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

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Dimerization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Nuclear translocation of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). GAPDH and LaminA/C were used as cytoplasm and nuclear protein loading controls. ( D ) Immunofluorescence images showing the impact of 14,15-EET and TPPU on the subcellular localization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells; bars =50 μm. ( E ) Scheme showing the two main RNA sensor-induced IRF3 activation pathways. ( F-G ) AC16 cells that were uninfected (CON) or infected with CVB3 were treated with 11,12-EET, 14,15-EET or TPPU (n=3). Shown are changes in IRF3 phosphorylation following the siRNA-mediated down regulation of MDA5 (F), or TLR3 (G). ( H ) Activity of a IFNβ-luciferase reporter construct in HEK293 cells expressing either a control vector (vector), pcMDA5, pcMAVS, pcTBK1 , or pcIRF3-5D and treated with solvent (CON) or 14,15-EET (1 μM) for 24 hours. ( I ) Impact of the siRNA-mediated downregulation of TBK1 on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Primary antibodies were as follows: sEH (1:1000,10833-1-AP, Proteintech Group, Shanghai, China), IRF3 (1:1000, 4302, Cell Signaling Tech, MA, US), phospho-IRF3 (Ser396) (1:1000, 29047s, Cell Signaling Tech, MA, US), TBK1(1:1000, A3458, ABclonal Biotechnology, Wuhan, China), phospho-TBK1(Ser172) (1:1000, 5483, Cell Signaling Tech, MA, US), GSK3β(1:1000, A11731, Abclonal Biotechnology, Wuhan, China), phospho-GSK3β(Tyr216) (1:1000, ab75745, Abcam, Cambridge, UK), IFNβ (1:1000, 97450S, Cell Signaling Tech, MA, US), His-tag (1:1000, AE003, Proteintech Group, Shanghai, China), Flag-tag (1:1000, 20543-1-AP, Proteintech Group, Shanghai, China), MDA5 (1:1000, 21775-1-AP, Proteintech Group, Shanghai, China), TLR3(1:1000, 17766-1-AP, Proteintech Group, Shanghai, China), GAPDH (1:10000,10491-1-AP, Proteintech Group, Shanghai, China), Tubulin (1:2000, 11224-1-AP, Proteintech Group, Shanghai, China), β-actin (1:10000, 66009-1-lg, Proteintech Group, Shanghai, China), LaminA/C (1:1000, A0249, Abclonal Biotechnology, Wuhan, China).

Techniques: Infection, Translocation Assay, Immunofluorescence, Activation Assay, Activity Assay, Luciferase, Construct, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Impact of the siRNA-mediated downregulationof GSK3β on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Impact of the EET antagonist; EEZE (1 μM) on the phosphorylation of GSK3β in AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Co-immunoprecipitation of TBK1 with a GSK3β-Flag fusion protein from HEK293 cells treated with 14,15-EET (1 μM) or TPPU (10 μM) in the absence or presence of EEZE (n=3). ( D ) Impact of the wild-type GSK3β and the Y216F and Y216D GSK3β mutants on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3). ( E-F ) Impact of GSK3β mutation alone and in combination with 14,15-EET on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3-4). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Primary antibodies were as follows: sEH (1:1000,10833-1-AP, Proteintech Group, Shanghai, China), IRF3 (1:1000, 4302, Cell Signaling Tech, MA, US), phospho-IRF3 (Ser396) (1:1000, 29047s, Cell Signaling Tech, MA, US), TBK1(1:1000, A3458, ABclonal Biotechnology, Wuhan, China), phospho-TBK1(Ser172) (1:1000, 5483, Cell Signaling Tech, MA, US), GSK3β(1:1000, A11731, Abclonal Biotechnology, Wuhan, China), phospho-GSK3β(Tyr216) (1:1000, ab75745, Abcam, Cambridge, UK), IFNβ (1:1000, 97450S, Cell Signaling Tech, MA, US), His-tag (1:1000, AE003, Proteintech Group, Shanghai, China), Flag-tag (1:1000, 20543-1-AP, Proteintech Group, Shanghai, China), MDA5 (1:1000, 21775-1-AP, Proteintech Group, Shanghai, China), TLR3(1:1000, 17766-1-AP, Proteintech Group, Shanghai, China), GAPDH (1:10000,10491-1-AP, Proteintech Group, Shanghai, China), Tubulin (1:2000, 11224-1-AP, Proteintech Group, Shanghai, China), β-actin (1:10000, 66009-1-lg, Proteintech Group, Shanghai, China), LaminA/C (1:1000, A0249, Abclonal Biotechnology, Wuhan, China).

