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. 2009 Oct 23;106(45):19090–19095. doi: 10.1073/pnas.0904849106

Fig. 5.

Fig. 5.

Pdx1 regulates multiple genes involved in maintaining ER homeostasis. (A) Quantitative RT-PCR confirmation of ER-related genes downregulated in Mouse PancChip 6.1 cDNA microarray analyses of AdshPdx1-infected Min6 cells compared with either uninfected or AdshLuc-infected controls. (n = 3; *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001 relative to AdshLuc-infected cells). (B) Upper, Schematic of the genomic sequence 1.5 kb upstream of the mouse Atf4 promoter highlighting two regions of evolutionary conservation with the human sequence and the Pdx1 consensus sites contained within (adapted from rVista 2.0). Lower, Pdx1 directly binds the Atf4 promoter as assessed by chromatin immunoprecipitation (ChIP) from Min6 cells. (C) Pdx1 also occupies the Wfs1 promoter in Min6 cells as assessed by quantitative ChIP, consistent with the high-throughput ChIP on chip data. (D) ChIPs performed on primary islets freshly isolated from 8-week-old mice demonstrate Pdx1 occupancy at the two conserved regions of the Atf4 promoter and the Wfs1 promoter in vivo. Primers designed to amplify a known Pdx1-binding region of the insulin promoter and a piece of the albumin promoter were used as positive and negative controls, respectively. n = 4 individual experiments, data are mean ± s.e.m, *, P < 0.05 relative to IgG control pulldown. (E and F) Pdx1 and Atf4 mRNA (E) and protein (F) levels in Min6 cells transfected with either a pool of four siPdx1 duplexes (siPdx1) or a nontargeting control pool (siNT). n = 3 replicates per condition; **, P < 0.01. In (F), nuclear extracts were analyzed by Western blot, using histone H3 as a loading control. (G) qPCR analysis of Atf4, known Atf4 targets and Wfs1 expression in islets isolated from individual 12-week-old Pdx1+/+ or Pdx1+/− mice. n = 4–6 mice per group; *, P < 0.05; **, P < 0.01.