ASK1 expression is directly repressed by Blimp-1. (A) RT-QPCR analysis of mRNA levels of Blimp-1 and ASK1 in mature B cells isolated from peripheral human blood mononuclear cells (PBMC-CD19⁺) and plasma cells isolated from bone marrow aspirates from patients with MM (MMBM-CD138⁺). Results were compared with the internal control GAPDH mRNA and further normalized to PBMC-CD19⁺. Data are mean ± SD (n = 5). (B) Immunoblotting shows the expression of Blimp-1 and ASK1 in SKW6.4 and H929 cells. Tubulin was the loading control. (C) Knockdown of Blimp-1 increases the expression of ASK1 mRNA. Human MM cell lines IM9, U266, and H929 transduced with a lentiviral vector (Blimp-1i or ctrli) for 4 days were subjected to the analysis of ASK1 mRNA expression by RT-QPCR. GAPDH mRNA was used as the internal control, and the results were normalized to the ctrli sample. Data are mean ± SD (n = 3). (D) Induction of Blimp-1 represses ASK1. Blimp-1 was induced by treatment with CdCl₂ (5μM) and 4-OHT (3μM) in WI-IL2Blimp1 fused with estrogen ligand binding domain stable transfectants for 24 hours. Induced or uninduced cells were used to prepare RNA (RT-QPCR analysis) and nuclear extract and total-cell lysates (immunoblot analysis). RT-QPCR results were compared with the uninduced samples. Data are mean ± SD (n = 3). (E) Chromatin from H929 cells was immunoprecipitated with anti–Blimp-1 or rabbit IgG (control), and the precipitated chromatin samples were then amplified by QPCR with the use of primer sets spanning the 2 putative Blimp-1 binding sites (ASK1 site 1 and site 2) in the ASK1 promoter, the known Blimp-1 site in the CIITA promoter, and other control loci. Results were normalized to input DNA. (F) Blimp-1 suppresses ASK1 transcription. Raji cells were transfected by electroporation with the indicated doses of Blimp-1 expression plasmid, RL-tk, for normalization control, and the firefly luciferase reporter constructs driven by −1362 to 100 bp relative to the transcriptional start site of ASK1 containing either a WT or mutated (Mut) Blimp-1 binding site. Cells were harvested after 24 hours and subjected to a luciferase assay. Luciferase was normalized to each luciferase reporter control group that had not been transfected with the Blimp-1 expression vector. Results are mean ± SD from 3 independent experiments. *P < .05, **P < .01, ***P < .001. UTR indicates untranslated region.