Figure 4.

Impairment of MDA5-dependent intracellular poly(I:C) responsiveness in P2’s fibroblasts and rescue by WT MDA5. (A) IFIH1 PCR products from fibroblast cDNA, from P2 and two healthy controls (C1, C2), amplified with a forward primer binding to part of exon 6 and a reverse primer binding to part of exon 10. The result shown is representative of two independent experiments. (B) Sanger sequencing results for IFIH1 from P2’s fibroblast cDNA. (C) cDNA TOPO cloning and sequencing results demonstrating completely aberrant splicing of IFIH1 in fibroblasts from P2. At least 50 transcripts were sequenced for the patient and the control. The result shown is the sum of two independent experiments. (D) IFIH1 mRNA levels in SV40-fibroblasts from two healthy controls (C1, C2), P2, and MDA5 KO SV40-fibroblast, with primers spanning exons 5–6, exons 8–9, and exons 9–10, respectively; GUS was used as an expression control. Mean values and SD from three independent experiments, each with technical duplicates, are shown. (E) Western blot for MDA5 in SV40-fibroblasts from two healthy controls (C1, C2), P2, and MDA5 KO SV40-fibroblasts. One antibody recognizing aa 78–555 of MDA5 (M) and another recognizing the N-terminus of MDA5 (N) were used. GAPDH was used as a loading control. The data shown are representative of at least three independent experiments. (F) IFNB mRNA in primary fibroblasts from three healthy controls (Ctrls) and P2, without stimulation (NS), or after stimulation with intracellular poly(I:C) (PIC E-transfect: 10 ng/0.2 million cells, by nucleofection) for 9 and 20 h. P values were obtained by Student’s t test by comparing P2’s fibroblasts with all control fibroblasts after 20 h of stimulation with PIC E-transfect. *, P < 0.05. (G) IFNB and IFNL1 mRNA levels in SV40-fibroblasts from three healthy controls (Ctrls), P2, MDA5 KO SV40-fibroblasts, or TLR3^−/− patients, not stimulated (NS), stimulated for 4 h with extracellular 25 µg/ml poly(I:C), or infected with VSV M51R at an MOI of 1 for 18 h. GUS was included for normalization. Mean values and SD from three independent experiments are shown. (H) Production of IFN-β and IFN-λ in SV40-fibroblasts from two healthy controls (Ctrls), P2, MDA5 KO SV40-fibroblasts, or SV40-fibroblasts from a TLR3^−/− patient, 24 h after stimulation with 0.2 µg/ml T7-GFP, 25 µg/ml 5′ppp-dsRNA, or 25 µg/ml poly(I:C), with or without Lipofectamine (Lipo), as assessed by ELISA. Mean values and SD from three independent experiments are shown. (I) MDA5 was detected by immunoblotting with an anti-MDA5 antibody, for SV40-fibroblast cells from one healthy control (C4), and from P2 without plasmid transfection (NT) or after transfection with the empty vector (Vec) or with WT IFIH1. (J) IFNB mRNA, in the absence of stimulation (NS), or after 6 h of stimulation with intracellular poly(I:C) (10 ng/0.2 million cells), as assessed by RT-qPCR, in SV40-fibroblasts from two healthy controls and P2, without plasmid transfection or after transfection with the empty vector (Vec) or with WT MDA5. Mean values ± SD were calculated from two controls tested in one experiment, representative of three independent experiments performed.