Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A) CRITR-seq vector with a type III interferon reporter. Functional gRNA is constitutively transcribed from the U6 promoter. gRNA sequence is also present in the reporter mRNA transcribed from the IFNL1 promoter, serving as a barcode indicating which edit occured in the transcribing cell. SIN LTR = self-inactivating long terminal repeat, LNGFR = low-affinity nerve growth factor receptor, WPRE = woodchuck hepatitis virus posttranscriptional regulatory element. (B) Model of amplicon sequencing and data analysis of CRITR-seq screen. Amplicon sequencing is performed on the gRNA-containing regions of the genomic DNA and polyadenylated mRNA. The mRNA/gDNA ratio for each gRNA sequence represents the normalized IFNL1 transcription levels for that guide. Graph represents a distribution of all gRNAs based on their mRNA/gDNA ratio, with individual examples highlighted as colored points plotted based on their hypothetical Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) score. (C) Workflow for the CRITR-seq screen in this study. 3 libraries were generated independently, starting from PCR-amplifying the gRNAs out of the GeCKO library . A clonal line of Cas9-expressing A549 cells was transduced with lentivirus carrying the CRITR-seq vector at an MOI of 1.5, with an assumption that most gRNAs would not affect the interferon induction phenotype, so very little epsitasis would complicate our measurements. After allotting 10 days for gene editing, edited cells were infected with NS1 mut influenza A virus at an MOI of 2 based on qPCR titer.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Plasmid Preparation, Functional Assay, Sequencing, Virus, Amplification, Genome Wide, CRISPR, Knock-Out, Generated, Expressing, Transduction, Infection

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A) Three CRITR-seq libraries were generated by cloning the GeCKO library gRNA sequences into the CRITR-seq vector three independent times. Amplicon sequencing was performed on the region containing the gRNA in the plasmid library, the genomic DNA of the transduced A549 cells, and the mRNA from the transduced cells after 8 hour infection by NS1 mut . gRNAs were identified for reads matching the CRITR-seq vector for the 10 nucleotides upstream and 13 nucleotides downstream of the gRNA sequence, requiring perfect matching. (B-D) Distribution of gRNAs by count in the plasmid (B), genomic DNA (C), and RNA (D) libraries.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Generated, Cloning, Plasmid Preparation, Amplification, Sequencing, Infection

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A) Distribution of gRNAs across all 3 replicates of the CRITR-seq screen, based on mRNA/gDNA ratio. gRNAs with less than 25 reads in genomic DNA in any replicate were excluded from analysis. Read counts across replicates were normalized based on the non-targeting gRNAs from each replicate. (B) Distribution of genes based on the median mRNA/gDNA ratio for surviving guides targeting that gene across all 3 replicates. Individual genes from the RIG-I signaling pathway, shown in (C), are plotted based on their MAGeCK robust ranking aggregation score for depletion in the mRNA. MAGeCK statistical scores were calculated using non-targeting gRNAs as the null distribution, with depletion in the mRNA as the alternative hypothesis. (C) RIG-I signaling pathway. Genes/proteins in blue are considered essential for interferon induction through this pathway, while genes/proteins in green may be partially redundant. IRF7 was excluded from this analysis because in epithelial cells it is expressed at very low levels prior to interferon signaling and likely does not contribute to interferon transcription at this early time point , . (D) mRNA/gDNA ratios from (B), subsetted by gene category. “Proviral” genes were identified by Li et al. in a CRISPR screen for genes required for influenza infection in A549 cells. “Early” genes are a subset of the proviral genes annotated to be involved in viral entry, nuclear import, viral transcription/replication, or nuclear export. Proviral and Early genes tested are listed in Table S1. RIG-I pathway genes are those shown in (C). Solid lines represent the median; dotted lines represent the first and third quartiles. * indicates ANOVA p<0.05, post-hoc Tukey’s test q<0.05.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: CRISPR, Infection

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A) Same distribution of genes from CRITR-seq screen as in , but with the top 3-ranked genes enriched in the mRNA highlighted. MAGeCK statistical scores here were calculated with enrichment in the mRNA as the alternative hypothesis, using non-targeting gRNAs as null distribution. (B) A549 cells were transfected with Cas9-gRNA ribonucleoprotein complexes with gRNAs targeting the indicated genes. After passaging 10 days to allow for gene editing, cells were infected with NS1 mut at a genome-corrected MOI of 2. RNA was harvested at 8 hours post-infection for qPCR analysis of IFNB1 and IFNL1 transcripts. NTC = non-targeting control. (C-D) A549 cells were treated with siRNA for 9 days and then infected with NS1 mut at a genome-corrected MOI of 1 (C) or WT WSN at an infectious MOI of 1 (D). RNA was harvested 8 hours post-infection for qPCR analysis. The same results were obtained for the WSN NS1 stop virus (with WSN genetic background for all segments), shown in . Validation of NELFB knockdown is shown in . (E) A549 cells were treated with siRNA for 9 days before harvesting RNA for qPCR. n.d. = not detected. Validation of NELFB knockdown is shown in . (F) A549 cells were treated with siRNA for 9 days and transfected with 50 ng poly(I:C) for 8 hours before harvesting RNA for qPCR. Validation of NELFB knockdown is shown in . Biological replicates are shown as individual data points, with lines representing the means. One-tailed t-test (B) with increased expression as the alternative hypothesis, or two-tailed t-tests (C-F) were performed to compare each treatment with the non-targeting control. n=3 (B,D-F) or n=4 (C). * indicates p<0.05 after Benjamini-Hochberg multiple hypothesis correction.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Transfection, Passaging, Infection, Control, Virus, Biomarker Discovery, Knockdown, One-tailed Test, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A) RNA from experiment performed in and , with HA transcripts measured by qPCR. The same results were obtained for the WSN NS1 stop virus, shown in . NTC = non-targeting control. (B) A549 cells were treated with siRNA for 9 days. Cells were infected with PB1 455:350 at an infectious MOI of 1, with or without 100 nM baloxavir acid added at time of infection. RNA was harvested 8 hours post infection for analysis by qPCR. Validation of NELFB knockdown is shown in . (C) An A549 IFNL1 reporter cell line was treated with siRNA for 9 days and infected with NS1 mut at a genome-corrected MOI of 1. 13 hours post infection, cells were stained for the viral protein M2 and fixed for flow cytometry. Graph shows distribution of M2 staining normalized for unit area, for one representative replicate. Full flow data shown in . (D) A549 cells were treated with either of 2 different NELFB -targeting siRNAs, a non-targeting control, or no siRNA, with 2 biological replicates per treatment. After 9 days of siRNA treatment, cells were infected with NS1 mut at a genome-corrected MOI of 2. RNA was harvested 8 hours post infection, and 5’ RACE was performed on polyadenylated mRNAs, followed by sequencing. Cap-snatched sequence length refers to the number of nucleotides between the template switch oligo sequence and the +1 position of the flu mRNA sequence. Violin plots contain box plots for each sample, and the median of all samples is represented by the gray dotted line. Violin plot whiskers extend to the most extreme points in the dataset, excluding the top 2% of lengths. Validation of NELFB knockdown phenotype is shown in . (E) A549 cells were treated with siRNA for 4 days and infected with PB1 455:350 at an infectious MOI of 1. RNA was harvested 8 hours post infection for analysis by qPCR. (F) A549 cells were treated with siRNA 9 days before infection with WT WSN at an infectous MOI of 5. Media was replaced with fresh IGM 2 hours post infection. 14 hours post infection, viral supernatant was collected and cells were lysed for RNA extraction. Reverse transcription was performed using universal influenza primers for the RNA from the supernatant, and random hexamer primers for the RNA from the cell lysate. Further qPCR analysis is shown in . For panels A-B and E-F, biological replicates are shown as individual data points, with lines representing the means. Two-tailed t-tests were performed to compare targeted siRNA with the non-targeting control. n=4 (A, left) or n=3 (A, right; B; E-F). Benjamini-Hochberg multiple hypothesis correction was performed for panels B and F. * indicates p<0.05.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Virus, Control, Infection, Biomarker Discovery, Knockdown, Staining, Flow Cytometry, Sequencing, RNA Extraction, Reverse Transcription, Random Hexamer, Two Tailed Test

