Fig 2: Pharmacological inhibition of the insulin and type I receptors induces cell death a SNU-1 were cultured in suspension, whereas MKN74 and NUGC3 were plated onto coverslips, in untreated FCS-containing medium and the indicated concentration of BMS-754807 for three days. Cells were assayed for histone H3 Ser 10 phosphorylation (khaki arrows) by immunofluorescence and counterstained with DAPI. Asterisks indicate that the proportion of cells in the mitotic-phase of the cell cycle is significantly lower after incubation with BMS-754807 (one-way ANOVA; SNU-1, p = 0.0009; MKN74, p = 0.0046; NUGC3, p = 0.0023). NS indicates data that are not significantly different. Pale pink arrows indicate apoptotic cells. b SNU-1 were incubated in untreated FCS-containing with BMS-754807 (BMS) for three or six days. Cleaved caspase-3 or cleaved PARP were analysed by immunofluorescence (pink arrows). Asterisks indicate that the proportion of cells with detectable cleaved PARP is statistically significantly different after incubation with BMS-754807 (one-way ANOVA; p < 0.01). c Metastatic adenocarcinoma cells were isolated from patients and incubated in 20% untreated FCS-containing medium and different concentrations of BMS-754807 for three days after which cleaved PARP and GAPDH were analysed by western transfer. The protein bands were quantified by densitometry and corrected for GAPDH. Asterisks indicate concentrations of BMS-754807 at which there was statistically significantly more cleaved PARP in its presence than in its absence (one-way ANOVA; p < 0.001). d Cells were incubated in full, untreated FCS-containing medium with BMS-754807 for three days and phosphorylated (Phospho.) insulin Tyr 1150/1151 and type I IGF Tyr 1135/1136 receptors (IGF receptors), Akt Ser 473 and cleaved PARP analysed as above, quantified and corrected for corresponding total protein or GAPDH, respectively. Representative images are shown as inserts. Asterisks indicate concentrations of BMS-754807 at which there was statistically significantly less phosphorylated protein, or more cleaved PARP than in its absence (one-way ANOVA; p < 0.001). e SNU-1 and NUGC3 were incubated in serum-free medium for two hours and then in serum-free medium and the indicated concentrations of BMS-754807 in the absence or presence of ligand (lig.) for 15 min. Phosphorylated (Phospho.) insulin Tyr 1150/1151 and type I IGF Tyr 1135/1136 receptors (IGF receptors), Akt Ser 473 or GAPDH were measured. Total corresponding protein is shown underneath the phosphorylated protein images

Fig 3: Depleting PTOV1 inhibits migration and invasion and promotes cell apoptosis. a and b Representative images (a) and quantification (b) of cell migration the indicated cells analyzed by wound healing assay. c and d Representative images (c) and quantification (d) of invade cells analyzed by chamber invasion assay. e and f Representative images (e) and quantification (f) of cell apoptosis of the indicated cells treated with or without drugs analyzed by Annexin V/PI staining. g Western blotting analysis of Caspase-3 (Casp-3), Cleaved Casp-3 and Bcl-2. GAPDH served as a loading control. Error bars represent mean ± SD from least 3 independent experiments. *, P < 0.05; #, P < 0.01; $, P < 0.001, two-tailed, unpaired t-test

Fig 4: SAHA and HCI-2509 combination altered cell cycle progression, induced apoptosis and inhibited migration capacity in vitro(A) Distribution of cell cycle phases in TC32 and CADO-ES cell lines after 24 h of SAHA, HCI-2509 or combination treatment by flow cytometry. Percentages of the most affected phase are depicted in each condition. (B) Apoptosis induction analysis of population positive for cleaved Caspase 3 in TC32 and CADO-ES cell lines after 24h and 48h of SAHA, HCI-2509, alone or in combination, at different concentrations. (C) Migratory capacity analysis of transwell migration assay after 48 h of pre-treated TC32 and CADO-ES cell lines. Percentage of migratory cells is shown for each drug treatment respect to the control (DMSO) in both cell lines. All values show mean ± s.d. of three biological independent replicates. Statistical tests: significant analysis of variance, Tukey post-hoc test <0.001 (***), 0.01 (**) and 0.05 (*).

Fig 5: WEE1i-ATRi combination increases M-phase entry, replication fork instability, and apoptosis in CCNE1Amp cancer cells(A) Detection of ?H2AX-positive cells in S phase of WEE1i-ATRi-treated CCNE1Amp, CCNE1Gain, and CCNE1Low cells. Cells were treated with DMSO (control) or 200 nM WEE1i, 1 µM ATRi, or both for 8 h and then fixed and stained with ?H2AX and PI for flow cytometry. ?H2AX-positive cells in S phase were quantified. Representative images of OVCAR3 (left) and quantified data (right) are shown (both versus WEE1i monotherapy: OVCAR3, p < 0.0001; KLE, p < 0.0001; OVCAR8, p < 0.0001; MFE280, p = 0.8151; WO-20, p = 0.9997; OVKATE, p = 0.8630; SNU685, p = 0.2171; n = 3; mean ± SD).(B) Representative immunoblots for ?H2AX in OVCAR3 (left) and KLE (right) cells after they were treated as in (A) for the indicated time. Actin is loading control.(C) Quantification of pRPA32(S33) by flow cytometry of the indicated cells after treatment similar to (A) after 24 h (n = 3; mean ± SD; WEE1i versus both shown by an asterisk).(D) Flow cytometry quantification of apoptotic cells by Annexin V-APC (allophycocyanin) and propidium iodide (PI) staining of the indicated cells after treatment similar to (A) for 48 h (n = 3; mean ± SD; WEE1i versus both: MFE280, p = 0.0022; OVKATE, p = 0.0084; OVCAR3, p = 0.0005; OVSAHO, p = 0.0001; WO-20, p = 0.0002; SNU685, p < 0.0001).(E) Representative immunoblots for cleaved caspase-3 in OVCAR3 (upper) and KLE (lower) treated for the indicated time as in (A).(F) Experimental design for replication fork analysis. OVCAR3 and KLE cells were pretreated with a drug as in (A) for 30 min and pulse labeled with 5-chloro-2'-deoxyuridine (CIdU) (red) followed by 5-iodo-2'-deoxyuridine (IdU) (green) for 15 min each in the continuous presence of inhibitors.(G) Quantification of replication fork speed (length of track/duration of both pulses). At least 200 intact, unidirectional tracks were counted for each condition (mean ± SEM; WEE1i versus both shown by an asterisk).(H) Quantification of fork asymmetry as calculated by long green length/short green length replication initiation tracks. At least 130 intact initiation tracks were counted for each condition (mean ± SEM; WEE1i versus both shown by an asterisk).(I) Quantification of inter-origin distance (IOD) for firing calculated by the distance between two nearby origins on the same fiber. At least 100 intact tracks were counted per condition (mean ± SEM; WEE1i versus both shown by an asterisk).Individual samples are presented as data points, and data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. Representative data are shown (A–E) for one of 3 biologically independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.