HDAC11 regulates CCL2 expression by recruiting PU.1.

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HDAC11 regulates CCL2 expression by recruiting PU.1. HDAC11 regulates CCL2 expression by recruiting PU.1. (A) Real-time RT–PCR analysis of CCL2 mRNA expression in NIH 3T3 cells transfected with plasmids expressing PU.1, HDAC11, or an empty vector. GAPDH served as an internal control. (B) 293T cells were cotransfected with Flag-tagged vector, HDAC11 WT or mutant HDAC11 H143A, and PU.1 plasmids. Flag antibody immunoprecipitation (IP) and PU.1 antibody IB were performed to determine whether PU.1 interacts with mutant HDAC11. Flag-tagged HDAC11 H143A plasmid was generated from Flag-tagged HDAC11 using the Quikchange II XL site-directed mutagenesis kit (Agilent Technologies). (C) Real-time RT–PCR analysis of CCL2 mRNA expression in NIH 3T3 cells transfected with empty vector, or plasmids expression HDAC11 WT or HDAC11 H143A. GAPDH served as an internal control. (D) Schematic representation of luciferase (luc) reporter plasmids for various portions of the CCL2 promoter. (E) NIH 3T3 cells were transfected with indicated expression and luc reporter plasmids. Luciferase activity was measured in extracts 48 h after transfection. Data are expressed as mean ± SD of triplicates and are representative of at least two independent experiments. (F) ChIP analysis of PU.1 enrichment at the CCL2 promoter upon deletion of HDAC11. PEMs isolated from HDAC11 KO and WT mice were subjected to ChIP assays with the indicated antibodies. Data were normalized to input samples for the amount of chromatin. Normal IgG was used as negative control. (G, H) U251 cells with stable knockdown of HDAC11 expression (shHDAC11) or control were stimulated with MOG35–55 peptide, then transfected with PU.1 plasmid (PU.1) or empty vector (vector). 48 h later, real-time RT–PCR analysis of CCL2 mRNA expression, and luciferase reporter assays of CCL2 promoter (CCL2-P-322) activity were performed. Immunoblots show stable expression of HDAC11 in U251 cells. All data represent mean ± SD from three experiments, and P-values were calculated by t test, *P < 0.05. Lei Sun et al. LSA 2018;1:e201800039 © 2018 Sun et al.