Journal: BMC Pharmacology & Toxicology

Article Title: GAS-STING signaling plays an essential pathogenetic role in Doxorubicin-Induced Cardiotoxicity

doi: 10.1186/s40360-022-00631-0

Figure Lengend Snippet: Suppressed DOX-induced phosphorylation of IRF3, TBK1 and p65 was observed by immunoblot analysis following STING knockdown in HL-1 cells following DOX treatment (1 μm for 24 h). Quantitative data represent the relative ratio to total IRF3, TBK1 or p65 ( n = 6)

Article Snippet: Antibodies against cGAS (Cell Signaling Technology), STING (Cell Signaling Technology), TBK1 (Cell Signaling Technology), Phospho-TBK1 (Cell Signaling Technology), IRF3 (Cell Signaling Technology), Phospho-IRF3 (Cell Signaling Technology), NF-κB p65(Cell Signaling Technology), Phospho-NF-κB p65 (Cell Signaling Technology), Caspase-3 (Protein (Abcam) were adopted to assess protein expression via immunoblotting.

Techniques: Western Blot

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: The M35 protein forms homodimers after crystallisation and in cell lysates. (A) Ribbon representations of the M35 protein crystal structure. M35_S (aa 2-452) was crystallised and its structure solved at 1.94 Å. M35 monomers are depicted in red (aa 7-343 and aa 377-441) or orange (aa 8-345 and aa 376-440), respectively. Visible N and C termini (bold) and the ends of each protein chain are labelled accordingly. The structure is depicted from three perspectives. (B) Size-exclusion chromatography followed by multi-angle light scattering (SEC- MALS) of purified NStr-M35_S protein. LS: light scattering. (C) Co-immunoprecipitation of M35 in cell lysates. HEK293T were co-transfected with indicated expression plasmids for M35-V5/His and M35-HAHA, M34-V5/His and M35-HAHA (negative control), or single constructs filled up with EV. An anti-V5-immunoprecipitation (IP) was performed 24 h later. Input and IP samples were analysed by SDS-PAGE and immunoblotting with HA- and V5-specific antibodies. Detection of GAPDH served as loading control, # unspecific band. One representative of two independent experiments is shown. (D) Native PAGE of M35 in cell lysates. HEK293T cells were co-transfected with expression plasmids for eGFP-IRF3 (control) or M35-V5/His or the corresponding EV, and for Flag-MAVS (stimulated conditions) or the respective EV (unstimulated conditions). Cells were lysed 20 h later and analysed in parallel by native (upper panel) or SDS-PAGE (lower panel) followed by immunoblotting and detection with GFP-, V5-, Flag- and GAPDH-specific antibodies as indicated. Lysates of M35-V5/His-expressing cells were diluted 1:4 in lysis buffer to adjust the signal strength in the native immunoblot. One representative of three independent experiments is shown.

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Size-exclusion Chromatography, Purification, Immunoprecipitation, Transfection, Expressing, Negative Control, Construct, SDS Page, Western Blot, Clear Native PAGE, Lysis

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: Identified M35 loss-of-function mutations impair the homodimerization of M35. (A) Immunofluorescence assay of M35 derivatives. HEK293T cells transfected with expression constructs for M35-V5/His WT, Δβ, or R69A or the corresponding EV were subjected to immunofluorescence labelling with a V5-specific antibody. Nuclei were stained with Hoechst. The scale bar represents 10 µm. Images are representative of at least two independent experiments. (B) Native PAGE of M35 derivatives. Native (upper panel) and SDS-PAGE (lower panel) followed by immunoblotting were performed as described before by co-transfecting HEK293T with expression plasmids for eGFP-IRF3, or M35-V5/His WT, Δβ, or R69A or the respective EV, and for Flag-MAVS (stimulated conditions) or the respective EV (unstimulated conditions), and analysis with GFP-, V5-, Flag- and GAPDH-specific antibodies. Lysates with M35-WT and M35-Δβ were diluted as indicated in lysis buffer to adjust the signal strength in the native immunoblot. One representative of three independent experiments is shown.

