Journal: Autophagy

Article Title: Salicylic acid and the viral virulence factor 2b regulate the divergent roles of autophagy during cucumber mosaic virus infection

doi: 10.1080/15548627.2021.1987674

Figure Lengend Snippet: Autophagy is induced during CMV infection. ( A ) Representative images of the GFP-ATG8a marker in healthy and CMV infected plants with and without concanamycin A (ConA) treatment. Chloroplasts are shown in magenta. Images are confocal Z-stacks. Scale bar: 20 μm. The inset shows a representative of large irregular GFP-ATG8a structures found with CMV. Scale bar: 5 μm. ( B ) GFP-ATG8a foci were counted from similar images as in ( A ) using ImageJ. ( n = 10). ( C ) Western blot analysis of free GFP levels derived from GFP-ATG8a in mock and CMV infected plants. Ponceau S (PS) staining verified comparable protein loading. The average and standard deviation from a quantification of the ratio between fusion to free GFP using ImageJ is shown below the western blot, and is based on four independent experiments. ( D ) Transcript levels for NBR1, ATG8a and ATG8e in mock and CMV infected plants were determined by RT-qPCR. ( n = 4). ( E ) Representative western blot of NBR1 levels in mock and CMV inoculated WT and atg5 plants. Ponceau S (PS) staining verified comparable protein loading. A quantification of NBR1 protein levels using ImageJ from three independent western blots normalized using PS stained Rubisco is shown below the western blot. Statistical significance (* P < 0.05; ** P < 0.01) was revealed by Student´s t -test (compared to WT).

Article Snippet: Quantitative RT-PCR analysis (qPCR) was performed with Maxima SYBR Green/Fluorescein qPCR Master Mix (Thermo Fisher Scientific, K0241) using the CFX Connect TM Real-Time PCR detection system (BIO-RAD) with gene-specific primers listed in Table S1.

Techniques: Infection, Marker, Western Blot, Derivative Assay, Staining, Standard Deviation, Quantitative RT-PCR

Journal: Autophagy

Article Title: Salicylic acid and the viral virulence factor 2b regulate the divergent roles of autophagy during cucumber mosaic virus infection

doi: 10.1080/15548627.2021.1987674

Figure Lengend Snippet: Autophagy impacts various aspects of CMV resistance. ( A ) CMV infection rate in WT and atg7 plants determined by presence/absence of viral symptoms three weeks after mechanical sap inoculation (8 independent replicates of 9 plants). ( B ) Vertical transmission of CMV in seeds from infected WT and atg7 plants was estimated by RT-PCR from 10 pools of 20 seedlings per plant ( n = 5 independent plants). ( C ) CMV RNA levels relative to PP2A in WT and atg5 plants determined by RT-qPCR at 10, 18 and 30 DAI ( n = 4). ( D ) A representative western blot of CMV 2b protein levels from infected WT and atg5 plants. Ponceau S (PS) staining verified comparable protein loading. ( E ) Four independent western blots as in (D) were used for quantification of 2b levels in infected WT and atg5 plants using ImageJ. PS-stained Rubisco was used to normalize loading. ( F and G ) Relative CMV RNA levels were determined by RT-qPCR at 14 ( F ) and 28 ( G ) DAI in WT, atg5, sid2, atg5 sid2, NahG, atg5 NahG, npr1 and atg5 npr1 plants. ( n = 4). Statistical significance (** P < 0.01, * P < 0.05) was revealed by Student´s t -test.

Article Snippet: Quantitative RT-PCR analysis (qPCR) was performed with Maxima SYBR Green/Fluorescein qPCR Master Mix (Thermo Fisher Scientific, K0241) using the CFX Connect TM Real-Time PCR detection system (BIO-RAD) with gene-specific primers listed in Table S1.