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: A549 IFNL1 reporter cells were treated for 9 days with siRNA and infected, or not, with NS1 mut at a genome-corrected MOI of 1. 13 hours post infection, cells were stained for the viral protein M2 and fixed for flow cytometry. (A) Individual replicates shown. The threshold for IFNL1 + or M2 + cells was set at the fluorescence level for which an average of 0.1% of uninfected cells would be called as positive events. Interferon-positive events colored in orange. For visualization, data was subsetted to 5000 events to show equivalent numbers between conditions. (B) Mean fluorescence intensity (MFI) of M2 staining. Two-tailed t-test was performed to compare targeted siRNA with the non-targeting control, n=3, * indicates p<0.05.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Infection, Staining, Flow Cytometry, Fluorescence, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: A549 cells were treated with either of 2 different NELFB -targeting siRNAs, a non-targeting control, or no siRNA, with 2 biological replicates per treatment. After 9 days of siRNA treatment, cells were infected with NS1 mut at a genome-corrected MOI of 2. RNA was harvested 8 hours post infection for 5’ RACE and sequencing and qPCR analysis. qPCR results shown here confirm NELFB knockdown and a consistent phenotype of increased viral RNA and interferon production in the samples treated with NELFB siRNA. Biological replicates are shown as individual data points, with lines representing the means. Two-tailed t-tests were performed to compare each sample with the untreated condition and with the non-targeting control, n=2. * indicates p<0.05 after Benjamini-Hochberg multiple hypothesis correction.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Control, Infection, Sequencing, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A-B) Same experiment as in , with different RNAs measured by qPCR. A549 cells were treated with siRNA for 9 days before infection with WT WSN at an infectious MOI of 5. Media was replaced with fresh IGM 2 hours post infection. 14 hours post infection, viral supernatant was collected and cells were lysed for RNA extraction of both. Reverse transcription was performed using universal influenza primers for the RNA from the supernatant, and random hexamer primers for the RNA from the cell lysate. Biological replicates are shown as individual data points, with lines representing the means. Two-tailed t-tests were performed to compare targetign siRNA with the non-targeting control, n=3. Benjamini-Hochberg multiple hypothesis correction was performed for panel A. * indicates p<0.05.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Infection, RNA Extraction, Reverse Transcription, Random Hexamer, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A-C) Wild-type A549 cells (A-B) or a RIG-I -knockout cell line derived from a single cell clone (C) were treated with siRNA for 9 days. Cells were infected with NS1 mut at a genome-corrected MOI of 1, with or without 100 nM baloxavir acid added at time of infection. RNA was harvested 8 hours post infection for qPCR analysis. NTC = non-targeting control. We note the RIG-I -knockout cell line has some residual RIG-I expression observed by Western blot after flu infection . HA qPCR measurements in these cells are shown in . (D) Flow cytometry experiment from , with zsGreen IFNL1 reporter results shown. Left: % IFNL1 + cells determined as the percent of cells positive for zsGreen expression. Right: mean fluorescence intensity (MFI) of zsGreen for IFNL1 + cells. Full flow data shown in . (E) Cells from (D) were divided into bins based on levels of M2 staining. % IFNL1 + cells plotted for each bin. Bins with less than 100 events were removed from plotting and analysis. Points represent biological replicates, with lines indicating the means. Two-tailed t-tests were performed comparing the NELFB siRNA samples with the non-targeting control for each treatment condition (A-D) or for each M2 expression bin (E), n=3. * indicates p<0.05 after Benjamini-Hochberg multiple hypothesis correction.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Knock-Out, Derivative Assay, Infection, Control, Expressing, Western Blot, Flow Cytometry, Fluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: We transfected A549 cells with Cas9 ribonucleoprotein using a gRNA targeting RIG-I . After performing dilution cloning to isolate single cells, the clonal-derived line was infected with NS1 mut at a genome-corrected MOI of 2 for 24 hours. Infected and uninfected A549 or A549 RIG-I knockout cells were lysed for SDS-PAGE and Western blot.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Transfection, Cloning, Derivative Assay, Infection, Knock-Out, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A-B) A549 cells were infected, or not, with PB1 455:350 , at an infectious MOI of 1, with or without 100 nM baloxavir acid or 100 nM pimodivir added 2 hours prior to infection. RNA was harvested 14 hours post infection for qPCR analysis. (C-D) A549 cells were infected with a high-defective population of WT WSN at an MOI of 2 based on qPCR titer. For drug treatments, cells were treated with 10 nM baloxavir acid and/or 50 μg/mL cycloheximide (CHX) at time of infection. RNA was harvested 9 hours post infection for qPCR analysis. Points represent biological replicates, with lines indicating the means. For A and B, two-tailed t-tests were performed comparing each treatment with the uninfected sample and with the infected sample without inhibitors, n=3. For C and D, two-tailed t-tests were performed comparing presence and absence of baloxavir, for each CHX condition, n=3. * indicates p<0.05 after Benjamini-Hochberg multiple hypothesis correction.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Infection, Two Tailed Test

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) The left graph shows the X-ray crystal structure of a p50 / p65 heterodimer bound to DNA as published in (PDB 1kvx), while the right graph shows the entire p65 protein structure including the disordered C-terminal half as calculated by alphafold ( https://alphafold.ebi.ac.uk/entry/Q04206 ). Residues required for dimerization (Phe (F) 213, Leu (L) 215) or DNA binding (Glu (E) 39) are indicated in both structures. (B) Scheme of the HA-tagged p65-miniTurbo fusion proteins that were used to reconstitute p65-deficient HeLa cells under the control of a tetracycline-sensitive promoter. F213 and L215 in p65 wildtype (wt) were mutated to Asp (FL / DD) for dimerization-deficient p65 or E39 to Ile (E / I) for DNA-binding-deficient p65. (C) Principle of proximity-based biotin tagging. (D) Pools of HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected (using branched Polyethyleneimine, PEI)) with the constructs shown in (B) and their expression was induced with doxycycline (1 µg / ml) for 17 h. At the end of this incubation, intracellular biotinylation was induced by adding 50 µM biotin for 70 minutes as indicated. Additionally, half of the samples were treated with IL-1α (10 ng / ml) for the last 60 minutes. Cell cultures expressing HA-miniTurbo only (empty vector, EV) or receiving only doxycycline or biotin served as negative controls (indicated by gray font). Parental HeLa cells (p) were included as further controls. Left panel: Cells were lysed and proteins were analyzed by Western blotting for the expression of p65-HA-miniTurbo and HA-miniTurbo using anti p65 and anti HA antibodies. Equal loading was confirmed by probing the blots with anti β-actin antibodies. Right panel: Biotinylated proteins from the same samples were purified on streptavidin agarose beads and biotinylation patterns were visualized by Western blotting using streptavidin-horseradish peroxidase (HRP) conjugates (representative images from two independent experiments). (E) Biotinylated proteins from the experiment shown in (C) and from a second biological replicate were identified by mass spectrometry. Volcano plots show the ratio distributions of Log 2 -transformed mean protein intensity values on the X-axes obtained with wild type p65 or the p65 mutants compared to the empty vector controls in the presence or absence of IL-1α treatment. Y axes show corresponding p values from t-test results. Strong enrichment of the bait p65 / RELA proteins together with the core canonical NF-kB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). (F) Specific proteins binding to p65 / RELA wild type were defined by significant enrichment (LFC ≥ 2, -log 10 p ≥ 1.3) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only (see ). This set of proteins was intersected with proteins enriched in cells expressing p65 mutant proteins (LFC ≥ 2, -log10 p ≥ 1.3). Venn diagrams show the numbers of p65 / RELA interactors and their overlaps before and after IL-1α-treatment, with values in the lower left corners indicating total numbers of interactors. (G) The six protein sets shown in (E) were subjected to parallel overrepresentation pathway analysis using Metascape software . The Venn diagrams show the overlap of the top 100 enriched pathway terms. For IL-1α samples, only 92 terms were enriched. Values in the lower left corners indicate total numbers of unique pathways. (H) The table shows the most strongly enriched pathway categories associated with the p65 / RELA wild type or mutant interactomes. Numbers in brackets indicate the total numbers of p65 / RELA interactors per condition that were subjected to overrepresentation analysis according to (E, F). The mass spectrometry data and bioinformatics analysis results are provided in Supplementary Table 1. See also and . rtTA, reverse tetracycline-controlled transactivator.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Binding Assay, Control, CRISPR, Transfection, Construct, Expressing, Incubation, Plasmid Preparation, Western Blot, Purification, Mass Spectrometry, Transformation Assay, Mutagenesis, Software

