Figure 1. Satins upregulates the expression of Jmjd3 in macrophages through NF-κB.

(A) Statins activate NF-κB pathways in RAW 264.7 cells. (a) Heatmap of differentially expressed genes with or without statin (lovastatin, 7 + 200 µM mevalonate; 2 days). (b) Pathways identified by gene set enrichment analysis (GSEA) of differentially expressed genes in (a). (c) The details of most highly represented pathway, TNFA signaling vis NF-κB. (B) Methyl-β-cyclodextrin (MCD) activate NF-κB pathways in RAW 264.7 cells. (a) Heatmap of differentially expressed genes with or without MCD (5 mM, 1 hr). (b) Pathways identified by GSEA of differentially expressed genes in (a). (c) The details of most highly represented pathway, TNFA signaling vis NF-κB. (C) Statins and MCD upregulate Jmjd3 in RAW 264.7 cells. (a) Reverse transcriptase quantitative PCR (RT-qPCR) of genes with or without MCD (5 mM; 1 hr). (b) Jmjd3 gene expression in MCD- or statin-treated RAW 264.7 macrophages. (c) Effect of NF-κB inhibitors, MG-132 (5 µM) and BAY11-7082 (10 µM) on Jmjd3 expression in MCD-treated RAW macrophages. (d) Western blotting of JMJD3 protein expression and (e) levels of H3K27Me3 in macrophages treated with 5 mM MCD (1 hr). The pCREB was used as internal control for cholesterol depletion and actin a loading control. Original blots are in source data. (D) Statin/MCD upregulates Jmjd3 in bone marrow-derived macrophages (BMDMs). (a) Jmjd3 gene expression in statin-treated BMDMs (10 µM pravastatin + 200 µM mevalonate; 2 days). (b) Effect of NF-κB inhibitors, MG-132 [5 µM] and BAY11-7082 [10 µM], on Jmjd3 expression in MCD-treated BMDMs. (c) Jmjd3 expression in cholesterol repletes MCD-treated macrophages. Graphs are representative of 3 independent experiments with 3 replicates per condition and are presented as means ± standard deviation (SD). Statistical analysis was performed using unpaired, two-tailed Student’s t-test. An asterisk (*) or double asterisks (**) indicate a significant difference with p < 0.05 and p < 0.001, respectively. A hashtag (#) indicates a significant difference between MCD without or with inhibitors, p < 0.05.

Figure 1—figure supplement 1. Cellular cholesterol contents regulate NF-κB pathway.

RAW macrophages were treated with methyl-β-cyclodextrin (MCD) or statins, and RNA-seq was performed as in Figure 1. TF regulatory pathways of activated genes were analyzed using Metascape; top TFs employed by either lovastatin (A) (7 + 200 µM mevalonate) or MCD (B) (5 mM, 1 hr). The genes downregulated by statin is analyzed in (C). No TF is identified in MCD downregulated genes.

Figure 1—figure supplement 2. The expression of Jmjd3 activated by methyl-β-cyclodextrin (MCD) requires NF-κB activity.

Effect of NF-κB inhibitors, JSH23 (10 µM) and SC514 (10 µM), on Jmjd3 expression in MCD-treated RAW macrophages. Graphs are representative of 3 independent experiments with 3 replicates per condition and are presented as means ± standard deviation (SD). Statistical analysis was performed using unpaired, two-tailed Student’s t-test. Double asterisks (**) indicates a significant difference p<0.001.

Figure 1—figure supplement 3. Levels of H3K27Me3 are decreased in macrophages treated with statin or methyl-β-cyclodextrin (MCD) (lovastatin, 7 + 200 µM mevalonate; 2 days) or MCD (5 mM, 1 hr).

The pCREB was used as internal control for cholesterol depletion and actin a loading control. Original blots are in source data.