Fig. 3: C19MC-silenced cells and innate immune response.
From: The Chromosome 19 miRNA cluster guards trophoblasts against overacting innate immunity
a Viral resistance in BeWo-dA11 cells. The cells were inoculated with the VSV virus at multiplicity of infection (MOI) of 0.02 and 0.1 for 5 h. The viral load was assessed by RT-qPCR of the viral protein VSV-G. Data shown as mean ± SD. * p < 0.02; ** p < 0.001, n = 3. b A heat map of the ~100 top differentially expressed genes in BeWo-Cas9 vs -dA11, unstimulated (Ctrl) or exposed to 10 μg/ml poly(I:C) overnight. Three libraries per line were sequenced. c Volcano plots depicting differentially expressed genes in BeWo-Cas9 vs -dA11, unstimulated (left panel) or exposed to 10 μg/ml poly(I:C) (right panel). d RT-qPCR analysis for selected ISGs. The bars represent the fold induction by poly(I:C) (10 µg/ml). Data are displayed using a square root scale y-axis to account for the large differences between the cell lines. Data are mean ± SD, n = 3. * p < 0.05, ** p < 0.001, *** p < 0.0005, **** p < 0.0001, ns not significant. e RT-qPCR analysis for interferon-β (IFNB) and interferon-λ (IFNL). Cells were stimulated by 10 µg/ml of poly(I:C). Data are mean ± SD, n = 3, *p < 0.05, ** p < 0.001. f IFNL1 levels in cell culture medium. Cells were stimulated by 10 µg/ml of poly(I:C). The concentration of supernatant cytokines was determined using multiplex MSD ELISA assay. Data are mean ± SD, n = 3. *p < 0.05, **p < 0.005. g The concentration of IFNL1 (left panel) and IFNL2 (right panel) in BeWo-Cas9 and -dA11 supernatants, measured by ELISA after exposure to 10 µg/ml of poly(I:C). Data are mean ± SD, n = 6. ns not significant, **p < 0.01, ***p < 0.001. h Reporter gene activation by poly(I:C). Cells were transfected with luciferase reporters for IFNB, IFNL1, interferon-stimulated response element (ISRE) plus a Renilla-Luc control, then exposed to 10 μg/ml poly(I:C) overnight. Fold change was calculated vs unexposed BeWo-WT. Data are mean ± SD, *** p < 0.001, n = 3. i A scatter plot depicting the correlation among poly(I:C)-induced ISG transcript in BeWo-A11 vs BeWo-WT (y-axis) and U2OS-WT vs U2OS-C19MC (x-axis). The graph shows mRNAs with ≥2-fold change and p < 0.01 in both cell groups. The correlation coefficient (Pearson) is depicted in the graph.
