Application: WB
Species: human
Sample: PASMC
Figure 6. PIM1 (proviral integration site for Moloney murine leukemia virus 1)transcription was regulated by MrgD (Mas-related G-protein–coupled receptor member D) through AKT (protein kinase B)-mediated MAZ (MYC-associated Zinc-finger protein) binding. A, The Genecards, JASPAR, and PROMO databases and single-cell sequencing (SCS) were analyzed using a Venn diagram, and 1 candidate transcription factor, MAZ, was confirmed. B, Analysis of MAZ protein expression by Western blotting in human pulmonary artery smooth muscle cells (PASMCs) overexpressing MrgD (n=6 independent experiments). C, Analysis of MAZ mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) in human PASMCs overexpressing MrgD (n=6 independent experiments). D, Analysis of PIM1 protein expression by Western blotting in human PASMCs with MrgD or MAZ knockdown under hypoxic conditions (n=6 independent experiments). E, Analysis of PIM1 mRNA expression by RT-PCR in human PASMCs with MAZ knockdown (n=6 independent experiments). F, Luciferase activity was measured in HEK293T cells (human embryonic kidney 293 cells expressing the SV40 large T-antigen) following infection with either MAZ or PIM1 (n=6 independent experiments). G, The luciferase activity after different promoter deletions of PIM1 in HEK293T cells infected with pcDNA3.1 or MAZ was measured (n=6 independent experiments). H, Chromatin immunoprecipitation (ChIP) assay to determine MAZ binding sites to PIM1 promoters in human PASMCs (n=6 independent experiments). I, Analysis of AKT, AMPK (AMP-activated protein kinase), and PKA (protein kinase A) phosphorylation protein expression by Western blotting in human PASMCs with MrgD knockdown (n=6 independent experiments). J, Analysis of MAZ by Western blotting in human PASMCs treated with MK-2206 2HCI (10 μmol/L), metformin (10 mmol/L), or 8-bromo-cAMP (0.1 mmol/L; n=5 independent experiments). K, Analysis of AKT phosphorylation and MAZ protein expression by Western blotting in human PASMCs with MrgD knockdown and treatment with MK-2206 2HCI (10 μmol/L; n=6–8 independent experiments). L, Schematic representation of the role of MrgD in pulmonary hypertension (PH). MrgD deficiency upregulates MAZ through AKT phosphorylation, which promotes MAZ binding to the PIM1 promoter, induces the proliferation of PASMCs and collagen production, and results in PH. All quantitative data are represented as mean±SEM. Unpaired Student t test (E), nonparametric Mann-Whitney U test for comparisons (C, I, and J), 2-way ANOVA with Tukey multiple comparison test (B, D, G, and K), and a Welch ANOVA followed by a 2-step linear step-up procedure of Benjamini, Krieger, and Yekutieli (F).
(1)
