Fig 2: MAZ is required for MYB induction during the exit from quiescence. (A) Experimental timeline. The proliferation of isolated primary PBLs was induced with a combination of leucoagglutinin and IL2 and maintained by the presence of IL2 during 12 days. Cells were then made quiescent by IL2 starvation and induced to re-enter the cell cycle 48 h later by adding leucoagglutinin and IL2. (B) Immunoblot analysis of p130 phospho-S672; p130 total; a combination of pRB phospho-S780, phospho-S795 and phospho-S807/811; pRB total; and TFIIH (loading control) in nuclear extracts from these cells. (C) RT-qPCR analysis of MYB, MAZ and E2F1 mRNA expression in lymphocytes stimulated for the indicated periods of time to re-enter the cell cycle. mRNA amounts were normalized to GUSB expression. Data are shown as means ± SEM (n = 3) relative to 0 h. *P < 0.05, **P < 0.01, ***P < 0.001 versus 0 h; two-way ANOVA with Bonferroni post-test. (D) Immunoblot analysis of phospho-MAZ (S460) and MAZ total levels (employing the MAZ-123 antibody) in nuclear extracts from these cells. (E) Experimental timeline as in (A). Proliferating lymphocytes were transduced with lentivirus expressing GFP and either a control shRNA or MAZ-specific shRNAs sh59 or sh60 2 days before IL2 starvation. (F and G) Lymphocytes transduced with lentivirus expressing shRNAs as indicated were forced to become quiescent by IL2 starvation for 48 h. (E) MAZ knockdown was confirmed by RT-qPCR analysis at this time. mRNA amounts were normalized to GUSB expression (means ± SEM, n = 3). ***P < 0.001, ****P < 0.0001 versus Control; one-way ANOVA with Bonferroni post-test. (F) Transduced, quiescent cells were treated with leucoagglutinin plus IL2 for 1 h to re-enter the cell cycle (Activated) or left untreated (Resting). MYB mRNA levels were determined by RT-qPCR analysis and normalized to GUSB expression (means ± SEM, n = 3). ***P < 0.001 versus Control Activated; ####P < 0.0001 versus Control Resting; two-way ANOVA with Bonferroni post-test.

Fig 3: The combination of AKT and CDK inhibitors is effective against HCC cells(A) HepG2 cells were treated with palbociclib (PB) (10 µM) or MK-2206 (MK) (5 µM) as single agents or with the combination of the two (PB 5 µM + MK 5 µM). A fourth group of cells received no treatment (NT). Viability and apoptosis were evaluated 72 h after the beginning of treatment. Data are represented as mean ± SD. *p value =0.05; **p value =0.01; ***p value =0.001; ****p value =0.0001. (B) Western blot analysis and quantification of RB1, AKT proteins and their phosphorylated forms in HepG2-treated cells. The values are normalized on the GAPDH protein levels. (C) Tumor nodule volumes in DEN-treated TG221 mice of the following groups: (1) palbociclib (PB) (n = 6); (2) MK-2206 (MK) (n = 6); (3) palbociclib + MK-2206 (PB + MK) (n = 5); (4) vehicle (CTRL) (n = 13). Experimental therapies started at 6 months, when all mice presented one or more tumor nodules in their livers. Single tumor nodules were monitored by ultrasound at the beginning and end of treatment. (D) Mice weight was monitored during treatment to check drug toxicity. Data are represented as mean ± SD.

Fig 4: IFN? activates STAT3, but does not inhibit cyclin E expression or pRb phosphorylation at S795, in the absence of STAT1.STAT1-KO NSPCs were treated with IFN? (100U/ml) and collected on DIV 2, 3, and 5 for western blot analysis. Representative blots for phosphorylated and total STAT3 A: cyclins D1, D2, D3, E and cdk2, B: and for total pRb and associated pRb phosphorylation sites, and C: are shown. Signal intensity for each band was normalized to GAPDH as a loading control. Quantitation of signal intensity is shown as the average from 3 independent biological replicates with SEM. Statistical analysis was applied using repeated measures one-way ANOVA with Bonferroni multiple comparisons post-hoc analysis (*p < 0.5, ****p < 0.0001).

Fig 5: AKT1 knockout clones exhibit impaired senescence and SASP production (A). Beta-galactosidase staining of 1 µM Palbociclib (PALBO) or DMSO-treated clonal cell lines for 7 days, quantified in (B). (C–E). Induction of IL1, IL6, and IL8 in clonal cell lines by RT-qPCR after 7 days of treatment with PALBO at 1 µM or DMSO as a control (F). Representative Western blot of cells treated with PALBO or DMSO for 7 days for individual and total AKT phosphorylation. (G) Representative flow cytometry histograms of PI-stained cells for cell cycle analysis treated with PALBO or DMSO for 24 h. (H) Percentage of cells arrested in G1 phase after 24 or 48 h of PALBO treatment, quantified by flow cytometry after PI staining. (I) Representative Western blot of cells treated with PALBO after 7 days for retinoblastoma proteins phosphorylated on residue Ser780 (pRb) vs. total retinoblastoma protein (Rb). (J). Representative flow cytometry plots of CFSE-stained cells to evaluate proliferation, after treatment with DMSO and PALBO for 7 days, or PALBO for 7 days with subsequent drug removal for 72 h prior to analysis, quantified in (K). Error bars represent SEM. Significance determined by Student’s t-test, or two-way ANOVA with multiple comparisons (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, n.s. = no significance). The uncropped blots are shown in Supplementary Materials.