HSF1 Attenuates LPS-Induced Acute Lung Injury in Mice by Suppressing Macrophage Infiltration

Figure 5

HSF1 downregulated the transcription of MCP-1/CCR2. (a, b, d) The binding of HSF1 to HSE in MCP-1 and CCR2 promoters was assessed using EMSA. (a, b) EMSA was used to detect the binding of the HSF1 protein with HSE1 (a) and HSE2 (b) in the MCP-1 promoter region in vitro. The biotin probe specific for HSE1 or HSE2 was bound by nuclear extracts of RAW 264.7 cells, which could be blocked by an HSF1 antibody. (d) EMSA was used to detect the binding of the HSF1 protein with HSE1 in the CCR2 promoter region in vitro. The biotin probe specific for HSE1 was bound by nuclear extracts, which could be blocked by an HSF1 antibody. Lane 1: negative control; lane 2: biotin-labeled HSE probe preincubated with nucleoproteins (HSF1-HSE complexes); lane 3: supershift analysis using anti-HSF1 antibodies; lane 4: competition using a 200-fold excess of the unlabeled competitive probe; lane 5: competition using a 200-fold excess of the unlabeled mutant probe. The HSF1/HSE complex is indicated by red arrowheads, the supershift band is indicated by blue arrowheads, and unbound HSE probe is indicated by black arrowheads. (c, e) Luciferase reporter assay analysis of HSF1 regulation on the promoter transcription activity of MCP-1 (c) and CCR2 (e) in RAW 264.7 cells transfected with the HSF1 overexpression vector (mHSF1) or empty expression vector pcDNA3.1. After 48 h of transfection, the cell lysate was extracted, and the firefly luciferase activity was measured and normalized to the Renilla luciferase activity. , pcDNA3.1 versus mHSF1 (intragroup comparison); ^#, versus mHSF1 + MCP-1-wt (or mHSF1 + CCR2-wt); ^##, versus mHSF1 + MCP-1-wt (or mHSF1 + CCR2-wt). Data are representative of at least three independent experiments. values were determined using two-tailed Student’s -test for comparing two groups and one-way ANOVA for comparing multiple groups.

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