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Immunofluorescence, Transfection, Expressing, Construct, Staining, Clear Native PAGE, SDS Page, Western Blot, Lysis

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: Presence of M35 impairs binding of IRF3 to the host’s IFNβ enhancer upon stimulation of PRR signalling. (A) Chromatin immunoprecipitation (ChIP) assay. iMEFs stably expressing M35-myc/His or the corresponding EV were stimulated by transfection of poly(I:C) or mock-treated. After 6 h, formaldehyde (FA) was applied to cross-link interactions and cells were harvested. Chromatin was isolated, fragmented for processing, and subjected to immunoprecipitation with an IRF3- specific antibody. The precipitated material was decrosslinked, DNA was purified and analysed by qPCR alongside 1% of input material. (B) Immunoblot of chromatin samples from iMEFs. iMEFs were processed as described in (A) and analysed by immunoblotting with myc-, pIRF3-, and IRF3- specific antibodies, and fibrillarin-specific antibodies. Fibrillarin served as a loading control for the nuclear fraction. Shown is one representative of three independent experiments. (C) ChIP for recruitment of IRF3 to the Ifnb1 promoter in presence or absence of M35. ChIP was performed as described in (A) with an IRF3-specific and an IgG control antibody, and samples were analysed for enrichment of the IFNβ enhancer sequence by qPCR. A primer set targeting the promoter of Il6 upstream of a predicted IRF3 binding site was used as negative control. Shown are combined data from two independent experiments.

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Stable Transfection, Expressing, Transfection, Isolation, Immunoprecipitation, Purification, Western Blot, Sequencing, Negative Control

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: SLAM-seq for characterisation of the dependency of ISD-stimulated transcripts on IRF3 or of IFNβ-stimulated transcripts on canonical type I IFN-IFNAR1/IFNAR2 signalling in MEFs. (A) Determination of IRF3-dependent and IFNAR1-responsive transcripts. Primary MEFs of WT, IRF3 -/- or IFNAR1 -/- mice were stimulated by transfection of 5 µg/mL ISD for 4 h or mock- transfected, or stimulated with 100 U/mL of murine IFNβ for 3 h or left untreated. Transcripts were labelled in the last 2 h of stimulation by incubation with 200 µM of 4-thiouridine (4sU) and analysed by SLAM-seq. Samples were prepared and analysed in quadruplicate. (B-C) Heatmaps showing the log fold-changes (log FC; blue: down-, red: up-regulation) in the indicated cell lines for (B) the 28 genes IRF3-dependent genes detected after ISD stimulation or (C) the 2,888 IFNAR1-responsive genes detected after IFNβ treatment. Transcripts with an FDR ≤ 0.01 were considered statistically significant. The green marks on the left indicate overlaps with IFNα- responsive genes in human fibroblasts or conserved (core) between ten different species , brown marks show significant regulation in the different cells. Genes were clustered according to Euclidean distances with Ward’s clustering criterion. (D) Venn diagram showing overlaps of genes regulated in an IRF3-specific manner in response to ISD treatment (IRF3-dependent genes), regulated upon IFNβ in WT MEFs (independent of regulation in IFNAR1 -/- cells) or regulated by IFNβ only in WT but not IFNAR1 -/- MEFs (IFNAR1-responsive genes). (E) Correlation plot showing spearman correlation (blue: negative, red: positive correlation) for pairwise comparisons of log FC for indicated treatments and cell lines for IRF3-dependent genes. (F-G) Heatmaps showing the log FC (blue: down-, red: up-regulation) of (F) IRF3-dependent or (G) IFNAR1-responsive genes in WT cells after IFNβ or ISD treatment compared to controls, and in untreated knockout cell lines compared to WT. Genes significantly differentially expressed (FDR ≤ 0.01) in the IFNAR1 -/- or IRF3 -/- compared to WT MEFs are marked on the left (blue: down-, red: up- regulation).