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Parental HeLa cells or pools of HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected with empty vector (EV) encoding HA-miniTurbo (HA-mTb) or with p65 / RELA wild type (wt) fused C-terminally to HA-mTb (p65(wt)-HA-mTb) as described in the legend of . The expression of the constructs was induced with increasing concentrations of doxycycline for 17 h as indicated. At the end of the incubation, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Cell extracts were analyzed by Western blotting for the expression of the p65-HA-mTb fusion protein or HA-mTb using polyclonal antibodies raised against the C-terminus of p65 / RELA (sc-372) or a monoclonal antibody raised against N-terminal amino acids 1-286 of p65 / RELA (sc-8008), or an anti HA antibody, respectively. Note that the fusion protein is better recognized with the N-terminal antibody preparations. (B) HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected with the indicated constructs and their expression was induced with doxycycline at 1 µg / ml for 17 h. On the next day, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Total RNA was isolated and analyzed by RT-qPCR for expression of the indicated genes. Bar graphs show means ± s.d. from two biologically independent experiments. (C) Cells were transfected as in (A) and expression of the p65 / RELA fusion protein was induced 20 h later with doxycycline (10 ng / ml) for 4 h. In last period of this incubation, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Cells were lysed and cytosolic (C), soluble nuclear (N1) and insoluble, chromatin nuclear fractions (N2) were analyzed by Western blotting for the expression and distribution of p65(wt)-HA-mTb. Antibodies against RNA polymerase II, tubulin and β-actin were used to control purity of fractions and equal loading.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: CRISPR, Transfection, Plasmid Preparation, Expressing, Construct, Incubation, Western Blot, Isolation, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Biotinylated proteins from the experiments shown in and from a second biological replicate were identified by mass spectrometry in the presence or absence of IL-1α treatment of cells. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild type p65 in the presence of doxycycline and biotin (wt) compared to the empty vector control (EV) or compared with conditions in which only biotin (wt(bio)) or doxycycline (wt(dox)) were added to the cell cultures, to determine false positive values in the absence of expression of fusion protein but facilitated biotinylation, or in the absence of biotinylation but induced expression of the fusion protein, respectively. X-axes show mean ratio value and Y-axes show p values from t-test results. Strong enrichment of the bait p65 / RELA proteins together with the core canonical NF-kB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). (B) Specific proteins binding to p65 / RELA wild type were defined by significant enrichment (LFC ≥ 2, -log 10 p ≥ 1.3) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only as shown in (A). Venn diagrams show the total numbers of specific p65 / RELA interactors and their overlaps before and after IL-1α-treatment. The intersecting 279 (without IL-1α) and 310 (with IL-1α) interactors were pooled, resulting in the set of 366 specific p65 / RELA interactors that was used for further downstream analyses. Numbers in the left lower corner of the boxes indicate the total number of detected interactors.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Mass Spectrometry, Transformation Assay, Plasmid Preparation, Control, Expressing, Binding Assay

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Protein interaction network of the 46 known p65 / RELA interactors found by miniTurboID. Edge widths visualize the evidence for experimental interactions deposited in the STRING database . Nodes are colored in red and are arranged according to the enrichment found by proximity labeling in our study. (B) Venn diagram of p65 / RELA interactors in IL-1α or untreated cells revealing a total of 366 unique p65 / RELA interactors, of which 320 (87.4 %) have no documented protein interaction entries in STRING. (C) Overlap of the RELA interactome with 1639 human TFs and 801 epigenetic regulators . (D) Graphs visualizing the top 10 enriched epigenetic regulators. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild type p65 / RELA (wt) or with p65 / RELA mutants (FL/DD, E/I) compared to empty vector controls (EV). Only 9 reader proteins were found. (E) Association of enriched epigenetic regulators with known epigenetic complexes according to the annotation provided by . Numbers in brackets show identified components per complex. (F) Venn diagram showing the overlap of enriched TFs in basal or IL-1α-stimulated conditions. (G) Volcano plots visualizing all TFs significantly enriched with wt p65 / RELA (LFC ≥ 2, -log 10 p ≥ 1.3) compared with empty vector control (EV) and the changes obtained with p65 mutants in basal conditions. (H) Distribution of TF families found to be associated with p65 / RELA in basal and IL-1α-stimulated conditions according to the annotation provided by (I) IL-1α-dependent enrichment of all TF belonging to ZBTB and ZNF families as identified by miniTurboID. (J) The top 10 pathway terms according to GO (BP, CC, MF), KEGG, Reactome, STRING clusters and WikiPathways data base entries and the top 10 subcellular localizations associated with the 366 p65 / RELA interactors. Annotations, number of components and false discovery rates (FDR) were retrieved using the STRING plugin of Cytoscape . The mass spectrometry data sets and bioinformatics analysis results are provided in Supplementary Table 1.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Labeling, Transformation Assay, Plasmid Preparation, Control, Mass Spectrometry

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Final list of top ranking high confidence interactors p65 / RELA selected for further studies. The heatmap shows the Log 2 transformed mean protein intensity values from technical triplicates of the two biological independent miniTurboID experiments, the enrichment ratio values compared to the empty vector (HA-miniTurbo) control (EV) and the regulation by IL-1α. With the exception of N4BP3, all proteins were identified by at least two peptides. (B) Graph showing that the top 38 p65 / RELA interactors are largely devoid of known protein interactions based on STRING entries. According to STRING, only two factors (CEBPD and FOSL1) interact with p65 /RELA. Node borders visualize the main functional annotations. (C) HeLa cells were transiently transfected for 48 h with 20 nM of siRNAs mixtures for 38 HCI and p65 / RELA, a siRNA targeting luciferase, transfection reagent alone or were left untreated (untr.). Half of the cells per plate were treated for 1 h with IL-1α (10 ng / ml) at the end of the incubation. cDNAs were transcribed in lysates and amplicons for three NF-kB target genes, two housekeeping genes and all 38 HCI p65 / RELA interactors were pre-amplified by linear PCR and then quantified by qPCR. Based on Ct values, mRNA levels were quantified and normalized against GUSB . The effects of knockdowns were calculated separately for basal and IL-1α-inducible conditions against the luciferase siRNA. The heatmap shows hierarchically Kmeans clustered mean ratio values derived from three biologically independent siRNA screens. As a positive control, RELA knockdowns were performed in parallel. Green colors highlight p65 / RELA interactors selected for further analysis. (D) The miniTurboID enrichment of six p65 / RELA interactors (green colors) chosen from (C) is shown. The complete set of data of the screen is provided in Supplementary Table 2. See also .

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Transformation Assay, Plasmid Preparation, Control, Functional Assay, Transfection, Luciferase, Incubation, Amplification, Derivative Assay, Positive Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Scheme illustrating the arrangement of siRNAs and controls on individual cell culture plates and the performance of RT-qPCR measurements in cell extracts without prior RNA purification. A linear PCR amplification step was included to pre-amplify specific transcripts. (B) Confirmation of knockdown of 38 HCI and of RELA mRNAs by RT-qPCR as shown in (A). Bar graphs show mean changes ± s.d. relative to the luciferase siRNA controls (siLuci) from three biologically independent experiments.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Cell Culture, Quantitative RT-PCR, Purification, Amplification, Knockdown, Luciferase

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: Proximity-ligation assays coupled to immunofluorescence (IF) were performed with HeLa cells or Δp65 HeLa cells lacking endogenous p65 / RELA to demonstrate interactions of p65 / RELA with TFE3 (A), TFEB (B), GLIS2 (C) and ZBTB5 (D) using pairs of antibodies as indicated. PLA-spots are colored in red, while p65 IF is colored in green. Nuclear DNA is counterstained with Hoechst (blue signals). The images show representative fluorescence raw data and the violin plots on the right show quantification from the numbers of cells indicated in brackets. Samples omitting one of the two antibodies or both primary antibodies (ctr) served as negative controls. Solid lines indicate medians and dashed lines indicate 1 st and 3 rd quartiles. Asterisks indicate results from Kruskal-Wallis tests compared to the parental control (****p ≤ 0.0001). obtained by one-way ANOVA.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Ligation, Immunofluorescence, Fluorescence, Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to analyze the influences of novel p65 / RELA interactors on basal p65 / RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. (B) HeLa cells were transiently transfected for 48 hours with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 hour at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a -log 10 p value ≥ 1.3. Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in untreated, basal conditions, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). (C) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all constitutively expressed genes and the corresponding changes in the gene subsets defined in that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. (D) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), and gene sets significantly up- or down-regulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of (B) and are marked in red. Coefficients of correlation (Pearson’s r), corresponding p values and coefficients of determination (r 2 ) rare indicated for all comparisons. The complete set of data is provided in Supplementary Table 3.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to analyze the influences of novel p65 / RELA interactors on IL-1α-regulated p65 / RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. (B) HeLa cells were transiently transfected for 48 h with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 hour at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a -log 10 p value ≥ 1.3. Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in IL-1α-treated samples, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). (C) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all IL-1α-regulated genes and the corresponding changes in the gene subsets defined in that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. Asterisks indicate significant changes as determined by a two-tailed Mann-Whitney test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). (D) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), IL-1α-regulated genes (blue), and gene sets significantly up- or down-regulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of (B) and are marked in red. Coefficients of correlation (Pearson’s r), corresponding p values and coefficients of determination (r 2 ) rare indicated for all comparisons. The complete set of data is provided in Supplementary Table 3.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to project the protein interactions of all target genes defined by knockdowns of p65 / RELA or its interactors in IL-1α-stimulated cells into combined functional networks. (B) Table summarizing the numbers of mapped IDs (= nodes) corresponding to the gene groups shown in , their protein interactions (= edges) and the protein interaction network enrichment p values as derived from STRING. (C) Cytoscape-derived PPI networks. Nodes are colored and arranged according to the deregulation of the corresponding genes by knockdown of p65 / RELA or its interactors. Edges visualize known protein interactions, including the small number of interactions reported for p65 / RELA, S100A8 / 9, and TFE3 / TFEB. No interactions were found for ZBTB5 and GLIS2.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Functional Assay, Derivative Assay, Knockdown