Techniques: Infection, Transmission Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining

Journal: Autophagy

Article Title: Salicylic acid and the viral virulence factor 2b regulate the divergent roles of autophagy during cucumber mosaic virus infection

doi: 10.1080/15548627.2021.1987674

Figure Lengend Snippet: Autophagy degrades 2b within and outside of the infection context. ( A ) Colocalization analysis of GFP-ATG8a and 2b-RFP in roots of transgenic Arabidopsis after DMSO and ConA treatment for 6 h. ( B and C ) 2b-RFP transgenic seedlings were treated with DMSO (control), ConA or AZD for 12h followed by western blot detection of 2b using anti-RFP. Ponceau S staining shows loading. Representative blot is shown in (B) and a quantification of 2b levels relative to PS stained Rubisco using ImageJ from three independent experiments with the dashed line indicating DMSO levels (C). ( D and E ) Accumulation of 2b in transgenic WT and atg5 plants was analyzed by western blotting. Representative blot is shown in (D) with PS-stained Rubisco indicating loading. Quantification of 2b levels using ImageJ from three independent experiments with the dashed line indicating WT levels (E). ( F ) Expression levels of the 2b transgene in WT and atg5 backgrounds related to (D and E) was determined by RT-qPCR. ( n = 4). ( G ) 2b-dependent virulence measured as relative fresh weight loss caused by transgenic 2b expression compared to non-transgenic plants in WT and atg5 backgrounds. ( n = 9). A representative plant image is shown below. ( H ) Colocalization analysis in N. benthamiana leaves co-expressing 2b-RFP or CP-RFP with GFP-ATG8a. The Z-stack images were acquired 48 h post agroinfiltration. Scale bar: 20 μm. ( I ) Co-IP (Co-immunoprecipitation) analysis of 2b-RFP with GFP-ATG8a from N. benthamiana ( N.b .) and Arabidopsis. Co-expression of GFP was used as control in N. benthamiana and 2b-RFP expression alone in Arabidopsis. Shown is the anti-RFP input signal, as well as anti-GFP and anti-RFP signals from the GFP-based IP samples. ( J ) Co-IP analysis of 2b with GFP-ATG8a from infected Arabidopsis plants. Non-infected GFP-ATG8a and infected GFP expressing plants were used as control. Shown are the input and IP samples probed with anti-GFP and anti-2b.

Article Snippet: Quantitative RT-PCR analysis (qPCR) was performed with Maxima SYBR Green/Fluorescein qPCR Master Mix (Thermo Fisher Scientific, K0241) using the CFX Connect TM Real-Time PCR detection system (BIO-RAD) with gene-specific primers listed in Table S1.

Techniques: Infection, Transgenic Assay, Control, Western Blot, Staining, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Autophagy

Article Title: Salicylic acid and the viral virulence factor 2b regulate the divergent roles of autophagy during cucumber mosaic virus infection

doi: 10.1080/15548627.2021.1987674

Figure Lengend Snippet: CMV protein 2b dampens SA-induced autophagy. ( A ) GFP-ATG8a foci were counted from mock and CMV infected GFP-ATG8a and GFP-ATG8a NahG plants after ConA and DMSO treatment using ImageJ ( n = 10). ( B ) Western blot analysis of GFP-ATG8a from mock and infected control and NahG plants. The experiment was repeated twice. ( C ) Transcript levels of ATG8a, ATG8e and NBR1 were determined by RT-qPCR from 2b transgenic plants shown in ( n = 4). ( D ) The number of GFP-ATG8a foci were analyzed in roots of 10 days old seedlings without and with stable expression of 2b after 10 h ConA treatment. Counting was performed using ImageJ on confocal Z-stack projections ( n = 15). Representative images are shown with scale bar: 20 μm. ( E ) Short-day grown 4-week-old GFP-ATG8a and GFP-ATG8a expressing 2b-RFP plants were sprayed with 1 mM SA. GFP-ATG8a foci were quantified 12 h later using confocal imaging and ImageJ software. Control plants were sprayed with corresponding 0.1 % EtOH ( n = 9). ( F ) Representative images of GFP-ATG8a 12 h after SA treatments in control and 2b-RFP plants. Shown is GFP in green and chloroplasts in magenta. Scale bar: 20 μm. ( G ) SA-induced free GFP release from GFP-ATG8a was analyzed in control and 2b-RFP plants by western blot using anti-GFP. The western blot experiment was repeated twice with similar results. PS staining of the membrane was used to verify loading. ( H ) RLUC-ATG8a activity was analyzed after co-expression with CMV proteins and GUS control in N. benthamiana leaves at 3 DAI. FLUC was used as an internal control for RLUC activity, and the quantitative data is presented as a ratio of FLUC to RLUC-ATG8a (higher ratio = increased ATG8a degradation). n = 4. ( I ) As (H), but co-expressed proteins were combinations of 2b and Arabidopsis ATG3 as indicated. Statistical significance (** P < 0.01) was revealed by Student´s t -test.

