Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Schematic representation of mechanistic strategies of barcoding. (A–C) Barcodes can be introduced to a template using adaptors through direct ligation (A) , using RT- or PCR primers at the reverse transcription or PCR amplification step (B) , and using hybridizing molecular inversion probes (C) . (D) Schematic representation of the difference between “barcodes” and “sample indexes”. Barcodes aim to correct sequencing errors. For example, a misreading nucleotide, guanosine (G) can be corrected in final consensus sequences for a pool of Sample 1 (top panel). Sample indexes are used to multiplex different sequencing amplicons generated from different pools of samples (Sample 1, 2, and 3) (bottom panel). Panel (A) is modified based on in and panel (C) is modified based on in .

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Ligation, Reverse Transcription, Amplification, Sequencing, Multiplex Assay, Generated, Modification

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Systematic comparison of barcoding strategies used in the category of molecular barcodes.

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Comparison, Software, Sequencing, Multiplex Assay, CRISPR, Plasmid Preparation, Microarray, Binding Assay, Amplification, Extraction, Ligation, DNA Sequencing, Multiplexing, Generated, Reverse Transcription, Staining, Flow Cytometry, High Throughput Screening Assay, Inhibition, Blocking Assay, Conjugation Assay, RNA Sequencing Assay, Transmission Assay, Incubation, Diagnostic Assay, Next-Generation Sequencing, Infection

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: bioRxiv

Article Title: Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance

doi: 10.1101/2020.06.30.179796

Figure Lengend Snippet: Developing a GRN model to assess indirect regulatory effects of MLL-AF4. ( A ) Schematic illustrating concept of MLL-AF4 targeting of TF genes, leading to subsequent TF protein expression and downstream regulation of indirect targets. ( B ) Pie chart of MLL-AF4 target genes in SEM cells (defined by nearest annotated promoter in ChIP-seq), describing overlap with MLL-AF4 ChIP-seq target genes from patient samples. ( C ) DEGs from nascent RNA-seq after 96 hours’ MLL-AF4 siRNA knockdown. Differential expression is defined as FDR < 0.05. (n=3). Shaded area represents genes bound by MLL-AF4 ChIP-seq. ( D ) Illustration of workflow used to generate a GRN from nascent RNA-seq and ChIP-seq data. ( E ) Visualization of whole network. Nodes in red represent genes downregulated upon MLL-AF4 KD, while blue represents upregulated genes. Circle size represents degree centrality. ( F ) Top 20 genes of the MLL-AF4 GRN by degree centrality. Lines (edges) indicate predicted interaction from protein (source) to gene locus (target), with arrowheads pointing towards downstream nodes. ( G ) DEGs after MLL-AF4 knockdown that are unbound by MLL-AF4, as highlighted in ( C ). Shaded areas represent proportion of genes bound by MAZ, ELF1 or RUNX1 ChIP-seq, as indicated.

Article Snippet: TaqMan probes were used for RUNX1 (Hs00231079_m1); CASP9 (Hs00609647_m1), BCL2 (Hs00608023_m1), CDK6 (Hs01026371_m1), PROM1 (Hs01009257_m1), MYC (Hs0015348_m1), ELF1 (Hs00608023_m1), GNAQ (Hs01586104_m).

Techniques: Expressing, ChIP-sequencing, RNA Sequencing, Knockdown, Quantitative Proteomics

Journal: bioRxiv

Article Title: Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance

doi: 10.1101/2020.06.30.179796

Figure Lengend Snippet: Core GRN factors are correlated with MLL-AF4 DNA binding profiles. ( A ) ChIP-seq tracks for MLL-N, AF4-C, RUNX1, ELF1, MAZ, H3K27ac and ATAC-seq over GNAQ . ChIP-seq data normalized to 1×10 7 reads. Spearman’s rank correlations comparing RUNX1, ELF1 and MAZ signal with MLL-N signal over all TSS sites are shown. ( B ) Heatmaps of ChIP-seq datasets as well ATAC-seq in SEM cells. Left – Heatmaps generated over MLL-AF4 peaks with rows ordered on level of MLL-N and AF4-C reads. Right – Heatmaps generated over RUNX1 peaks with rows ordered on RUNX1 read count. ( C ) Meta-gene plots of ChIP-seq signal of expressed TSSs, from 3 kb up and downstream (left), or 80 kb downstream (right). Profiles shown include TSS not bound by MLL-AF4 (grey), MLL-AF4 bound TSS non-spreading (blue), MLL-AF4 bound TSS with spreading greater than 5 kb (red), and MLL-AF4 bound TSS spreading greater than 50 kb (purple). ( D ) Left – Boxplots showing number of TF ChIP-seq peaks/kb over gene bodies, grouped by percentage MLL-AF4 coverage of the gene body. Right – Scatter plot of number of TF ChIP-seq peaks in a gene body against gene body width (kb). Local regression lines (LOESS) describe the relationship between number of peaks and gene width, with grey ribbon representing 95% confidence intervals. Local regression lines calculated for genes with no MLL-AF4, MLL-AF4 non-spreading, MLL-AF4 spreading greater than 5 kb, and MLL-AF4 spreading greater than 50 kb.