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to use p65 / RELA ChIPseq data for delineating chromatin recruitment of RELA together with its interactors on the basis of DNA motifs and three possible scenarios of interactions. (B) Windows of 1000 base pairs surrounding experimentally determined p65 / RELA ChIPseq peaks were searched for motifs of RELA and REL using matrices from the JASPAR data base. P values indicated significant enrichment compared to the whole genome. The Venn diagram shows the overlap and inserts show motif compositions. (C) Venn diagrams indicating the overlap of motifs found for RELA or the RELA interactors TFE3, TFEB or GLIS2 in chromosomal regions assigned to p65 / RELA ChIPseq peaks. P values indicated significant enrichment compared to the whole genome. Inserts show motif compositions. (D) All target genes that were significantly up- or downregulated under basal or IL-1α-stimulated conditions as shown in or were collected and were examined for their association with a p65 / RELA ChIPseq peak. The pie charts show the numbers of RELA, TFE3, TFEB and GLIS2 motifs detected in siRNA RELA target genes with an annotated p65 / RELA peak in their promoters or enhancers. (E) Overlap of all genes with a p65 / RELA peak in promoters or enhancers and at least one motif for the indicated transcription factors in IL_1a-stimulated conditions. (F) Genome browser view of the TNFAIP3 locus with p65 / RELA ChIPseq peaks, activated enhancers and promoters (H3K27ac), accessible chromatin (ATACseq) and mRNA production (RNAseq) before and after 1 h of IL-1α stimulation. Data sets were from GSE64224, GSE52470 and GSE134436 and are aligned to HG19 ( ; ). p65 / RELA binding regions of 1000 bp under p65 / RELA peaks and identified TF motifs are indicated by horizontal lines. (G) HeLa cells were left untreated or were starved for 24 h in HBSS. Half of the cells was treated with IL-1α (10 ng / ml) for 1 h before the end of the experiment. ChIP-qPCR was performed with the indicated antibodies or IgG controls and a primer pair covering the TNFAIP3 promoter region (marked with an arrow in ). Floating bar plots show percent input plus the mean of all values from three independent biological replicates performed with two technical replicates. The complete set of data is provided in Supplementary Table 4.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Binding Assay, ChIP-qPCR

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: Venn diagrams indicating the overlap of RELA motifs with motifs of ZBTB factors that were found by miniTurboID to interact with RELA, in chromosomal regions assigned to p65 / RELA ChIPseq peaks. P values indicated significant enrichment compared to the whole genome. Inserts show motif compositions.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques:

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Workflow and timeline for SARS-CoV-2 neutralizing antibodies identification The overall scheme shows three different phases for the identification of SARS-CoV-2 neutralizing antibodies (nAbs). Phase 1 consisted in the enrolment of COVID-19 patients (n = 14) from which PBMCs were isolated. Memory B cells were single-cell sorted (n = 4,277), and after 2 weeks of incubation, antibodies were screened for their binding specificity against the S protein trimer and S1/S2 domains. Once S protein-specific monoclonal antibodies (mAbs) were identified (n = 1,731) phase 2 started. All specific mAbs were tested in vitro to evaluate their neutralization activity against the authentic SARS-CoV-2 virus, and 453 nAbs were identified. nAbs showing different binding profiles on the S protein surface were selected for further functional characterization and to identify different neutralizing regions on the antigen. Phase 3 starts with the characterization of the heavy and light chain sequences of selected mAbs (n = 14) and the engineering of the Fc portion of three most promising candidates. The latter were also selected for structural analyses that allowed the identification of the neutralizing epitopes on the S protein. Finally, the most potent antibody was tested for its prophylactic and therapeutic effect in a golden Syrian hamster model of SARS-CoV-2 infection.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Isolation, Incubation, Binding Assay, In Vitro, Neutralization, Activity Assay, Virus, Functional Assay, Infection

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Gating strategy for single-cell sorting and monoclonal antibodies screening for S protein S1 + S2 subunits binding and neutralization of binding (NoB) activity, related to Figure 2 (A) Starting from top left to the right panel, the gating strategy shows: Live/Dead; Morphology; CD19 + B cells; CD19 + CD27 + IgD - ; CD19 + CD27 + IgD - IgM - ; CD19 + CD27 + IgD - IgM - S-protein + B cells. (B) The graph shows supernatants tested for binding to the SARS-CoV-2 S-protein S1 + S2 subunits. Threshold of positivity has been set as two times the value of the blank (dotted line). Darker dots represent mAbs which bind to the S1 + S2 while light yellow dots represent mAbs which do not bind. (B) The graph shows supernatants tested by NoB assay. Threshold of positivity has been set as 50% of binding neutralization (dotted line). Dark blue dots represent mAbs able to neutralize the binding between SARS-CoV-2 and receptors on Vero E6 cells, while light blue dots represent non-neutralizing mAbs.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: FACS, Binding Assay, Neutralization, Activity Assay

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Characterization and distribution of SARS-CoV-2 S protein-specific nAbs, related to Figure 2 (A) The bar graph shows the distribution of nAbs binding to different S-protein domains. In dark red, light blue and gray are shown antibodies binding to the S1-domain, S2-domain and S-protein trimer respectively. The total number (n) of antibodies tested per individual is shown on top of each bar. (B) The bar graph shows the distribution of nAbs with different neutralization potencies. nAbs were classified as weakly neutralizing (> 500 ng/mL; pale orange), medium neutralizing (100 – 500 ng/mL; orange), highly neutralizing (10 – 100 ng/mL; dark orange) and extremely neutralizing (1 – 10 ng/mL; dark red). The total number (n) of antibodies tested per individual is shown on top of each bar.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Binding Assay, Neutralization

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Functional characterization of potent SARS-CoV-2 S protein-specific nAbs (A–C) Graphs show binding curves to the S protein in its trimeric conformation, S1 domain, and S2 domain. Mean ± SD of technical triplicates are shown. Dashed lines represent the threshold of positivity. (D–F) Neutralization curves for selected antibodies were shown as percentage of viral neutralization against the authentic SARS-CoV-2 wild type (D), D614G variant (E), and the emerging variant B.1.1.7 (F). Data are representative of technical triplicates. A neutralizing COVID-19 convalescent plasma and an unrelated plasma were used as positive and negative control, respectively. (G–I) Neutralization potency of 14 selected antibodies against the authentic SARS-CoV-2 wild type (G), D614G variant (H), and the emerging variant B.1.1.7 (I). Dashed lines show different ranges of neutralization potency (500, 100, and 10 ng/mL). In all graphs, selected antibodies are shown in dark red, pink, gray, and light blue based on their ability to recognize the SARS-CoV-2 S1 RBD, S1 domain, S protein trimer only, and S2 domain, respectively.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Functional Assay, Binding Assay, Neutralization, Variant Assay, Negative Control

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Neutralization activity of selected nAbs against SARS-CoV-2, SARS-CoV, and MERS-CoV pseudotypes, related to Figure 3 (A–D) Graphs show the neutralizing activities of 14 selected nAbs with different SARS-CoV-2 S-protein binding profiles against SARS-CoV-2, SARS-CoV-2 D614G, SARS-CoV and MERS-CoV pseudotypes respectively. Dashed lines represent the threshold of positivity. Mean ± SD of technical duplicates are shown. In all graphs selected antibodies are shown in dark red, pink, gray and light blue based on their ability to recognize the SARS-CoV-2 S1-RBD, S1-domain, S-protein trimer only and S2-domain respectively.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Neutralization, Activity Assay, Protein Binding

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Characterization of Fc-engineered candidate nAbs, related to Figure 7 (A) the graph shows binding curves of J08, I14 and F05 MUT and WT to the FcγR2A. (B and C) graphs show binding curves of J08, I14 and F05 MUT and WT to the FcRn at pH 6.2 (B) and 7.4 (C). (D and E) Graphs show the ADNP and ADNK induced by J08, I14 and F05 MUT and WT versions; all the experiments were run as technical duplicates. In every experiment a control antibody (CR3022) and an unrelated protein were used as positive and negative control respectively. (F–H) Graphs show binding curves to the S-protein in its trimeric conformation, S1-domain and S2-domain. Mean of technical triplicates are shown. (I–K) Neutralization curves against the authentic SARS-CoV-2 wild type, the D614G variant and the B.1.1.7 emerging variant for J08-MUT, I14-MUT and F05-MUT shown in blue, green and red respectively. Data are representative of technical triplicates.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Binding Assay, Negative Control, Neutralization, Variant Assay