Article Snippet: Quantitative RT-PCR analysis (qPCR) was performed with Maxima SYBR Green/Fluorescein qPCR Master Mix (Thermo Fisher Scientific, K0241) using the CFX Connect TM Real-Time PCR detection system (BIO-RAD) with gene-specific primers listed in Table S1.

Techniques: Infection, Western Blot, Control, Quantitative RT-PCR, Transgenic Assay, Expressing, Imaging, Software, Staining, Membrane, Activity Assay

Journal: Autophagy

Article Title: Salicylic acid and the viral virulence factor 2b regulate the divergent roles of autophagy during cucumber mosaic virus infection

doi: 10.1080/15548627.2021.1987674

Figure Lengend Snippet: Autophagy-enhanced CMV resistance involves RNA silencing and is uncoupled from disease symptom tolerance. (A ) CMV RNA levels relative to PP2A were determined by RT-qPCR at 14 DAI in WT, atg7, dcl2 dcl4 and atg7 dcl2 dcl4 plants ( n =4). ( B ) CMV RNA levels relative to PP2A were determined by RT-qPCR at 14 DAI in WT, atg5, ago1 and atg5 ago1 plants ( n = 4). ( C) and ( D ) The relative fresh weight of CMV infected plants compared to mock were determined in the same genotypes as in ( A) and ( B ) at 28 DAI. ( n =10). Representative images of mock and CMV infected plants at 28 DAI are shown to the right ( E) and ( F ). ( G ) PR1 transcript levels were analyzed in the indicated genotypes using RT-qPCR at 18 DAI with CMV ( n = 4). Statistical significance (** P < 0.01, * P < 0.05) was revealed by Student´s t -test. Letters indicate genotypes with statistically different levels (ANOVA P < 0.05, Tukey-HSD test).

Article Snippet: Quantitative RT-PCR analysis (qPCR) was performed with Maxima SYBR Green/Fluorescein qPCR Master Mix (Thermo Fisher Scientific, K0241) using the CFX Connect TM Real-Time PCR detection system (BIO-RAD) with gene-specific primers listed in Table S1.

Techniques: Quantitative RT-PCR, Infection

Journal: Cell host & microbe

Article Title: Inhibiting the Plasmodium eIF2α kinase PK4 prevents artemisinin-induced latency

doi: 10.1016/j.chom.2017.11.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GSK2606414 , MedChemexpress , Cat#HY-18072.

