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. 2013 Oct 17;4(10):e870. doi: 10.1038/cddis.2013.403

Figure 4.

Figure 4

Calumenin (Calu)-15 promotes the transcription of growth differentiation factor-15 (GDF-15). (a) Quantitative real-time PCR analysis of the relative mRNA expression levels of the indicated genes in the HeLa cells stably expressing Calu-15–EGFP (n=3). ABHD2, α-/β-hydrolase domain containing protein 2; NELL2, neural epidermal growth factor-like 2; ITPR1, inositol 1,4,5-triphosphate receptor, type 1; PML, promyelocytic leukemia; EGFP, as a control. Data are mean±S.E.M.; ***P<0.001 (unpaired two-tailed Student's t-test). (b) Lysates of HEK293T cells expressing Calu-15–2 × EGFP or 2 × EGFP were immunoblotted (western blotting (WB)) with the indicated antibodies. Right panel, the graph shows the relative protein level of GDF-15 (n=4). Data are mean±S.E.M.; ***P<0.001 (unpaired two-tailed Student's t-test). (c) Analysis of GDF-15 promoter activity by dual-luciferase reporter assay. The left panel shows the schematic structures of plasmids with different lengths of GDF-15 promoter followed by luciferase gene. The right graph indicates the relative luciferase activity in Calu-15–EGFP-overexpressing HeLa cells transfected with the indicated plasmids (n=4). EGFP, as a control. Data are mean±S.E.M.; **P<0.01, ***P<0.001 (unpaired two-tailed Student's t-test). (d) Chromatin immunoprecipitation (ChIP) assay of HeLa cells expressing Calu-15–EGFP or EGFP. (e) Quantification of the GDF-15 promoter activity in HeLa cells expressing Calu-15–2 × EGFP wild type (WT), T73A, or T73E (n=3). Data are mean±S.E.M.; *P<0.05 (unpaired two-tailed Student's t-test)