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet:

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Virus, Recombinant, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Antibody Labeling, Bicinchoninic Acid Protein Assay, Clone Assay, Random Hexamer Labeling, Plasmid Preparation, Mutagenesis, Luciferase, Software

Journal: bioRxiv

Article Title: TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

doi: 10.1101/2021.05.23.445113

Figure Lengend Snippet: (A) Upper panels: Graphical representations of the Ebola virus genome, and of the tetra-cistronic minigenome (4cis) encoding Renilla luciferase ( Rluc ) used in the transcription- and replication-competent (trVLP) assay. Lower panel: Schematic representation of the EBOV trVLP assay. Plasmids expressing ISGs of interest were co-transfected with viral RNP proteins into p1 target cells. Alternatively, p1 target cells were pre-treated with IFN-I prior infection with EBOV trVLPs (detailed description on Methods). (B) U87-MG-Tim1 cells were transfected with plasmids expressing EBOV’s RNP components and pre-treated with increasing amounts of Universal type-I IFN-α, IFN-α2a or IFN-ß1a, 16-24 hours prior to EBOV trVLP infection. Reporter activities were measured 24 hours later. (C) U87-MG-TIM1 and HEK293T-TIM1 cells were transfected with plasmids expressing EBOV NP, VP35, VP30 and a HA-tagged version of L-polymerase, before being treated with increasing amounts of IFN-I prior to EBOV trVLP infection as in . Cells were lysed 24 hours post-transfection and lysates analysed by western blot for the expression of the EBOV RNP proteins and HSP90. (D) ISG Screen Validation. Normal distribution of EBOV trVLP Rluc values in the presence of individually over-expressed ISGs on p1 (left panel, blue dots) and p2 (right panel, red dots) target cells, normalized to Fluc levels on the same well. The number (n) of ISGs within standard deviation (s.d. or z score) ranges is shown in the boxes. (E) HEK293T-TIM1 cells were transfected with plasmids expressing the EBOV RNP proteins together with selected ISGs. Cells lysates were analysed 48 hours later by western blot as in (C). (F) HEK293T-TIM1 cells were transfected with individual ISGs and EBOV RNP-expressing plasmids as in (E), and tested for cellular viability 48 hours later using Cell-Titer Glo luminescence-based assay (Promega). As positive control for toxicity (red bar), cells were treated for 48h with a concentration of MG132 (25µM) sufficient to reduce in 50 percent the ATP levels in the supernatant. Values are represented as percentage of luminescence obtained in control wells transfected with GFP (grey bar), and toxicity cut-off represented as a dashed line. (G) Fold activation of Firefly luciferase NF-kB, ISG56/IFIT1 or ISRE reporters (top, middle or bottom panels, respectively) in HEK293T cells transiently transfected with selected individual ISGs compared to control GFP vector. Cells were harvested either 24 hours (blue) or 48 hours (grey) post-transfection.

Article Snippet: Cell lysates and pull-down samples were subjected to SDS-PAGE and Western blots performed using antibodies against HSP90 (Santa Cruz), TRIM25 (Abcam), EBOV-NP, EBOV-VP35, HA-tag (Rockland) and ZAP (Abcam).

Techniques: Virus, Luciferase, Expressing, Transfection, Infection, Western Blot, Biomarker Discovery, Standard Deviation, Luminescence Assay, Positive Control, Concentration Assay, Control, Activation Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

doi: 10.1101/2021.05.23.445113

Figure Lengend Snippet: (A) EBOV trVLP normalized reporter activity on HEK293T-TIM1 cells transfected with EBOV RNP proteins and either GFP (grey) or TRIM25 (blue) prior to infection with increasing amounts of EBOV trVLPs (p1 target cells, left panel). Supernatants from p1 cells were harvested 24 hpi and used to infect p2 target cells (right panel), and reporter activities measured 24 hours later. (B) HEK293T-TIM1 cells were transfected with plasmids expressing either GFP (grey) or TRIM25 (blue), and transduced with BlaVP40-EBOV-GP virus like particles 24 hours later. Viral particle entry was determined 24 hours post-transduction by measuring the percentage of cells with cleaved CCF2-AM dye by flow cytometry. (C) Quantification of viral RNA transcripts present on cell lysates (left panel) and supernatants (right panel) of HEK293T-TIM1 cells transfected and infected as in (A). Strand-specific reverse transcription primers were used on total RNA extracted from cells to generate cDNAs for minigenomic RNA (vRNA), complementary RNA (cRNA), and mRNA, which were subsequently analysed by RT-qPCR. Random hexamer primers were used to generate cDNAs from total viral RNA extracted from supernatants, and qPCR analysis performed using primers/probe sets targeting 5’ trailer region of the 4cis genome or VP40 RNA. (D) U87-MG- and HEK293T-based CRISPR cells lines were treated with increasing amounts of IFN-I, and lysed 24 hours later for analysis. Protein levels of HSP90 and TRIM25 were determined by western blot on LacZ CRISPR control cells and corresponding TRIM25 CRISPR KO cell lines. (E) EBOV trVLP reporter activities on U87-MG LacZ CRISPR-TIM1 (grey) and U87-MG TRIM25 CRISPR KO-TIM1 (blue) target cells (p1) transfected with EBOV RNP proteins, and pre-treated with increasing amounts of IFN-I prior to infection. (F) Relative quantification of intracellular and supernatant trVLP RNA levels on U87-MG LacZ CRISPR-TIM1 (grey) and U87-MG TRIM25 CRISPR KO-TIM1 cells (blue) from (E). Random hexamer primers were used to generate cDNAs and RT-qPCR analysis was performed using primers/probe sets targeting trVLP 4cis genome trailer region (vRNA and cRNA, left panel), or VP40 RNA (intracellular vRNA, cRNA and mRNA, middle panel; minigenomic RNA in the supernatant, right panel). Data shown as fold change compared to control (no IFN) based on absolute copy numbers. (G) EBOV trVLP reporter activities on U87-MG LacZ CRISPR-TIM1 (solid lines) and U87-MG TRIM25 CRISPR KO-TIM1 (dashed lines) target cells transfected with EBOV RNP proteins and pre-treated with increasing amounts of IFN-α2a (blue) or IFN-ß1b (red) prior to infection. (H) U87-MG LacZ CRISPR (grey) and U87-MG TRIM25 CRISPR KO cells (blue) were treated with increasing amounts of IFN-I, and transduced the following day with a VSV-G pseudo-typed lentiviral vector expressing GFP (CSGW). The percentage of GFP-positive cells was determined 24 hours later by flow cytometry. (I) HEK293T LacZ CRISPR (grey) and HEK293T TRIM25 CRISPR KO (blue) were used as producer cells (p0) of EBOV trVLPs and reporter activities measured 48 hours post-transfection. Supernatants from p0 cells were used to infect HEK293T-TIM1 target cells (p1), and reporter activities determined 24 hours later. All the represented EBOV trVLP RLuc reporter activities are normalized to control FLuc values obtained in the same lysates. *p > 0.05, **p > 0.01 and ***p > 0.001 as determined by two-tailed paired t-test. All error bars represent ± SEM of at least three independent experiments.

Article Snippet: Cell lysates and pull-down samples were subjected to SDS-PAGE and Western blots performed using antibodies against HSP90 (Santa Cruz), TRIM25 (Abcam), EBOV-NP, EBOV-VP35, HA-tag (Rockland) and ZAP (Abcam).