Techniques: Recombinant, Mutagenesis, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β KO mice exhibit altered responses to HSV-1 skin infection. a Wild type (WT; n = 5 ♀ and 13 ♂) and IL-36β KO mice ( n = 7 ♀ and 12 ♂) were infected with HSV-1 on the flank and skin regions along HSV-1 infected dermatomes collected 6 days post-infection. Viral DNA loads were determined by QPCR. Data are pooled from three independent experiments and shown as means ( ± SD). * p < 0.05 (One-Way ANOVA). b , c Viral ICP4 protein levels were examined by Western blotting ( b ) and quantified using ImageJ software ( c ). GAPDH was used as loading control. Data are representative of three independent experiments and shown as means (±SD) in c (WT: n = 2 female and 4 male; IL-36β KO mice: n = 3 female and 3 male). * p < 0.05 (one-way ANOVA). d Wild type and IL - 36β KO mice were infected with HSV-1 ( n = 3 per group) on the flank and skin RNA isolated 6 days post-infection. Heat-map of genes differentially expressed in the two strains is shown. e Pathway associations of genes identified as differentially expressed in d . f Expression of Oas1 , Eif2ak2, Isg15, Ifitm3, Ifitm2, and Ifit3 mRNAs were examined in wild type and IL-36β KO HSV-1 infected skin ( a ). The mRNAs were normalized against GAPDH and are shown as relative expression compared to female wild-type mice (means ± SD). * p < 0.05; ** p < 0.005 (one-way ANOVA). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Infection, Western Blot, Software, Control, Isolation, Expressing

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β promotes activation of STAT1 and STAT2 during HSV-1 skin infection. a , b Expression of Stat1 ( a ) and Stat2 ( b ) mRNAs were examined in wild type and IL-36β KO HSV-1 infected skin (Fig. ). No statistically significant differences were detected. c Quantification of total STAT1 and pSTAT1 in wild type and IL-36β KO HSV-1 infected skin by western blotting and ImageJ analysis (WT, n = 5; KO, n = 5). d STAT2 and pSTAT2 levels in wild type and IL-36β KO HSV-1 infected skin were determined by western blotting and ImageJ analysis (WT, n = 3; KO, n = 4). c , d Representative data from one of three independent experiments involving male mice is shown. Quantitative data are shown as means ± SD. * p < 0.05; ** p < 0.01 (one-way ANOVA). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Activation Assay, Infection, Expressing, Western Blot

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: STAT1 is activated in epidermal keratinocytes during HSV-1 infection. Wild type and IL-36β KO mice were infected with HSV-1 and skin along the dermatome collected 5 days later. Consecutive skin sections were examined by H&E and pSTAT1 immunohistochemistry. Early ( a – h ), intermediate ( i – p ), and advanced ( q – x ) lesions are shown. Black, blue, and red arrows point to lesion edges and positive pSTAT1 nuclei in keratinocytes and leukocytes, respectively. Black and blue scale bars represent 200 and 50 μm, respectively

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Infection, Immunohistochemistry

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β induces STAT1- and STAT2-dependent antiviral immunity in keratinocytes. a Human keratinocytes were pre-treated with medium only or IL-36β before infection with HSV-1 (MOI = 0.01). Levels of HSV-1 ICP4 protein were determined by Western blotting and ImageJ analyses using GAPDH as control. b Mouse primary keratinocytes were pre-treated with medium only or IL-36β, followed by HSV-1 infection (MOI = 0.01), and ICP4 levels examined by western blotting. c Wild type ( + / + ) and IL-36β KO (−/−) mouse primary keratinocytes were infected with 0.01 MOI HSV-1 and ICP4 examined by western blotting. d Wild type and STAT1 −/− primary male mouse keratinocytes were treated with medium only or IL-36β followed by HSV-1 infection (MOI = 0.01) for 24 h. Levels of HSV-1 ICP4 and host Mx1 were examined by western blotting. e Wild type and STAT2 −/− primary male mouse keratinocytes were examined after IL-36β pre-treatment and HSV-1 infection using western blotting. a – e Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group); ** p < 0.01. Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Infection, Western Blot, Control