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Transfection, Incubation, Knock-Out

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: Presence of M35 modulates expression of IRF3-dependent genes. (A) Determination of the global effect of M35’s presence on gene expression. iMEFs stably expressing M35-HAHA or a corresponding EV were stimulated by transfection of 5 µg/mL ISD, mock-transfected or left untreated and incubated for indicated times. Transcripts were labelled in the last 90 min of stimulation by incubation with 200 µM of 4-thiouridine (4sU). Total transcripts were analysed by SLAM-seq. Samples were prepared and analysed in triplicate. (B) Depicted are log FCs of transcripts of EV (x axis) vs. M35-HAHA (y axis) iMEFs after indicated times of ISD stimulation compared to mock-transfection. (C) Expression kinetics of selected transcripts upon PRR stimulation in EV and M35-HAHA iMEFs. Total RNA counts are given in transcripts per million (tpm). Differences between transcript levels in ISD-stimulated EV and M35-HAHA iMEFs with FDR < 0.01 were considered statistically significant, ns non-significant. (D) Volcano plot showing differential expression of total cellular transcripts in EV compared to M35- HAHA iMEFs in untreated conditions as log FC (x axis), plotted against -log 10 of the FDR (y axis, with significantly (FDR < 0.01) regulated transcripts above the dashed horizontal line). Numbers indicate total up- (log FC > 0) or down-regulated (log FC < 0) transcripts in the respective sections. (E) Heatmaps showing the log FC (blue: down-, red: up-regulation) in the indicated SLAM-seq samples for the 28 IRF3-dependent genes. Genes differentially expressed (FDR ≤ 0.01) in M35-expressing compared to EV iMEFs or in IFNAR1 -/- or IRF3 -/- compared to WT MEFs are marked at the left (blue: down-, red: up-regulation). (F) Response of IRF3-dependent genes upon infection with MCMV with or without M35. Immortalised BMDMs (iBMDMs) pre- treated with 1 µM ruxolitinib (IFNAR signalling inhibitor) were infected with MCMV M35stopRevertant (REV) or MCMV M35stop (M35stop) at MOI of 0.1 or mock infected. Cells were harvested 4 h post infection for RT-qPCR analysis. Relative fold induction of Ifnb1 , Ifna4 , Ifit3 , and Rsad2 transcripts was calculated based on the housekeeping gene Rpl8 , and values were normalised to REV-infected samples. Data is shown as mean ±SD and combined from two ( Ifna4 ) or three ( Ifnb1 , Ifit3 , Rsad2 ) independent experiments. Significance compared to infection with REV was calculated by Student’s t -test (unpaired, two-tailed), ns non-significant, * p < 0.05, ** p < 0.01.

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Expressing, Stable Transfection, Transfection, Incubation, Infection, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: M35 binds to specific host promoters and interferes with IRF3-dependent gene expression. Upon infection of a host cell with MCMV, pathogen-associated molecular patterns (PAMPs) are sensed by pattern recognition receptors (PRRs) and activate the transcription factors NF-κB and IRF3. NF-κB induces expression of proinflammatory cytokines, NF-κB and IRF3 together induce expression of Ifnb1 , and IRF3 regulates expression of further type I interferons and induces a subset of the interferon-stimulated genes (ISGs). Released type I interferons activate the type I interferon receptor (IFNAR). IFNAR signalling induces assembly of different transcription factors complexes, mainly interferon-stimulated gene factor 3 (ISGF3) which further drives expression of various ISGs. During MCMV infection, the viral tegument protein M35 is released and rapidly shuttles to the nucleus. M35 binds to IRF3-targeted recognition elements in host promoters and thus antagonizes recruitment of IRF3, resulting in inhibition of IRF3-driven gene expression.