Techniques: Activity Assay, Transfection, Infection, Expressing, Transduction, Virus, Flow Cytometry, Reverse Transcription, Quantitative RT-PCR, Random Hexamer, CRISPR, Western Blot, Control, Quantitative Proteomics, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

doi: 10.1101/2021.05.23.445113

Figure Lengend Snippet: (A, B, H) U87-MG- and/or HEK293T-based CRISPR cells lines were treated with increasing amounts of IFN-I, and lysed 24 hours later for protein analysis. Protein levels of HSP90 (A, B and H), RIG-I (A), FL-MAVS/mini-MAVS (B) and TBK1 (H) were determined by western blot on LacZ CRISPR control cells and corresponding CRISPR knock-out (KO) cells lines. (C, I) HEK293T-based CRISPR cell lines depicted in the figures were transfected with plasmids expressing EBOV RNP proteins and TIM-1 together with either GFP (grey bars) or TRIM25 (blue bars), and later infected with a fixed amount of EBOV trVLPs. 24 hpi cells were lysed and trVLP reporter activities measured. (D) EBOV trVLP reporter activities on U87-MG LacZ CRISPR (grey) and U87-MG RIG-I CRISPR KO (blue) target cells (p1), transfected with TIM-1 and EBOV RNP proteins, and pre-treated overnight with increasing amounts of IFN-I prior to infection. Luciferase activities measured 24 hours post-infection. (E) EBOV trVLP reporter activities on U87-MG LacZ CRISPR (grey), U87-MG FL-MAVS KO (blue) and FL-MAVS/miniMAVS CRISPR DKO (red) target cells (p1), transfected with TIM-1 and EBOV RNP proteins, and pre-treated overnight with increasing amounts of IFN-I prior to infection. Luciferase activities measured 24 hours post-infection. (F) Protein levels of HSP90 and MAVS determined by western blot lysates from HEK293T LacZ control cells, and HEK293T-MAVS DKO cells lines engineered to stably express CRISPR-resistant variants of both MAVS isoforms (MAVS CR ), miniMAVS (M1A CR ) or FL-MAVS (M142A CR ). (G) HEK293T LacZ CRISPR and engineered HEK293T-MAVS DKO cell lines from (F) were co-transfected with plasmids expressing EBOV RNP components and TIM-1 together with either GFP (grey bars) or TRIM25 (blue bars), and later infected with EBOV trVLPs. Reporter activities were measured 24 hours later. (J) U87-MG LacZ CRISPR and U87-MG TBK1 CRISPR KO cells were transfected with RNP proteins and TIM-1, followed by a IFN-I pre-treatment prior to infection with a fixed amount of EBOV trVLPs. EBOV trVLP reporter activities in p1 were measured 24 hours after infection. (K) Fold activation of a firefly luciferase NF-kB reporter in the depicted HEK293T-based CRISPR cells lines transiently transfected with TRIM25 compared to control GFP vector. Cells were harvested 48 hours post-transfection and FLuc reporter values normalised to control Renilla luciferase activity in the same lysates. All the represented EBOV trVLP Renilla reporter activities are normalized to control Firefly luciferase values obtained in the same lysates. *p > 0.05, **p > 0.01 and ***p > 0.001 as determined by two-tailed paired t-test. All error bars represent ± SEM of at least three independent experiments.

Article Snippet: Cell lysates and pull-down samples were subjected to SDS-PAGE and Western blots performed using antibodies against HSP90 (Santa Cruz), TRIM25 (Abcam), EBOV-NP, EBOV-VP35, HA-tag (Rockland) and ZAP (Abcam).

Techniques: CRISPR, Western Blot, Control, Knock-Out, Transfection, Expressing, Infection, Luciferase, Stable Transfection, Activation Assay, Plasmid Preparation, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

doi: 10.1101/2021.05.23.445113

Figure Lengend Snippet: (A) EBOV trVLP normalized reporter activity on HEK293T-TIM1 cells transfected with EBOV RNP proteins and either GFP (grey) or ZAP-L (red) prior to infection with increasing amounts of EBOV trVLPs (p1 target cells, left panel). Supernatants from p1 cells were harvested and used to infect p2 target cells (right panel), and reporter activities measured 24 hours later. (B) HEK293T- and U87-MG-based CRISPR cells lines were treated with increasing amounts of IFN-I, and lysed 24 hours later for analysis. Protein levels of HSP90 and ZAP were determined by western blot on LacZ CRISPR control cells and corresponding ZAP CRISPR KO cell lines. (C) EBOV trVLP reporter activities on U87-MG LacZ CRISPR-TIM1 (grey) and U87-MG ZAP CRISPR KO-TIM1 (red) target cells (p1), transfect with RNP proteins and pre-treated with increasing amounts of IFN-I prior to infection. Reporter activities measured 24 hours after infection. (D) Quantification of intracellular and supernatant viral RNA levels on U87-MG LacZ CRISPR-TIM1 (grey) and U87-MG ZAP CRISPR KO-TIM1 target cells (red) that were transfected with EBOV RNP proteins and pre-treated with IFN-I, prior to infection with EBOV trVLPs as in (C). Random hexamer primers were used to generate cDNAs and RT-qPCR analysis was performed using primers/probe sets targeting either the trailer region of the viral genome (vRNA and cRNA, left panel), or VP40 RNA (intracellular vRNA, cRNA and mRNA, middle panel; minigenomic RNA in the supernatant, right panel). Data presented as fold change compared to control (no IFN) based on absolute copy numbers. (E) EBOV trVLP normalized reporter activities on HEK293T LacZ CRISPR, HEK293T TRIM25 CRISPR KO and HEK293T ZAP CRISPR KO cells stably expressing TIM1, that were transfected with EBOV RNP plasmids together with GFP (grey), ZAP L (red) or TRIM25 (blue), prior to infection with a fixed amount of EBOV trVLPs (p1 target cells). Supernatants from p1 were then harvested and used to infect HEK293T-TIM1 cells (p2 target cells). (F) Relative quantification of EBOV L-Polymerase RNA transcripts on cell lysates of HEK293T LacZ CRISPR-TIM1 and HEK293T TRIM25 CRISPR KO-TIM1 cells transfected EBOV RNP plasmids in combination with either with GFP (grey) or ZAP-L (red), and infected with a fixed amount of EBOV trVLPs. Random hexamer primers were used to generate cDNAs from total RNA, and RT-qPCR analysis performed using a primers/probe sets targeting EBOV L-polymerase and gapdh . Data normalized to L-polymerase RNA levels on HEK293T LacZ CRISPR-TIM1 cells transfected with GFP based on ΔΔCt values. (G) EBOV trVLP normalized reporter activity on p2 target cells. HEK293T-TIM1 p1 cells were transfected with EBOV RNP proteins, and either GFP (grey) or ZAP-L (red) prior to infection with increasing amounts of wild-type EBOV trVLPs (trVLPwt, solid lines) or a variant with no CpG dinucleotides on the Renilla ORF of the 4cis genome (CpG low, dashed lines). Supernatants from p1 were harvested and used to infect HEK293T-TIM1 p2 target cells. (H) Quantification of viral RNA transcripts present intracellularly (upper and middle panels) and in supernatants (lower panel) of 293T-TIM1 cells transfected as in (G), and infected with a fixed amount of EBOV trVLP WT or CpG low. Random hexamer primers were used to generate cDNAs from total RNA and RT-qPCR analysis was performed as in (D). (I) EBOV trVLP normalized reporter activity on HEK293T-TIM1 p2 target cells. HEK293T LacZ CRISPR-TIM1 (grey) and HEK293T ZAP CRISPR KO-TIM1 cells (red) were transfected with EBOV RNP proteins and pre-treated with increasing amounts of IFN-I prior to infection with wild-type EBOV trVLPs (solid lines) or CpG Low EBOV trVLP (dashed lines). Supernatants from p1 were harvested and used to infect HEK293T-TIM1 p2 target cells. Reporter activities measured 24 hours post-infection. All the represented EBOV trVLP Renilla reporter activities are normalized to control Firefly luciferase values obtained in the same lysates. P > 0.05, **p > 0.01 and ***p > 0.001 as determined by two-tailed paired t-test. All error bars represent ± SEM of at least three independent experiments.

Article Snippet: Cell lysates and pull-down samples were subjected to SDS-PAGE and Western blots performed using antibodies against HSP90 (Santa Cruz), TRIM25 (Abcam), EBOV-NP, EBOV-VP35, HA-tag (Rockland) and ZAP (Abcam).