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β activates expression of IFNAR1 and IFNAR2. a Ifnar1 and Ifnar2 mRNA expression was analyzed by real-time PCR in male mouse primary keratinocytes treated with medium only or IL-36β as indicated. b Mouse IFNAR1 and IFNAR2 protein expression was examined by western blotting and ImageJ analyses. c Human keratinocytes were treated with medium only or IL-36β and expression of IFNAR1 and IFNAR2 mRNA determined by real-time PCR. d Expression of human IFNAR1 and IFNAR2 protein was examined by western blotting and ImageJ analyses. a – d Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group); ** p < 0.01. Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IRF1 is induced by IL-36β and has divergent impact on IFNAR expression. a , b IRF1 mRNA expression in mouse ( a ) and human ( b ) keratinocytes was examined following IL-36β treatment at indicated time-points. c Mouse wild type and IRF1 −/− primary keratinocytes were treated as indicated for 6 h and expression of Ifnar1 and Ifnar2 mRNA analyzed by real-time PCR. d Mouse wild type and IRF1 −/− keratinocytes were treated with medium only or IL-36β for 6 h and IFNAR protein expression examined by western blotting and ImageJ analyses. e IFNAR1 and IFNAR2 mRNA expression in human control (Ctrl) and IRF1 −/− keratinocytes was examined following medium only or IL-36β treatment for 6 h. f Protein levels of IFNAR1 and IFNAR2 in human control and IRF1 −/− keratinocytes were determined by western blotting 6 h post-treatment. a – f Quantitative data are shown as means ± SD. * p < 0.05; ** p < 0.01; # p > 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IRF1 is partially involved in immunity against HSV-1. a – e C57BL/6 J (WT) and IRF1 KO mice were infected with HSV-1 on the flank. a Survival was monitored for 16 days. b Weight was measured for 9 days. c Gastrointestinal dysfunction was examined at day 9 post-infection. Red arrow points to disease affected stomach. d Skin lesion sizes were measured (male mice; WT: n = 5; KO: n = 4). e HSV-1 DNA copy numbers in the skin were determined 6 days post-infection (male mice; WT: n = 7; KO: n = 6). f Mouse primary keratinocytes from wild type (WT) and IRF1 KO mice were sequentially treated with medium only or IL-36β as indicated and infected with HSV-1 ( n = 2 biologically independent samples per treatment group). Levels of ICP4 and Mx1 were evaluated by western blotting and ImageJ analyses. g Levels of ICP4 and Mx1 protein were evaluated by western blotting and ImageJ analyses following IL-36β treatment and HSV-1 infection of human control (Ctrl) and IRF1 KO keratinocytes ( n = 2 biologically independent samples per treatment group). a , c * p < 0.05; ** p < 0.01 (Mantel-Cox and Gehan-Breslow-Wilcoxon tests). d – g Quantitative data are shown as means ± SD. * p < 0.05; # p > 0.05 (one-way ANOVA). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Infection, Western Blot, Control

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β induced antiviral state is dependent upon IFNAR. a Human keratinocytes were transfected with control (Ctrl) or IFNAR1 gRNA/Cas9 expression plasmids, treated with IL-36β and infected with HSV-1. b Control (Ctrl) or IFNAR2 gRNA/Cas9 expression plasmid transfected human keratinocytes were treated with IL-36β and infected with HSV-1. a , b Levels of ICP4, Mx1, IFNAR1, IFNAR2, and GAPDH were determined using western blotting and ImageJ analyses. c Mouse primary keratinocytes were treated with IL-36β as indicated, incubated with neutralizing antibodies against IFNAR or isotype matched Ig and infected with HSV-1. Levels of ICP4, Mx1 and GAPDH were determined using western blotting and ImageJ analyses. a – c Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Transfection, Control, Expressing, Infection, Plasmid Preparation, Western Blot, Incubation

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β accelerates type I IFN signaling. Medium only or IL-36β treated keratinocytes were further treated with type I IFN (0.01 ng mL −1 ) as indicated. Phosphorylation of STAT1 and STAT2 was examined by western blotting and ImageJ analyses. Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group). a Human keratinocytes were analyzed. b Mouse primary keratinocytes were examined. Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Phospho-proteomics, Western Blot

