Evaluation of p53 response elements.

<p>(A) cisRED prediction of p53 response elements in promoter regions of indicated mouse genes. Discovery <i>p</i>-value is plotted against experimentally determined fold change in gene expression. Selected genes are highlighted. (B) ChIP-PCR analysis of predicted p53-response elements in promoter regions of <i>Ins2</i>, <i>Cdkn1a</i> and <i>Ccng1</i>. Cells were either untransfected (“control”) or transfected with recombinant tagged p53 (“p53-HaloTag”), and p53-bound DNA immunoprecipitated. PCR was then performed on Ins2, Cdkn1a, or Ccng1 regions containing predicted p53-response elements. The presence of blocking ligand helps determine the specificity of the interaction. (C) Fold enrichment of promoter binding is calculated over the corresponding blocking ligand control in the p53-HaloTag condition.</p

Effects of manipulation of p53 levels and activity on induction of <i>Ins2</i> by GW8510 treatments.

<p>(A) Experimental knockdown and over-expression of p53 in alpha cells and (B) its effects on <i>Ins2</i> induction by 3-day treatment with 1.65 µM GW8510. (C) Co-treatment with small molecule inhibitors of p53 and upstream targets in the p53 signalling pathway and their effect on <i>Ins2</i> induction following treatment with GW8510. (D) Proposed model for GW8510-mediated induction of insulin expression via activation of p53 transcriptional activity. All data represent the mean±SD of at least three experiments; *p<0.05, **p<0.01 and ***p<0.001.</p

GW8510 treatment effects on protein levels of p53 transcriptional targets and on post-translational modification status of p53 and MDM2.

<p>(A) Western blot analysis and (B) quantification of protein levels of direct p53 targets following a two-day time-course with 1.65 µM GW8510. (C) Western blot analysis and (D) quantification of total protein and post-translational modification levels following 2–5 days of treatment with 1.65 µM GW8510. Data represent the mean ± SD of 3 biological replicates; *p<0.01, **p<0.01 and ***p<0.001.</p

Cell-cycle effects of GW8510 treatment.

<p>(A) FACS-generated histograms of propidium iodide stained cells treated with either vehicle control or GW8510 for 3 days. (B) Quantification of cell-cycle distributions from gated cellular populations in A expressed as percentage of the total cellular population. Data represent the mean ± SD of two biological replicates. (C) and (D) M-phase immunofluorescence analysis and quantification using histone H3 phospho-Ser10 as a mitosis marker. Total cells were counted using Hoechst nuclear stain. Representative images are shown for Hoechst, histone H3 phospho-Ser10, and overlay at indicated GW8510 concentrations. Values are expressed as fold over vehicle-treated controls. Data represent the mean ± SD of at least 3 biological replicates; ***p<0.001.</p

Effects of GW8510 treatment on mouse alpha cells and dissociated human islet cells.

<p>Pancreatic gene expression was measured by quantitative real-time RT-PCR (qPCR) following a (A) 5-day dose-response and (B) time-course with 1.65 µM GW8510. (C) Insulin secretion measurements in dissociated human islets following 5-day compound treatment at indicated concentrations. (D) Immunofluorescence analysis quantification of total cell numbers, measured by nuclear count, and numbers of alpha and beta cells, measured by glucagon and insulin staining, respectively, following compound treatment. (E) Representative images shown. All data represent the mean±SD of at least three experiments; *p<0.05, **p<0.01 and ***p<0.001.</p

Summary of proteomic studies.

<p>*including 467 phosphorylated kinase peptides.</p><p>Total proteome results based on 24 SCX fractions over two SILAC experiments. pSTY peptide results are based on 12 SCX fractions over two SILAC experiments.</p

Longer-term lentiviral knockdown of <i>Brsk1</i> and <i>Camkk2</i> stimulates PDX1 and insulin protein expression in αTC1 cells.

<p>Cells were infected with lentivirus carrying expression cassettes that encode shRNAs against (A) <i>Brsk1</i>, (B) <i>Camkk2</i>, (C) <i>Stk11</i>, and were selected with puromycin for 10 days. <i>Pdx1</i> mRNA expression (white bars) was measured jointly with the expression of each of the shRNA-targeted genes (black bars). Data represent fold change (in log₂ scale), compared to the average of control empty vectors. Data represent mean and standard deviation of four independent experiments. The significance was determined by t-test. *<i>p</i><0.05 and **<i>p</i><0.01. (D–S) Nuclei (blue), insulin (green), and Pdx1 (red) immunofluorescence, and merged images in 10-day shRNA-expressing αTC1 cells: (D–G) control virus, (H–K) sh1_Camkk2, (L–O) sh1_Brsk1, (P–S) sh1_Stk11. Scale bar = 100 µm.</p

Gene-expression analysis of alpha and beta cell lines reveals higher metabolic activity in the alpha cell line.

<p>Gene sets with increased expression in (A) alpha or (B) beta cell lines were identified by performing gene-set enrichment analysis (GSEA) on gene-expression profiling data, resulting in an enrichment score profile for each gene set (green line). Individual members of each gene set (vertical black bars) are enriched in either alpha cells (blue) or beta cells (red). To validate the predicted differences in cellular respiration between alpha and beta cells, we determined (C) extracellular acidification rate (ECAR) and (D) oxygen consumption rate (OCR) of alpha cells (red), BRD7389-treated alpha cells (black), βTC3 cells (blue), and INS-1E cells (brown). Glucose (Glu), oligomycin (Oligo), 2-deoxyglucose (DOG), CCCP, and rotenone/antimycin A (Rot/Ant) were added at the indicated times. Data represent the average and standard deviation of 18 biological replicates.</p

Knock-down of <i>Brsk1</i> and <i>Camkk2</i> elicit the expression of key beta cell-specific genes in αTC1 cells.

<p>mRNA expression of beta and alpha cell-specific genes was measured using qPCR after knock-down of (A) <i>Brsk1</i> or (B) <i>Camkk2</i>. Fold changes were calculated compared to untransfected control αTC1 cells, and normalized to <i>Gapdh</i> expression. Significance was determined by t-test. *<i>p</i><0.05, ***<i>p</i><0.005. (C) αTC1 cells transfected with the indicated siRNA, or treated with the indicated compounds, were analyzed for Pdx1 protein expression by immunofluorescence. The percentage of Pdx1⁺ cells was calculated for each treatment. The significance was determined by t-test. *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001.</p