Techniques: Activity Assay, Transfection, Infection, CRISPR, Western Blot, Control, Random Hexamer, Quantitative RT-PCR, Stable Transfection, Expressing, Quantitative Proteomics, Variant Assay, Luciferase, Two Tailed Test

Journal: bioRxiv

Article Title: TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

doi: 10.1101/2021.05.23.445113

Figure Lengend Snippet: (A) Quantification of viral RNA transcripts present intracellularly (left panel) and in supernatants (right panel) of HEK293T-TIM1 cells transfected as in , and infected with a fixed amount of EBOV trVLPs. Strand-specific reverse transcription primers were used on total RNA extracted from cells to generate cDNAs for minigenomic RNA (vRNA), complementary RNA (cRNA), and mRNA, followed by RT-qPCR analysis. Random hexamer primers were used to generate cDNAs from total RNA extracted from supernatants, and qPCR analysis performed using primers targeting the 5’ trailer region of the 4cis minigenome or VP40 RNA. (B) EBOV trVLP reporter activities on U87-MG LacZ CRISPR-TIM1 (solid lines) and U87-MG ZAP CRISPR KO-TIM1 (dashed lines) target cells transfected with EBOV RNP proteins and pre-treated with increasing amounts of IFN-α2a (blue) or IFN-ß1b (red) prior to infection. (C) HEK293T LacZ CRISPR (grey) and HEK293T ZAP CRISPR KO (red) were used as producer cells (p0) for EBOV trVLPs, and reporter activities measured 48 hours post-transfection. Supernatants from p0 were used to infect HEK293T-TIM1 target cells (p1), and reporter activities determined 24 hours later. (D) Influenza A minigenome assay was performed in HEK293T LacZ CRISPR (grey), HEK293T ZAP CRISPR KO (red) and HEK293T RIG-I CRISPR KO (blue) cells transfected with Influenza polymerase components (NP, PB1, PB2 and PA) and increasing amounts of TRIM25. Normalized Fluc values are presented as percentage relative to a GFP control in each cell line. (E) HEK293T LacZ CRISPR, and HEK293T TRIM25 CRISPR KO cells were transfected with plasmids expressing the EBOV NP, VP35, VP30 proteins and a HA-tagged L Polymerase together with either pcDNA4 or ZAP-L. Cells lysates were analysed 48 hours later by western blot for the expression of HSP90, TRIM25, ZAP and the EBOV RNP complex proteins. (F) Relative quantification of intracellular viral RNA transcripts on cell lysates of HEK293T LacZ CRISPR-TIM1 and HEK293T TRIM25 CRISPR KO-TIM1 cells transfected EBOV RNP plasmids in combination with either with GFP (grey) or ZAP-L (red), and infected with a fixed amount of EBOV trVLPs. Random hexamer primers were used to generate cDNAs from total RNA, and RT-qPCR analysis performed using a primers/probe sets targeting VP40 (mRNA,cRNA and vRNA, left), Trailer (cRNA and vRNA, right) and gapdh . Data normalized to HEK293T LacZ CRISPR-TIM1 cells transfected with GFP based on ΔΔCt values. (G) Graphical representation of the ratio between observed and expected CpG dinucleotide frequencies in the full-length EBOV genomic RNA (grey), in the wild-type trVLP 4cis genome (dark blue) and in the trVLP genomic variant with no CpG dinucleotides in the Renilla reporter sequence (CpG low, light blue). (H) Graphical representation of CpG dinucleotides localization on full-length EBOV genome (upper panel), on EBOV trVLP 4cis genome (middle panel) and on a 4cis genome with no CpG on the Renilla ORF (lower panel). CpG dinucleotides present on intergenic regions are represented in blue, while the ones present on viral ORFs are represented in red. In yellow are represented the CpG dinucleotides present on the Renilla reporter gene. (I) U87-MG LacZ CRISPR and U87-MG KHNYN CRISPR KO cells were transfected with RNP proteins and TIM-1, followed by a IFN-I pre-treatment prior to infection with a fixed amount of EBOV trVLPs. EBOV trVLP reporter activities in p1 were measured 24 hours after infection. (J) Relative quantification of intracellular viral RNA levels on cellular lysates from (I). Random hexamer primers were used to generate cDNAs and RT-qPCR analysis was performed using qPCR primers/probe sets targeting the VP40 RNA (vRNA, cRNA and mRNA, left panel) or the 5’-trailer region of the trVLP 4cis minigenome (vRNA and cRNA, right panel). Data presented as fold change compared to control (no IFN) based on absolute copy numbers.

Article Snippet: Cell lysates and pull-down samples were subjected to SDS-PAGE and Western blots performed using antibodies against HSP90 (Santa Cruz), TRIM25 (Abcam), EBOV-NP, EBOV-VP35, HA-tag (Rockland) and ZAP (Abcam).

Techniques: Transfection, Infection, Reverse Transcription, Quantitative RT-PCR, Random Hexamer, CRISPR, Control, Expressing, Western Blot, Quantitative Proteomics, Variant Assay, Sequencing

Journal: bioRxiv

Article Title: TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

doi: 10.1101/2021.05.23.445113

Figure Lengend Snippet: (A) Cellular lysates from HEK293T LacZ CRISPR-TIM1, HEK293T NPC1 CRISPR KO-TIM1 and HEK293T NPC1 CRISPR KO-TIM1 cells with restored expression of NPC1 (+NPC1) were analysed by western blot for HSP90 and NPC1. (B) The cells lines used in (A) were transduced either with BlaVP40-EBOV-GP or BlaVP40-VSV-G virus-like particles, and 24 hours later the percentages of cells presenting cleavage of CCF2-AM dye were determined by flow cytometry as readout for viral particle entry. (C) EBOV trVLP normalized reporter activity on the upper mentioned cell lines (p1) transfected with EBOV RNP proteins and infected the following day with a fixed amount of EBOV trVLPs. Reporter activities were measured 24 hours later. (D) Quantification of intracellular viral RNA levels on HEK293T-TIM1 target cells 3 and 24 hours post-infection with a fixed amount of EBOV trVLPs. Prior to infection the cells were transfected with EBOV RNP expressing plasmids together with GFP (grey), TRIM25 (blue) or ZAP-L (red). Strand-specific reverse transcription primers were used on total RNA extracted from cells to generate cDNAs for minigenomic RNA (vRNA), complementary RNA (cRNA), and mRNA, followed by RT-qPCR analysis. (E) Relative quantification of NP-associated RNA on HEK293T LacZ CRISPR-TIM1 cells transfected with GFP (grey), TRIM25 (blue) or ZAP-L (red) prior to infection with wild-type or CpG low EBOV trVLPs. 3 hours post-infection cells were UV cross-linked, and EBOV NP from incoming virions was immunoprecipitated from lysates with an anti-NP antibody. Following proteinase K treatment, pulled-down RNAs were extracted with Qiazol / chloroform, random hexamer primers used to generate cDNAs, and RT-qPCR analysis performed using a primers/probe set targeting EBOV VP40 RNA. Values are presented as percentage of absolute RNA copy numbers on cells transfected with GFP. (F) HEK293T LacZ CRISPR, HEK293T ZAP CRISPR KO and HEK293T TRIM25 CRISPR KO cells stably expressing TIM1 were transfected with GFP (grey), TRIM25 (blue) or ZAP-L (red) as depicted in the panels, and later infected with wild-type (solid bars) or CpG low (striped bars) EBOV trVLPs. Relative quantification of NP-associated RNA was determined as in (E). (G) EBOV trVLP reporter activities on HEK293T-TIM1 target cells (p1), transfect with RNP proteins and pre-treated overnight with increasing amounts of T-705 (Favipiravir) prior to infection. Reporter activities measured 24 hours after infection. (H) Relative quantification of intracellular viral RNA levels on HEK293T-TIM1 p1 target cells pre-treated with increasing amounts of T-705 compound and infected with EBOV trVLPs as in (G). Random hexamer primers were used to generate cDNAs and RT-qPCR analysis was performed using qPCR primers/probe sets targeting the VP40 mRNA or gapdh as endogenous control. Data presented as fold change compared to control (no T-705) based on ΔΔCt values. (I) Graphical representation of CpG dinucleotides localization on a monocistronic genome containing a Renilla reporter gene flanked by the 5’-leader and 3’-trailer regions of EBOV genome (upper panel), and its Low-CpG variant (bottom panel) in which all CpGs in the Renilla ORF were silently mutated. CpG dinucleotides present on the trailer and leader regions are represented in blue, while in yellow are represented the CpG dinucleotides present on the Renilla reporter gene. (J) Normalized reporter activity of the monocistronic genomes on HEK293T-TIM1 target transfected with EBOV RNP proteins, and either GFP (grey) or ZAP-L (red) prior to infection with increasing amounts of wild-type monocistronic VLPs (WT, solid lines) or a variant with no CpG dinucleotides on the Renilla ORF (CpG low, striped lines). (K) HEK293T LacZ CRISPR-TIM1 cells were transfected with GFP (grey) or ZAP-L (red) and later infected with wild-type (solid bars) or CpG low (striped bars) monocistronic VLPs. Relative quantification of ZAP-associated RNA was determined as in (E). (L) HEK293T LacZ CRISPR and HEK293T NPC1 CRISPR stably expressing TIM1 were infected with either WT or ΔGP EBOV nanoluciferase trVLPs. EBOV trVLP nanoluc reporter activities were measured 48 hpi. (M) HEK293T TRIM25 CRISPR KO cells were transfected with TIM1 and either a CRISPR-resistant version of TRIM25 or YFP before infection with WT or ΔGP EBOV nanoluciferase trVLPs. EBOV trVLP nanoluc reporter activities were measured 48 hpi.

Article Snippet: Cell lysates and pull-down samples were subjected to SDS-PAGE and Western blots performed using antibodies against HSP90 (Santa Cruz), TRIM25 (Abcam), EBOV-NP, EBOV-VP35, HA-tag (Rockland) and ZAP (Abcam).

