CCND1 and ODC1 as indicated. (D) Real-time quantitative PCR of eIF4E, CCND1, and ODC1 was performed in Caski and HeLa cells treated with Ribavirin (Rb) and Palomid 529 (P529) for 48 h. Values represent mean ± SD of at least three experiments. GAPDH was employed as a loading control for qRT–PCR and western blot assays. Differences between groups were compared by one-way ANOVA with Dunnett’s post hoc test (*P > 0.05; ns: no significance).