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-1 promotes enhanced type I IFN signaling through IFNAR in human cells. a , b Gene editing was performed in human keratinocytes using control (Ctrl), IFNAR1 ( a ) or IFNAR2 ( b ) gRNAs and Cas9. Cells were treated with medium only or IL-36β, infected with HSV-1 and analyzed by western blotting. c Cells were pre-treated with medium only or IL-1α followed by IFN-α/β. STAT1/2 activation was examined by western blotting. Quantitative data are shown as means ± SD. * p < 0.05; ** p < 0.01 (one-way ANOVA, n = 2 biologically independent samples per treatment group). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Control, Infection, Western Blot, Activation Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: MARCH8 downregulation modulates profibrotic responses including myofibroblast differentiation

doi: 10.1152/ajpcell.00166.2023

Figure Lengend Snippet: Transforming growth factor-beta (TGF-β) downregulates MARCH8 at the transcriptional level. A-B, qPCR analysis showed that MARCH8 mRNA was reduced by TGF-β in dose-dependent ( A , 12 h) and time-dependent ( B , 5 ng/mL) manners in primary normal human lung fibroblasts (HLFs). GAPDH was used as the internal control, n = 3 replicates. * P < 0.05 vs. vehicle control group. C : primary normal HLFs were pretreated with actinomycin D (Act D, 1 μg/mL) for 30 min, followed by TGF-β treatment (5 ng/mL, 16 h) for Western blotting (WB) analysis of MARCH8. GAPDH was the loading control. D : the quantification of C based on three independent experiments. * P < 0.05. NS, not significant. E : primary normal HLFs were pretreated with the proteasome inhibitor MG132 (5 μM) for 30 min, followed by TGF-β treatment (5 ng/mL, 16 h) for WB analysis of MARCH8. F : the quantification of E based on three independent experiments. * P < 0.05 based on three independent experiments. G : primary normal HLFs were treated with TGF-β (5 ng/mL, 16 h), followed by 10 μg/mL cycloheximide (CHX) treatment for the indicated times. Whole cell lysates were collected for WB analysis of MARCH8 degradation. H : MARCH8 levels were plotted relative to those at time 0 of CHX treatment ( G ) after being quantified by NIH ImageJ software and normalized to Actin. Solid line indicates control group and dash line indicates TGF-β group. One-way ANOVA followed by Dunnett’s test was used for multiple comparisons.

Article Snippet: Small molecular inhibitors including actinomycin D (Act D, HY-17559), LY294002 (HY-10108), MG132 (HY-13259), SIS3 (HY-13013), SB230580 (HY-10256), and U0126 (HY-12031A) were purchased from MedChemExpress (Monmouth Junction, NJ).

Techniques: Control, Western Blot, Software

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β KO mice exhibit altered responses to HSV-1 skin infection. a Wild type (WT; n = 5 ♀ and 13 ♂) and IL-36β KO mice ( n = 7 ♀ and 12 ♂) were infected with HSV-1 on the flank and skin regions along HSV-1 infected dermatomes collected 6 days post-infection. Viral DNA loads were determined by QPCR. Data are pooled from three independent experiments and shown as means ( ± SD). * p < 0.05 (One-Way ANOVA). b , c Viral ICP4 protein levels were examined by Western blotting ( b ) and quantified using ImageJ software ( c ). GAPDH was used as loading control. Data are representative of three independent experiments and shown as means (±SD) in c (WT: n = 2 female and 4 male; IL-36β KO mice: n = 3 female and 3 male). * p < 0.05 (one-way ANOVA). d Wild type and IL - 36β KO mice were infected with HSV-1 ( n = 3 per group) on the flank and skin RNA isolated 6 days post-infection. Heat-map of genes differentially expressed in the two strains is shown. e Pathway associations of genes identified as differentially expressed in d . f Expression of Oas1 , Eif2ak2, Isg15, Ifitm3, Ifitm2, and Ifit3 mRNAs were examined in wild type and IL-36β KO HSV-1 infected skin ( a ). The mRNAs were normalized against GAPDH and are shown as relative expression compared to female wild-type mice (means ± SD). * p < 0.05; ** p < 0.005 (one-way ANOVA). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Infection, Western Blot, Software, Control, Isolation, Expressing

