Fig. 1: PARP9 promotes type I IFN production in human innate immune cells in response to poly I:C stimulation or RNA viral infection.

The RT-qPCR (a) and immunoblot (IB, b) analysis of PARP9 or MAVS at mRNA (a, n = 3 per group) and protein (b) levels in THP-1 macrophages treated with shRNA to knockdown expression of PARP9 (sequence P9-a and P9-b) or MAVS. A scrambled shRNA served as a control (sh-Ctrl). The β-actin served as the loading control. ELISA of IFN-α (c) IFN-β (d), and IL-6 (e) production from THP-1 macrophages treated with the indicated shRNA after a 10 h stimulation with 0.5 μg/ml of long poly I:C delivered by Lipofectamine 3000 (n = 3 per group). f Human myeloid cells and lymphoid cells were purified from PBMCs using a cell sorter. Total RNA was isolated from these primary cells induced or not to chip hybridization and microarray. The profile of PARP9 expression in different cells is indicated. The relative expression of PARP9 was compared by plotting the values extracted from the gene expression database. A value <1 indicated the absence of gene expression. g Immunoblot (IB) analysis of PARP9 and β-actin in plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), and pDCs and mDCs induced with IFN-α (100 U/ml) for 2 h. The RT-qPCR (h, n = 3 per group) and immunoblot (i) analysis of PARP9 or MAVS at mRNA (h) and protein (i) levels in human monocyte-derived dendritic cells (MDDC) treated with shRNA to knockdown expression of PARP9 (sequence P9-b) or MAVS. A scrambled shRNA served as a control (sh-Ctrl). ELISA of IFN-α (j) and IFN-β (k) production from human MDDC treated with the indicated shRNA after a 10 h stimulation with 0.5 μg/ml of long poly I:C (LPIC), dsDNA from HSV-1 virus (HSV-60, 2.5 μg/ml) or cGAMP (1.0 μg/ml) delivered by Lipofectamine 3000 or 12 h infection with Reovirus (Reo) at an MOI of 5 (n = 3 per group). l Immunoblot analysis of PARP9 expression dynamics in human MDDC treated with IFN-α (500 U/ml) for 0 h, 8 h, 16 h and 24 h. m ELISA of IFN-β production dynamics in human MDDC treated with the indicated shRNA after stimulation with 0.5 μg/ml of LPIC for indicated time (n = 3 per group). Each circle represents an individual independent experiment and small solid black lines indicate the average of triplicates for results in c–e, j, k and m. Mock, scrambled shRNA-treated cells without stimulation. Error bars indicate standard error of the mean for results in a, h. NS, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, and p value was calculated by unpaired two-tailed Student’s t test. Data are from one experiment with duplicate (f) or representative of three independent experiments (a–e, j–m). Exact p values (a, p = 0.000026, p = 0.378, p < 0.000001, p = 0.276, p = 0.929, p = 0.000028; c p = 0.0019, p = 0.00099, p = 0.00077; d p = 0.0011, p = 0.00057, p = 0.00035; e, p = 0.063, p = 0.00018; h p < 0.000001, p = 0.924, p = 0.346, p < 0.000001; j p = 0.00014, p = 0.000095, p = 0.000053, p = 0.000024, p = 0.826, p = 0.73; k p = 0.00025, p = 0.00013, p = 0.00018, p = 0.00019, p = 0.65, p = 0.65; m p = 0.492, p = 0.0066, p = 0.00033, p = 0.00009).