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Expressing, Infection, Inhibition

Journal: bioRxiv

Article Title: KRAB zinc finger proteins ZNF587/ZNF417 protect lymphoma cells from replicative stress-induced inflammation

doi: 10.1101/2023.03.08.531722

Figure Lengend Snippet: (a) Waterfall plot of Hallmark GSEA signatures ranked by NES issued from RNA-seq data of day 6 ZNF587/417 KD in U2932 cells. Enriched signatures (NES>0) are highlighted in green. No signature was found significantly depleted. The dotted line represents p-value cutoff <0.05. Hallmark labels found enriched in KZFP Low from are highlighted in blue. (b) Bar plot depicting the number of DE TE integrants (FDR <0.05, FC >2) at day 3, 6, and 10 of ZNF587/417 KD in U2932 cells. Control shRNA-transduced cells collected in parallel to shRNA.1 KD cells were used as controls for every time point. (c) Center: Venn diagrams of DE TEs (FDR <0.05) upon ZNF587/417 KD in U2932 cells at day 6 (left) and 10 (right) after LV transduction. The number of DE TEs shared between the two time-points is shown at the intersection of the two disks. The number of TEs differentially expressed in only one of the two cell lines is shown in the non-overlapping area of the disks. Stacked bar charts depicting the proportion of up- and downregulated TEs at day 6 (left) and 10 (right) in U2932 KD cells are shown on each side of the Venn diagrams Bottom: Scatterplot of log2 fold changes of DE TEs shared between day 6 (x-axis) and day 10 (y-axis) in U2932 KD cells (ERVs were highlighted in dark red and other TEs in gray dots). (d) Scatterplot of log2 fold changes of DE ERVs at day 10 in U2932 KD cells. ERV1 elements are highlighted with dark red dots, ERVL-MaLR with purple dots, and other ERVs with gray dots. ERVs with a ZNF587 and/or ZNF417 binding motif are pointed with a triangle. The top 3 binding motifs determined in H1 embryonic stem cells were used for both KZFPs. (e) Scatter plot of RNA-seq from ZNF417/587 U2932 KD versus (shScr) control cells at day 6 of KD, outlining DEGs (grey dots, FDR <0.05) and among them the genes reported as IRF3 targets by Grandvaux et al. (dark red dots). (f) Bar plot measuring the concentration of CXCL10 cytokine by immunoassay in cell culture supernatant of ZNF587/417 shRNA.1 KD (blue) and control cells (dark red) 6 days after LV transduction normalized by the number of cells in each condition (pg/ml/millions of cells). (g) Scatter plot of RNA-seq from ZNF417/587 OCI-Ly7 KD versus control cells at day 6 of KD, outlining DEGs (grey dots, FDR <0.05) and among them genes belonging to type I/II IFN and Inflammatory response Hallmark gene sets, Interferon Signaling Reactome, and cellular response to type I, II and III IFN gene ontology terms (blue dots). (h) Waterfall plot of Hallmark GSEA signatures ranked by NES issued from RNA-seq data of day 10 ZNF587/417 KD in U2932 cells. Enriched signatures (NES>0) are highlighted in green and depleted signatures (NES<0) in yellow. The dotted line represents p-value cutoff <0.05. Hallmark labels found enriched in KZFP-Low/-High groups from are highlighted in blue and dark red, respectively.

Article Snippet: U2932 cells were fixed and permeabilized using True-Nuclear TM Transcription Factor Buffer Set (Biolegend) following manufacturer’s instructions, incubated with primary anti-dsDNA antibody (1:100, Mybiosource), anti-phospho-STING Ser365 (1:400, CST), or anti-phospho-IRF3 Ser396 (1:100, CST) for 30 minutes, washed, and incubated with secondary anti-Mouse IgG Alexa Fluor 488 (1:100, Invitrogen) or anti-Rabbit IgG Alexa Fluor 568 (1:100, Invitrogen) for another 30 minutes.