Techniques: CRISPR, Expressing, Western Blot, Virus, Flow Cytometry, Activity Assay, Transfection, Infection, Reverse Transcription, Quantitative RT-PCR, Quantitative Proteomics, Immunoprecipitation, Random Hexamer, Stable Transfection, Control, Variant Assay

Journal: bioRxiv

Article Title: TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

doi: 10.1101/2021.05.23.445113

Figure Lengend Snippet: (A) Lysates of HEK293T-TIM1 cells transfected either with GFP or TRIM25, in combination with EBOV NP and/or EBOV VP35, were immunoprecipitated with an anti-TRIM25 antibody. Cellular lysates and pull-downs were analysed by western blot for HSP90, TRIM25, EBOV NP and VP35. (B) Panels show representative fields for the localization of EBOV NP and endogenous TRIM25 on HEK293T-TIM1 cells left untreated (Null), or transfected with EBOV NP protein alone, or in combination either with VP35 or all remaining RNP proteins (VP35, VP30 and L). Cells were stained with anti-TRIM25 (red) and anti-NP (green) antibodies, as well as with DAPI (blue). White arrows point to the localization of TRIM25 intracellular aggregates. (C) Schematic representation of functional domains within TRIM25 (upper panel). Lysates of HEK293T-TIM1 cells transfected with EBOV NP in combination with GFP, TRIM25 or mutants thereof, were immunoprecipitated with an anti-TRIM25 antibody. Input and pull-down samples were blotted for HSP90, TRIM25 and EBOV NP (lower panel). (*) indicates the detected heavy-chains (HC) from the antibody used in the pull-down. (D) EBOV trVLP normalized reporter activity on HEK293T-TIM1 cells transfected with EBOV RNP proteins in combination with GFP (grey), TRIM25 wild-type (blue), TRIM25 ΔRING (red) or TRIM25 ΔSPRY (orange) mutants, prior to infection with increasing amounts of EBOV trVLPs (p1 target cells). EBOV trVLP Rluc reporter activities were measure 24 hpi and normalized to control Fluc values obtained in the same lysates. EBOV trVLP Renilla reporter activities are normalized to control Firefly luciferase values obtained in the same lysates. *p > 0.05, **p > 0.01 and ***p > 0.001 as determined by two-tailed paired t-test. All error bars represent ± SEM of at least three independent experiments. (E) HEK293T-TIM1 cells were transfected either with GFP or TRIM25, in combination with EBOV NP and/or a plasmid expressing a HA-tagged Ubiquitin (HA-Ub). Lysates from these cells were immunoprecipitated with an anti-HA antibody (left panels) or an anti-NP antibody (right panels). Cellular lysates and pull-down samples were analysed by western blot for HSP90, TRIM25, EBOV NP and HA (ubiquitin). (F) Lysates from HEK293T cells co-transfected with EBOV NP and YFP or TRIM25 were immunoprecipitated with an anti-NP antibody, and pulled-down fractions treated with USP2 deubiquitinase enzyme. Cellular lysates and pull-downs were analysed by western blot for HSP90, TRIM25 and EBOV NP.

Article Snippet: Cell lysates and pull-down samples were subjected to SDS-PAGE and Western blots performed using antibodies against HSP90 (Santa Cruz), TRIM25 (Abcam), EBOV-NP, EBOV-VP35, HA-tag (Rockland) and ZAP (Abcam).

Techniques: Transfection, Immunoprecipitation, Western Blot, Staining, Functional Assay, Activity Assay, Infection, Control, Luciferase, Two Tailed Test, Plasmid Preparation, Expressing, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

doi: 10.1101/2021.05.23.445113

Figure Lengend Snippet: (A) Lysates of HEK293T-TIM1 cells transfected either with GFP or TRIM25, in combination with EBOV NP and/or EBOV VP35 were immunoprecipitated with an anti-NP antibody. Cellular lysates and pull-downs were analysed by western blot for HSP90, TRIM25, EBOV NP and VP35. (B) Lysates of HEK293T-TIM1 cells transfected either with EBOV NP and/or TRIM25 were left untreated (NT), or treated with RNase A prior to immunoprecipitation with an anti-TRIM25 antibody. Cellular lysates and pull-downs were analysed by western blot for HSP90, TRIM25 and EBOV NP. (C) Lysates of HEK293T LacZ CRISPR and HEK293T ZAP CRISPR KO cells transfected either with GFP or TRIM25 and/or EBOV NP were immunoprecipitated with anti-TRIM25 antibody. Cellular lysates and pull-down samples were analysed by western blot for HSP90, TRIM25, EBOV NP and ZAP. (D) HEK293T-TIM1 cells were transfected either with GFP, TRIM25 wild-type or TRIM25 ΔRING mutant, in combination with EBOV NP and/or a plasmid expressing a HA-tagged Ubiquitin (HA-Ub). Lysates from these cells were immunoprecipitated 48 hours later with an anti-HA antibody. Cellular lysates and pull-down samples were analysed by western blot for HSP90, TRIM25, NP and HA (ubiquitin). (E) HEK293T LacZ CRISPR and HEK293T ZAP CRISPR KO cells were transfected either with GFP or TRIM25, in combination with EBOV NP and/or Ub-HA. Lysates from these cells were immunoprecipitated 48 hours later with an anti-HA antibody. Cellular lysates and pull-down samples were analysed by western blot as in (D). (F) HEK293T LacZ control and HEK293T FL-MAVS/miniMAVS CRISPR DKO cells were transfected either with GFP or TRIM25 in combination with EBOV NP and/or HA-Ub. Lysates were immunoprecipitated with anti-HA antibody and analysed by western blotting as in (D). (G) HEK293T LacZ CRISPR control and HEK293T TRIM25 CRISPR KO cells were infected with a fixed volume of EBOV trVLPs concentrated on a 20% sucrose-cushion. Cell were lysed at the depicted time points after infection and lysates analysed by western blot for HSP90 and EBOV NP.

Article Snippet: Cell lysates and pull-down samples were subjected to SDS-PAGE and Western blots performed using antibodies against HSP90 (Santa Cruz), TRIM25 (Abcam), EBOV-NP, EBOV-VP35, HA-tag (Rockland) and ZAP (Abcam).

Techniques: Transfection, Immunoprecipitation, Western Blot, CRISPR, Mutagenesis, Plasmid Preparation, Expressing, Ubiquitin Proteomics, Control, Infection

Journal: bioRxiv

Article Title: TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

doi: 10.1101/2021.05.23.445113

Figure Lengend Snippet: (A) Upper Panel: schematic representation of TRIM25 with the localization of the RING dimerization mutants (light blue), E3-Ligase catalytic mutants (pink), Bbox2 & coiled-coil mutants (orange) and SPRY-domain and RNA-binding mutants (light green) generated on a TRIM25 CRISPR-resistant background. Lower Panel: EBOV trVLP normalized reporter activity on HEK293T TRIM25 CRISPR KO-TIM1 cells transfected with EBOV RNP proteins in combination with GFP (grey), CRISPR-resistant (CR) TRIM25 wild-type (dark blue), or mutants thereof (see upper panel), prior to infection with EBOV trVLPs (p1 target cells). EBOV trVLP Rluc reporter activities were measure 24 hpi and normalized to control Fluc values obtained in the same lysates. All error bars represent ± SEM of four independent experiments. *p > 0.05, **p > 0.01 and ***p > 0.001 as determined by Oneway-ANOVA. Statistics represented above graphic bars were calculated as multiple comparisons to TRIM25 wild-type, while the statistics within graphic bars are represented in function of multiple comparison to YFP. (B) HEK293T TRIM25 CRISPR KO cells were transfected with plasmids expressing the EBOV RNP proteins together with YFP, CRISPR-resistant TRIM25 or mutants thereof. Cell lysates were analysed 48 hours later by western blot for the expression of HSP90, TRIM25 and EBOV NP. (C) Lysates of HEK293T cells transfected with EBOV NP in combination with either YFP or CRISPR-resistant TRIM25 (or mutants thereof) were immunoprecipitated with a rabbit anti-TRIM25 antibody. Cellular lysates and pull-down samples were analysed by western blot for HSP90, TRIM25 and EBOV NP.

Article Snippet: Cell lysates and pull-down samples were subjected to SDS-PAGE and Western blots performed using antibodies against HSP90 (Santa Cruz), TRIM25 (Abcam), EBOV-NP, EBOV-VP35, HA-tag (Rockland) and ZAP (Abcam).

Techniques: RNA Binding Assay, Generated, CRISPR, Activity Assay, Transfection, Infection, Control, Comparison, Expressing, Western Blot, Immunoprecipitation