Fig 2: Analyses of HSP60 (A), TLR3 (B), MDA5 (C), and p-IRF3 (D) on the 10th culture day of HepG2 after treatment with a HSP60 overexpression vector for 24 h. Data are expressed as the mean ± SE of the four to six samples per group. ** p < 0.01 and *** p < 0.001 between the indicated groups. TLR3, toll-like receptor 3; MDA5, melanoma differentiation-associated gene 5; P-IRF3, phosphorylated-interferon regulatory factor 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig 3: The effect of miR-155 mimic in MCF-7 is length-dependent. (A) Schematic illustration of custom lengthened negative control and shortened miR-145 and miR-155 mimics. Not to scale. (B) Normalized confluence readings collected post-transfection of MCF-7 cells with 10 nM of mimic variants (as in Fig. 1). Error bars, SEM of nine scans per well (n = 2). (C) Western blot analysis of MCF-7 cells matched to B, collected 24 h post-transfection; protein samples were sequentially probed for the dsRNA-response sensors RIG-I and MDA5, interferon-induced phosphorylated STAT1-Tyr701 and total STAT1, apoptosis marker PARP (F: full-length; Cl: cleaved), and actin as a loading control. (D) Western blot analysis of dsRNA-response elements (as in C) in MCF-7 cells collected 24 h post-treatment with apoptosis inducing actinomycin D (10 nM), cyclohexamide (100 nM), or DMSO alone. (E) Volcano density plots (as in Fig. 1) of microarray-based differentially expressed genes of MCF-7 cells transfected the indicated mimic variants. The y-axes are on a common scale.

Fig 4: A type I IFN response is unleashed in ADAR1‐knockout cells upon expression of LGP2, related to Figs 2 and 3 ADAR1‐knockout HEK293 cells (clone 2) were cotransfected with an empty vector (EV) or a FLAG‐LGP2‐encoding vector (LGP2) combined with a vector encoding GFP‐tagged ADAR1 p110 or p150. Cells were harvested 48 h post‐transfection and the type I IFN response was monitored by measuring IFN‐β and IFIT1 transcript expression, relative to ACTB expression, by RT‐qPCR. Data are means ± s.d. from a representative of three biological replicate experiments.ADAR1‐knockout HEK293 cells (clone 2) were transfected as in (A). Protein lysates were analyzed by SDS‐PAGE followed by immunoblotting using the indicated antibodies (n = 3).MDA5‐knockout HEK293 cells, generated in Fig 1D, were transfected with an ADAR1‐targeting siRNA (siADAR1) or a control siRNA (siCtrl) and 8 h later with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of MDA5. Cells were harvested 72 h post‐siRNA transfection and RT‐qPCR analysis was used to monitor ADAR1 knockdown and MDA5 (IFIH1) expression. All transcripts were normalized to ACTB. Data are means ± s.d. from a representative of two biological replicate experiments. Source data are available online for this figure.

Fig 5: CypA suppresses ubiquitin-mediated proteasome degradation of MAVS.(A) Immunoblot analysis of lysates in 293T/CypA+ and 293T/CypA- cells transfected with Flag-RIG-I, Flag-MDA5 or Flag-MAVS for 24 hr and then treated with 100 μg/ml CHX for the indicated durations (top). The relative expression levels of RIG-I, MDA5 and MAVS were quantified (below). (B) Immunoblot analysis of lysates in 293T/CypA- cells transfected with Myc-CypA or control vector for 24 hr and then treated with 100 μg/ml CHX for the indicated time points (top). The relative expression levels MAVS were quantified (below). (C) Immunoblot analysis of lysates in 293T/CypA+ and 293T/CypA- cells incubated with SeV for the indicated times (top). The relative expression levels of MAVS were quantified (below). (D) Immunoblot analysis of lysates in 293T/CypA+ and 293T/CypA- cells treated for 6 hr with 100 μg/ml CHX, along with 10 μM NH4Cl, 10 μM MG132, or DMSO. (E) Immunoblot analysis of lysates in 293T/CypA- cells transfected for 24 hr with HA-Ub, along with Flag-CypA or control vector and then treated with 10 μM MG132 for 6 hr. (F) Immunoblot analysis of lysates in 293T/CypA- cells transfected for 24 hr with HA-Ub, along with Myc-CypA or control vector and then immunoprecipitated with anti-MAVS antibody. (G) Immunoblot analysis of lysates in 293T/CypA- cells transfected for 24 hr with HA-tagged deletion constructs of MAVS (amino acids remaining, above lanes) and point substitution constructs containing amino acids 360–540 (KK-AA, K371A plus K420A), along with Myc-CypA or control vector. Data are representative of at least three independent experiments.DOI: http://dx.doi.org/10.7554/eLife.24425.017