Figure 1.
Effects of GW8510 treatment on mouse alpha cells and dissociated human islet cells.
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.
Figure 2.
Involvement of the p53 pathway and quantification of p53 transcriptional activity and expression levels of p53 target genes following GW8510 treatment of alpha cells.
(A) Heat-map display of one of the enriched gene sets (INGA_p53_TARGETS) in gene-expression profiling of alpha cells treated with 3.3 µM GW8510 for five days. Relative expression values in three biological replicates are plotted by color. Red, high expression levels, blue, low expression levels. (B) Reproducibility of gene-expression changes following GW8510 treatment, measured by microarray and qPCR. (C) Cellular p53 activity measured using a dual-luciferase reporter system. Activity of the firefly luciferase p53-reporter construct was normalized to constitutively active co-transfected Renilla luciferase, and to positive and negative controls. (D) qPCR measurement of transcript levels of direct p53 targets following 3- and 5-day treatments with 1.65 µM GW8510. Data represent the mean±SD of at least three experiments; **p<0.01 and ***p<0.001.
Figure 3.
GW8510 treatment effects on protein levels of p53 transcriptional targets and on post-translational modification status of p53 and MDM2.
(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.
Figure 4.
Cell-cycle effects of GW8510 treatment.
(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.
Figure 5.
Evaluation of p53 response elements.
(A) cisRED prediction of p53 response elements in promoter regions of indicated mouse genes. Discovery p-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 Ins2, Cdkn1a and Ccng1. 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.
Figure 6.
Effects of manipulation of p53 levels and activity on induction of Ins2 by GW8510 treatments.
(A) Experimental knockdown and over-expression of p53 in alpha cells and (B) its effects on Ins2 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 Ins2 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.