Fig 1: RLRs, RIG-I and MDA-5 recognize JUNV dsRNA. (A,B,E) A549 cells were infected with rCandid at a MOI of 1.0. 48 hpi infected and uninfected cell monolayers were fixed, permeabilized, and stained with the anti-dsRNA antibody (9D5, green), anti-JUNV NP (AG12, magenta), anti-RIG-I (red) (A,B) or anti-MDA-5 (red) (E) and DAPI (blue) as described in the Materials and Methods section. Cells were then observed under a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens. All image analysis and processing was performed using the FIJI software. Unless indicated, laser emissions were the same for all samples and only the same linear adjustments for brightness and contrast were made when appropriate across all samples. Representative images of three separate experiment are shown. (B) Exposure of RIG-I in mock infected cells was increased to show the distribution pattern of RIG-I in uninfected cells. (C,F) Quantification of the colocalization between dsRNA and RIG-I (C) or MDA-5 (F) using the Pearson's correlation coefficient. (D,G) Quantification of the colocalization between NP and RIG-I (D) or MDA-5 (G). Data shown represents the average and Std of 100 infected cells from three separate experiments.
Fig 2: Poly(I:C) potentiates the expression of MDA5 in vitro.a The apoptotic rate of NHKs in response to Poly(I:C), detected by flow cytometry assay. b, c The RNA levels (b) and protein levels (c) of IFIH1 in NHKs with Poly(I:C) concentration rose, detected by qRT-PCR assay and Western-blot respectively. d The expression of MDA5 in NHKs in response to 0.6 μg/ml Poly(I:C) within 48 h, determined by Western-blot assay. e The expression of MDA5 in NHKs stimulated by 0.6 μg Poly(I:C) for 24 h, detected by immunofluorescence assay. Bar = 10 μm. The data are representative of three independently performed experiments. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.
Fig 3: NF-κB and IRF3 is decisive for secretion of CXCL10 and CXCL16.a The Western-blots of total P-65, phosphor-P65, total IRF3 and phosphor-IRF3 in NHKs in the indicated time in response to Poly(I:C). b, c The Western-blots of total P-65, phosphor-P65, total IRF3 and phosphor-IRF3 when interfering MDA5 (si-MDA5) (b) or interfering MAVS (si-MAVS) (c) in HaCaT cells prior to 24 h 0.6 μg/ml Poly(I:C) treatment. d, e The mRNA and secretion levels of CXCL10 and CXCL16 when interfering NF-κB by using NF-κB siRNA (si-NF-κB) (d) or interfering IRF3 by using IRF3 siRNA (si-IRF3) (e) for 24 h prior to 0.6 μg/ml Poly(I:C) stimulation for 24 h in HaCaT cells. The data are presented as the mean ± SD across three independently performed experiments. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001. ns, not significant.
Fig 4: The expression of MDA5 and anti-CMV IgM is higher in some progressive vitiligo patients.a The anti-CMV IgM levels in 56 vitiligo patients and 26 healthy controls. The patients with the positive anti-CMV IgM were indicated in red. b The VASI scores of vitiligo patients with positive anti-CMV IgM or negative anti-CMV IgM. n = 5. c The Western-blot of MDA5 in the epidermis of perilesional skin of vitiligo patients with positive or negative anti-CMV IgM, in the apparently healthy epidermis of vitiligo patients with positive anti-CMV IgM and in the epidermis of healthy controls. n = 3. d, e The expression of MDA5 (d) and IFN-β (e) in epidermis of perilesional skin of vitiligo patients with positive or negative anti-CMV IgM, in the apparently healthy epidermis of vitiligo patients with positive anti-CMV IgM and in the epidermis of healthy controls, by immunofluorescence assay. n = 5, Bar = 50 μm. The data are presented as the mean ± SD across three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.
Fig 5: MDA5-mediated IFN-β is central to the secretion of CXCL10 while CXCL16 is directly regulated by IRF3.a The mRNA and secretion levels of IFN-β in HaCaT cells in response to Poly(I:C). b The secretion levels of IFN-β when interfering any of MDA5, MAVS, NF-kB, IRF3 prior to 24 h treatment of Poly(I:C) in HaCaT cells. c The secretion levels of CXCL10 and CXCL16 when treated with Poly(I:C)-pretreated supernatant (Sup) with or without neutralizing the IFN-β by using the IFN-β neutralizing antibody (Neu), detected by ELISA assay. d, e The expression of total JAK, phosphor-JAK, total STAT1, phosphor-STAT1 and MDA5 in HaCaT cells exposed to the Poly(I:C)-pretreated supernatant (Sup), and simultaneously with or without neutralizing IFN-β (Neu) (d) or blocking JAK1-STAT1 pathway with the pretreatment of Tofacitinib (Tofa) (e). f The mRNA and secretion levels of CXCL10 and CXCL16 in HaCaT cells stimulated by recombined human IFN-β (rh IFN-β) or pretreated with Tofacitinib (Tofa), detected by ELISA. g Chromatin immunoprecipitation assay (ChIP) of IRF3 to CXCL16 promoter after the HaCaT cells were treated by Poly(I:C) for 18 h. h The diagram of the pathophysiological process in keratinocytes under the virus invasion. Data are presented as the mean ± SD across three independently performed experiments. **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01. ns, not significant.
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