Techniques: RNA Sequencing Assay, shRNA, Transduction, Binding Assay, Concentration Assay, Cell Culture

Journal: bioRxiv

Article Title: KRAB zinc finger proteins ZNF587/ZNF417 protect lymphoma cells from replicative stress-induced inflammation

doi: 10.1101/2023.03.08.531722

Figure Lengend Snippet: (a) Number of DEGs of RNA-seq analysis performed at day 3 (n=2), day 6 (n=3), and day 10 (n=2) of ZNF587/417 KD vs control U2932 cells. (b) Euler diagrams of the overlap of DEGs upon ZNF587/417 KD in U2932 cells at each time point (day 3, 6, and 10). (c) Scatter plot of log2 normalized counts of ZNF417/587 KD cells vs. control U2932 at day 6, outlining DEGs (grey dots) among which genes belonging to type I/II IFN and Inflammatory response Hallmark gene sets, Interferon Signaling Reactome, and cellular response to type I, II and III IFN gene ontology terms are depicted in blue. (d) Representative images and dot plot showing the mean intensity of cytosolic double stranded DNA per cell, measured on z-stack immunofluorescence images (n ≥ 80 cells per condition). Statistics: Two-sided Mann–Whitney U-test. (e,f) same as (d) for phosphorylated STING (e) and phosphorylated IRF3 (f) signal respectively. (g) Top: Venn diagrams showing the overlap of DEGs upon ZNF587/417 KD in U2932 and OCI-Ly7 at day 6. Bottom: Scatterplot of Log2 fold changes of DEGs shared between U2932 (x-axis) and OCI-Ly7 (y-axis) KD cells. Blue dots highlight the genes belonging to IFN-/Inflammatory response terms detailed in (c). The best-fit line in grey results from the linear regression analysis of U2932 log2 fold changes onto OCI-Ly7 log2 fold changes. (h) Top: Venn diagrams showing the overlap of DEGs upon ZNF587/417 KD in U2932 and OCI-Ly7 cells at day 6 and genes discriminating KZFP High and KZFP Low DLBCLs. Bottom: Scatterplot of log2 fold changes of DEGs shared between genes discriminating KZFP High /KZFP Low DLBCLs (x-axis) and U2932 KD cells (y-axis). The best-fit line in black results from the linear regression analysis of KZFP High vs KZFP Low log2 fold changes onto U2932 KD log2 fold changes. For this panel, DEGs were defined with a FDR <0.05 and a fold change >2. r: correlation coefficient. (i) Phagocytosis assay of U2932 pHrodo-labeled cells co-cultured with M1-polarized macrophages. Representative 24h course of pHrodo signal quantification using InCucyte time-lapse imaging of ZNF587/417 KD cells with two different shRNAs (shRNA.1 in blue and shRNA.2 in turquoise blue) and control cells (shScramble in dark red). Total pHrodo cell area per image acquired was calculated for each time-point and plotted as a time course for each condition. Right: representative images of respective conditions. Statistics: Two-way ANOVA followed by Bonferroni correction. Asterisks indicate significant differences at 24h time-point compared to controls.

Article Snippet: U2932 cells were fixed and permeabilized using True-Nuclear TM Transcription Factor Buffer Set (Biolegend) following manufacturer’s instructions, incubated with primary anti-dsDNA antibody (1:100, Mybiosource), anti-phospho-STING Ser365 (1:400, CST), or anti-phospho-IRF3 Ser396 (1:100, CST) for 30 minutes, washed, and incubated with secondary anti-Mouse IgG Alexa Fluor 488 (1:100, Invitrogen) or anti-Rabbit IgG Alexa Fluor 568 (1:100, Invitrogen) for another 30 minutes.

Techniques: RNA Sequencing Assay, Immunofluorescence, MANN-WHITNEY, Phagocytosis Assay, Labeling, Cell Culture, Imaging, shRNA