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: Cell reports

Article Title: Precise Temporal Regulation of Post-transcriptional Repressors Is Required for an Orderly Drosophila Maternal-to-Zygotic Transition

doi: 10.1016/j.celrep.2020.107783

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) , Jackson ImmunoResearch , Cat#111-035-114; RRID: AB_2307391.

Techniques: Recombinant, Protease Inhibitor, Sequencing, Modification, Western Blot, Bradford Protein Assay, Labeling, Random Hexamer, Software, Functional Assay