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β promotes activation of STAT1 and STAT2 during HSV-1 skin infection. a , b Expression of Stat1 ( a ) and Stat2 ( b ) mRNAs were examined in wild type and IL-36β KO HSV-1 infected skin (Fig. ). No statistically significant differences were detected. c Quantification of total STAT1 and pSTAT1 in wild type and IL-36β KO HSV-1 infected skin by western blotting and ImageJ analysis (WT, n = 5; KO, n = 5). d STAT2 and pSTAT2 levels in wild type and IL-36β KO HSV-1 infected skin were determined by western blotting and ImageJ analysis (WT, n = 3; KO, n = 4). c , d Representative data from one of three independent experiments involving male mice is shown. Quantitative data are shown as means ± SD. * p < 0.05; ** p < 0.01 (one-way ANOVA). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Activation Assay, Infection, Expressing, Western Blot

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: STAT1 is activated in epidermal keratinocytes during HSV-1 infection. Wild type and IL-36β KO mice were infected with HSV-1 and skin along the dermatome collected 5 days later. Consecutive skin sections were examined by H&E and pSTAT1 immunohistochemistry. Early ( a – h ), intermediate ( i – p ), and advanced ( q – x ) lesions are shown. Black, blue, and red arrows point to lesion edges and positive pSTAT1 nuclei in keratinocytes and leukocytes, respectively. Black and blue scale bars represent 200 and 50 μm, respectively

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Infection, Immunohistochemistry

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β induces STAT1- and STAT2-dependent antiviral immunity in keratinocytes. a Human keratinocytes were pre-treated with medium only or IL-36β before infection with HSV-1 (MOI = 0.01). Levels of HSV-1 ICP4 protein were determined by Western blotting and ImageJ analyses using GAPDH as control. b Mouse primary keratinocytes were pre-treated with medium only or IL-36β, followed by HSV-1 infection (MOI = 0.01), and ICP4 levels examined by western blotting. c Wild type ( + / + ) and IL-36β KO (−/−) mouse primary keratinocytes were infected with 0.01 MOI HSV-1 and ICP4 examined by western blotting. d Wild type and STAT1 −/− primary male mouse keratinocytes were treated with medium only or IL-36β followed by HSV-1 infection (MOI = 0.01) for 24 h. Levels of HSV-1 ICP4 and host Mx1 were examined by western blotting. e Wild type and STAT2 −/− primary male mouse keratinocytes were examined after IL-36β pre-treatment and HSV-1 infection using western blotting. a – e Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group); ** p < 0.01. Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Infection, Western Blot, Control