Techniques: Infection, Immunoprecipitation, Mutagenesis

Journal: Cell Death and Differentiation

Article Title: CD-NTase family member MB21D2 promotes cGAS-mediated antiviral and antitumor immunity

doi: 10.1038/s41418-023-01116-1

Figure Lengend Snippet: A Western blot analysis of p-STING, p-TBK1 and p-IRF3 in WT and Mb21d2 -/- peritoneal macrophages after ISD or cGAMP stimulation, or HSV-1 infection for indicated times. B Western blot analysis of p-TBK1 and p-IRF3 in sg Ctrl, sg KO, and sg KO reintroduced of MB21D2 HeLa cells after ISD or cGAMP stimulation, or HSV-1 infection for indicated times. C Microscopy images of IRF3 nuclear translocation in sg Ctrl or sg KO HeLa cells after ISD stimulation for 4 h (left). The percentage of cells with nuclear translocation was quantified (right), at least 100 cells from each group were analyzed, n = 3. Scale bar, 20 μm. D Microscopy images of IRF3 nuclear translocation in WT or Mb21d2 -/- MEFs after ISD stimulation for 4 h (left). The percentage of cells with nuclear translocation was quantified (right), at least 100 cells from each group were analyzed, n = 3. Scale bar, 20 μm. Data are shown as mean ± SD and were analyzed by unpaired two-tailed Student’s t test ( C , D ) (* p < 0.05, ** p < 0.01).

Article Snippet: The following antibodies were used: anti-cGAS (#31659), anti-cGAS (#83623), anti-TBK1 (#3013), anti-phospho-TBK1 (#5483), anti-IRF3 (#4302), anti- phospho-IRF3 (#4947), anti-Myc (#2276) were purchased from Cell Signaling Technology; anti-VSV-G (V5507), anti-MB21D2 (SAB2108874) were purchased from Sigma-Aldrich; anti-Flag (PA1-984B) was purchased from Thermo Fisher Scientific; anti-HA (sc-7392), anti-Actin (sc-58673) were purchased from Santa Cruz Biotechnology; FITC anti-mouse CD3 (#100203), PE anti-mouse IFN-γ (#505807), APC anti-mouse Perforin (#154403), PE/Cy7-anti-mouse CD80 (#104733), PerpCP/Cyanine5.5 anti-mouse CD86 (#105027), FITC anti-mouse CD11c (#117305) were purchased from BioLegend; PE anti-mouse CD62L (#561918), PerCP-Cy5.5 anti-mouse CD8α (#561109), APC anti-mouse CD69 (#560689), PE/Cyanine7 anti-human/mouse Granzyme B (#372213) were purchased from BD Biosciences; APC anti-mouse-MHC class II (ab93559) was purchased from Abcam.

Techniques: Western Blot, Infection, Microscopy, Translocation Assay, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: CD-NTase family member MB21D2 promotes cGAS-mediated antiviral and antitumor immunity

doi: 10.1038/s41418-023-01116-1

Figure Lengend Snippet: A HEK293T cells were respectively transfected with Myc-tagged cGAS, STING, RIG-I, MAVS, TBK1, or IRF3 together with Flag-tagged MB21D2 for 24 h. Coimmunoprecipitations and immunoblots were performed with indicated antibodies. B Coimmunoprecipitations and immunoblots of MB21D2 with cGAS in THP-1 cells after ISD stimulation for indicated times. C Microscopy images of cGAS and MB21D2 colocalization in HeLa cells. Pearson correlation coefficient (PCC) was analyzed by Image J. Scale bar, 5 μm. D Coimmunoprecipitations and immunoblots of purified His-tagged hcGAS FL or hcGAS-ΔN Mutant protein with purified MB21D2 protein. E Coimmunoprecipitations and immunoblots of purified His-tagged mcGAS FL or mcGAS-ΔN Mutant protein with purified MB21D2 protein. F Coimmunoprecipitations and immunoblots of purified His-tagged MB21D2 FL or MB21D2 Mutant protein with purified mcGAS protein.

Article Snippet: The following antibodies were used: anti-cGAS (#31659), anti-cGAS (#83623), anti-TBK1 (#3013), anti-phospho-TBK1 (#5483), anti-IRF3 (#4302), anti- phospho-IRF3 (#4947), anti-Myc (#2276) were purchased from Cell Signaling Technology; anti-VSV-G (V5507), anti-MB21D2 (SAB2108874) were purchased from Sigma-Aldrich; anti-Flag (PA1-984B) was purchased from Thermo Fisher Scientific; anti-HA (sc-7392), anti-Actin (sc-58673) were purchased from Santa Cruz Biotechnology; FITC anti-mouse CD3 (#100203), PE anti-mouse IFN-γ (#505807), APC anti-mouse Perforin (#154403), PE/Cy7-anti-mouse CD80 (#104733), PerpCP/Cyanine5.5 anti-mouse CD86 (#105027), FITC anti-mouse CD11c (#117305) were purchased from BioLegend; PE anti-mouse CD62L (#561918), PerCP-Cy5.5 anti-mouse CD8α (#561109), APC anti-mouse CD69 (#560689), PE/Cyanine7 anti-human/mouse Granzyme B (#372213) were purchased from BD Biosciences; APC anti-mouse-MHC class II (ab93559) was purchased from Abcam.

Techniques: Transfection, Western Blot, Microscopy, Purification, Mutagenesis