Fig. 2: The pro-inflammatory phenotype of RRP1-deficient macrophages and the phenotype rescue by RRP1 NOP52 domian.

RT-qPCR detection of IL1B (a), IL6 (b) mRNA levels in THP-1 cells upon RRP1 silencing and IL-1β stimulation for the indicated hours. RT-qPCR detection of IL-1b (c) and Il6 (d) mRNA levels in wild-type (WT) or RRP1 knock out (RRP1 KO) RAW 264.7 cells with IL-1β stimulation for the indicated hours. e RT-qPCR detection of Tnfα mRNA levels in WT or RRP1 KO RAW 264.7 cells with murine TNFα (10 ng/ml) stimulation for the indicated hours. f Cell death ratio of WT or RRP1 KO RAW 264.7 cells treated with TCZ. TCZ: Combination of murine TNFα (20 ng/ml), cycloheximide (CHX) (10 μg/ml) and Z-IETD-FMK (2 μM). See methods. Western blotting of the phosphorylation (p-)of the key inflammatory signal pathway molecules in WT and RRP1 KO RAW 264.7 cells respectively stimulated by murine IL-1β (50 ng/ml) (g), TNFα (10 ng/ml) (h), IL-6 (20 ng/ml) (i) for indicated hours. GAPDH serves as loading control. j Western blot (left) and quantitative analysis (right) of the Flag tagged full-length RRP1 (FL), Nop52 and C terminal (Cter) expression in RRP1 KO RAW 264.7 cells. Empty vector (Vector) was transfected as a control. The Flag band intensities were normalized to GAPDH. RT-qPCR detection of IL-1b (k), Il6 (l) mRNAs after over-expressing the Flag tagged full-length RRP1 (FL), Nop52 (Nop52) and C terminal (Cter) in RRP1 KO RAW 264.7 cells followed by IL-1β stimulation. WT and the RRP1 KO cells transfected with empty vector (Vector) were used as controls. Western blotting data are representative of three independent experiments. RT-qPCR and Western bloting quantitative data are presented as means ± SD of (a–e, j right, k, l, n = 3) biologically replicates from three independent experiments with student’s t test (two-tailed unpaired). Cell death data are presented as means ± SD of (n = 4) biologically replicates from three independent experiments with ANOVA-test (two way). Source data are provided as a Source Data file.