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β activates expression of IFNAR1 and IFNAR2. a Ifnar1 and Ifnar2 mRNA expression was analyzed by real-time PCR in male mouse primary keratinocytes treated with medium only or IL-36β as indicated. b Mouse IFNAR1 and IFNAR2 protein expression was examined by western blotting and ImageJ analyses. c Human keratinocytes were treated with medium only or IL-36β and expression of IFNAR1 and IFNAR2 mRNA determined by real-time PCR. d Expression of human IFNAR1 and IFNAR2 protein was examined by western blotting and ImageJ analyses. a – d Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group); ** p < 0.01. Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IRF1 is induced by IL-36β and has divergent impact on IFNAR expression. a , b IRF1 mRNA expression in mouse ( a ) and human ( b ) keratinocytes was examined following IL-36β treatment at indicated time-points. c Mouse wild type and IRF1 −/− primary keratinocytes were treated as indicated for 6 h and expression of Ifnar1 and Ifnar2 mRNA analyzed by real-time PCR. d Mouse wild type and IRF1 −/− keratinocytes were treated with medium only or IL-36β for 6 h and IFNAR protein expression examined by western blotting and ImageJ analyses. e IFNAR1 and IFNAR2 mRNA expression in human control (Ctrl) and IRF1 −/− keratinocytes was examined following medium only or IL-36β treatment for 6 h. f Protein levels of IFNAR1 and IFNAR2 in human control and IRF1 −/− keratinocytes were determined by western blotting 6 h post-treatment. a – f Quantitative data are shown as means ± SD. * p < 0.05; ** p < 0.01; # p > 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IRF1 is partially involved in immunity against HSV-1. a – e C57BL/6 J (WT) and IRF1 KO mice were infected with HSV-1 on the flank. a Survival was monitored for 16 days. b Weight was measured for 9 days. c Gastrointestinal dysfunction was examined at day 9 post-infection. Red arrow points to disease affected stomach. d Skin lesion sizes were measured (male mice; WT: n = 5; KO: n = 4). e HSV-1 DNA copy numbers in the skin were determined 6 days post-infection (male mice; WT: n = 7; KO: n = 6). f Mouse primary keratinocytes from wild type (WT) and IRF1 KO mice were sequentially treated with medium only or IL-36β as indicated and infected with HSV-1 ( n = 2 biologically independent samples per treatment group). Levels of ICP4 and Mx1 were evaluated by western blotting and ImageJ analyses. g Levels of ICP4 and Mx1 protein were evaluated by western blotting and ImageJ analyses following IL-36β treatment and HSV-1 infection of human control (Ctrl) and IRF1 KO keratinocytes ( n = 2 biologically independent samples per treatment group). a , c * p < 0.05; ** p < 0.01 (Mantel-Cox and Gehan-Breslow-Wilcoxon tests). d – g Quantitative data are shown as means ± SD. * p < 0.05; # p > 0.05 (one-way ANOVA). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Infection, Western Blot, Control

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β induced antiviral state is dependent upon IFNAR. a Human keratinocytes were transfected with control (Ctrl) or IFNAR1 gRNA/Cas9 expression plasmids, treated with IL-36β and infected with HSV-1. b Control (Ctrl) or IFNAR2 gRNA/Cas9 expression plasmid transfected human keratinocytes were treated with IL-36β and infected with HSV-1. a , b Levels of ICP4, Mx1, IFNAR1, IFNAR2, and GAPDH were determined using western blotting and ImageJ analyses. c Mouse primary keratinocytes were treated with IL-36β as indicated, incubated with neutralizing antibodies against IFNAR or isotype matched Ig and infected with HSV-1. Levels of ICP4, Mx1 and GAPDH were determined using western blotting and ImageJ analyses. a – c Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Transfection, Control, Expressing, Infection, Plasmid Preparation, Western Blot, Incubation

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β accelerates type I IFN signaling. Medium only or IL-36β treated keratinocytes were further treated with type I IFN (0.01 ng mL −1 ) as indicated. Phosphorylation of STAT1 and STAT2 was examined by western blotting and ImageJ analyses. Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group). a Human keratinocytes were analyzed. b Mouse primary keratinocytes were examined. Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Phospho-proteomics, Western Blot

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-1 promotes enhanced type I IFN signaling through IFNAR in human cells. a , b Gene editing was performed in human keratinocytes using control (Ctrl), IFNAR1 ( a ) or IFNAR2 ( b ) gRNAs and Cas9. Cells were treated with medium only or IL-36β, infected with HSV-1 and analyzed by western blotting. c Cells were pre-treated with medium only or IL-1α followed by IFN-α/β. STAT1/2 activation was examined by western blotting. Quantitative data are shown as means ± SD. * p < 0.05; ** p < 0.01 (one-way ANOVA, n = 2 biologically independent samples per treatment group). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Recombinant human IL-36β (Catalog #: 6834-ILB-025), mouse IL-36β (Catalog #: 7060-ML-010) protein, and Universal Type I IFN (Catalog #: 11200–1) were obtained from R&D Systems.

Techniques: Control, Infection, Western Blot, Activation Assay