Fig 2: In PDA cells, E47 directed an integrated program of gene expression that controlled cell-cycle progression. (A–E) KEGG cell-cycle pathway reflecting E47-dependent alterations in expression of cell-cycle control genes in each cell line based on RNA-seq data. (F) Western blot analysis after induction of E47 showed increased CDKN1A/p21CIP1 protein. (G) Western blot analysis after induction of E47 showed increased CDKN1B/p27KIP1. (H) shRNA knockdown of CDKN1B/p27KIP1 in PANC-1/E47 cells resulted in an increased number of Ki67-positive cells after induction of nuclear E47. Data represent the mean of 6 separate fields, each with a minimum of 100 cells analyzed by an unpaired t test. Error bars are ± SEM. (I) Left: Western blot analysis showed increased p53-pSER15 in response to 2 × 1610 cGy of γ-irradiation (20 min of exposure on 2 consecutive days using a Gammacell40 Exactor, Research Irradiator with cesium 137 radiation source) to induce the DDR. Cells were harvested 3 hours after the second exposure. Right: Western blot analysis showed no increase in p53-pSER15 in response to induction of E47. ∗∗P ≤ .01. ∗∗∗∗P ≤ .0001.

Fig 3: E47 differentially regulates proteosomal degradation of MYC and CDKN1B/p27KIP1. (A) MYC protein levels decrease after 72 hours of induction of E47 in all 5 cell lines. (B) Western blot of whole-cell extracts after 72 hours ± tamoxifen and 6 hours of treatment with 10 μmol/L MG132. The numbers below MYC and CDKN1B/p27KIP1 represent the ratio of (+) MG132 to (-) MG132 after normalization of each band to the vinculin loading control. (C) shRNA RB knockdown in BxPC-3/E47 cells does not affect MYC expression when compared with the effect of control vector. (D) Western blot analysis of whole-cell extracts after 72 hours ± tamoxifen and 72 hours of treatment with 1 μmol/L PD033291. The effect of PD33291 on cell growth is reflected by the difference in cell number with treatment. Data were analyzed using an unpaired t test. Error bars are ± SEM of 3 independent biological replicates. (E) Western blot analysis of whole-cell extracts from PANC-1/E47 cells after transduction of lentivirus expressing either wild-type (WT) MYC or T58A degradation-resistant MYC. (F) PANC-1/E47 were transduced with either WT MYC or T58A MYC and treated with tamoxifen for 72 hours to induce E47. After treatment, cells were immunostained with Ki67 antibody and counted. The data represent the percentage of KI67-positive cells. Six replicate wells, each with a minimum of 200 cells, were counted and analyzed by an unpaired t test. Error bars are ± SEM. ∗P ≤ .05, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001.

Fig 4: RB is required for E47-mediated cell-cycle arrest. (A) Transcripts encoding E2F target genes required for DNA replication are decreased. (B) Western blot analysis showed that phosphorylated RB (pSER807/811 and pSER608) decreased in response to E47. (C) Western blot analysis showing RB knockdown in BxPC-3/E47 cells. (D) BxPC-3 cells immunostained with Ki67 antibody after RB knockdown and induction of E47. Statistically significant increase in Ki67(+) cell numbers after knockdown of RB and induction of E47. Data represent the mean of 3 independent biological replicates analyzed by unpaired t test. Error bars are ± SEM. (E) Western blot analysis of BxPC-3/E47 whole-cell extracts from cells harboring shRB-1 that targets the human RB gene (panel B) after transduction with a second virus expressing hemagglutinin-tagged mouse Rb under control of the MSCV promoter. (F) Immunostaining of the BxPC-3 cells expressing shRB-1 that targets human RB and ± expression of MSCV-driven hemagglutinin-tagged mouse Rb after 72 hours of induction of E47. Cells were immunostained for Ki67 and counted. Data represent the mean of 6 separate fields, each with a minimum of 200 cells analyzed by unpaired t test. Error bars are ± SEM. (G) Western blot analysis showing that expression of CDKN1B/p27KIP1 and CDKN1A/p21CIP1 is unchanged after rescue of Rb expression. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001.

Fig 5: PF-573228 promotes GBM cell senescence. (A) Representative SA-β-gal staining from U87-MG and U251-MG cells, control or treated with PF-573228 10 µM for four days. The % of SA-β-gal positive cells significantly increases upon FAK inhibition (*** p < 0.001; n ≥ 3). Bar = 251 µm. (B) p27/CDKN1B and p21/CDKN1A mRNA levels did not change significantly between control cells or cells treated with PF-573228 (10 µM, two or four days; n = 3). (C) p27 protein levels were analyzed in control cells and cells treated PF-573228 for two or four days. β-actin was used as loading control. Quantification of p27 normalized to β-actin indicates that p27 significantly increases after two days of treatment with PF-573228, and after two and four days in U87-MG and U251-MG cells (* p < 0.05; n ≥ 4). (D) SKP2 protein levels were analyzed in control cells or cells treated PF-573228 for two or four days. β-actin was used as loading control. Plot represents SKP2 levels normalized vs. control, which decrease in PF-573228-treated cells (*** p < 0.001; n = 4).