Article Snippet: TaqMan probes were used for RUNX1 (Hs00231079_m1); CASP9 (Hs00609647_m1), BCL2 (Hs00608023_m1), CDK6 (Hs01026371_m1), PROM1 (Hs01009257_m1), MYC (Hs0015348_m1), ELF1 (Hs00608023_m1), GNAQ (Hs01586104_m).

Techniques: Binding Assay, ChIP-sequencing, Generated

Journal: bioRxiv

Article Title: Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance

doi: 10.1101/2020.06.30.179796

Figure Lengend Snippet: Canonical RUNX1 DNA binding motifs are not associated with MLL-AF4 binding profiles. ( A ) Left – Boxplots showing quantification of RUNX1 motifs/kb in a gene body by MLL-AF4 percent coverage of the gene body. Right – Scatter plot of number of RUNX1 motifs in a gene body against gene width (kb). Local regression lines (LOESS) added to describe the relationship between number of motifs and gene width, with grey ribbon representing confidence intervals. Local regression lines calculated for genes with no MLL-AF4, MLL-AF4 non-spreading, MLL-AF4 spreading greater than 5 kb, and MLL-AF4 spreading greater than 50 kb

Article Snippet: TaqMan probes were used for RUNX1 (Hs00231079_m1); CASP9 (Hs00609647_m1), BCL2 (Hs00608023_m1), CDK6 (Hs01026371_m1), PROM1 (Hs01009257_m1), MYC (Hs0015348_m1), ELF1 (Hs00608023_m1), GNAQ (Hs01586104_m).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance

doi: 10.1101/2020.06.30.179796

Figure Lengend Snippet: RUNX1 is a highly central and essential node in the MLL-AF4 network. ( A ) essentiality screen for RUNX1, MYC, MAZ and ELF1 from the Project Score database of the Cancer Dependency Map . Each datapoint represents a different cell line, with essentiality highlighted in red. Cell lines were categorized into the type of cancer that they model. Statistical analysis from the database is represented as Bayesian scores for which values above 0 indicate essentiality. ( B ) Schematic illustrating how the published CRISPR screen data is used. Briefly, genes identified as essential in HT-29 (colon adenocarcinoma) or HT-1080 (Fibrosarcoma) were classified as non-specific essential. Genes not essential in HT-29 or HT-1080 but essential in MOLM-13 (MLL-AF9) or MV4-11 (MLL-AF4) were annotated as essential specifically for either MOLM-13 or MV4-11, or both cell lines. ( C ) Association of Log 2 degree centrality with CRISPR essentiality groups as outlined in ( B ). Leukemia-specific essential genes were further categorized as specific to MOLM-13, MV4-11 or both. ( D ) Degree centrality plotted against stress centrality of SEM MLL-AF4 GRN nodes. ( E ) Mean absolute stress fold changes in response to in silico deletion of GRN nodes and subsequent recalculation of stress centrality. Stress fold change is calculated on a per gene basis in the GRN by contrasting stress scores before and after in silico deletion. Top 20 nodes by greatest stress fold change are shown. ( F ) Stress fold change responses for individual transcription factors plotted against degree centrality for in silico deletion of RUNX1, MAZ, ELF1 and MYC. Blue datapoints indicate positive stress fold change responses, while red indicate a negative response.

Article Snippet: TaqMan probes were used for RUNX1 (Hs00231079_m1); CASP9 (Hs00609647_m1), BCL2 (Hs00608023_m1), CDK6 (Hs01026371_m1), PROM1 (Hs01009257_m1), MYC (Hs0015348_m1), ELF1 (Hs00608023_m1), GNAQ (Hs01586104_m).

Techniques: CRISPR, In Silico

Journal: bioRxiv

Article Title: Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance

doi: 10.1101/2020.06.30.179796

Figure Lengend Snippet: MLL-AF4 cooperates with RUNX1 in FFL and cascade circuits to regulate downstream targets. ( A ) DEGs from nascent RNA-seq after 96 hours’ RUNX1 siRNA knockdown. Differential expression is defined as FDR < 0.05. (n=3). Shaded area represents genes bound by RUNX1. ( B ) Venn diagram showing overlap between MLL-AF4 knockdown DEGs and RUNX1 knockdown DEGs ( A ). ( C ) GO biological process enrichment for overlap between MLL-AF4 and RUNX1 knockdown DEGs ( B ). Size of points is proportional to the significance of the enrichment, while point color represents fold enrichment over expected number of genes. ( D ) Top 20 genes of the RUNX1 GRN by degree centrality. Lines (edges) indicate predicted interaction from protein (source) to gene locus (target), with arrowheads pointing towards downstream nodes. ( E ) A schematic showing potential FFL (left) and cascade (right) motifs. FFLs are categorized into subtypes including coherent type 1 (C1-FFL), coherent type 3 (C3-FFL), incoherent type 1 (I1-FFL) and incoherent type 3 (I3-FFL). Solid lines represent direct interactions, dashed lines represent indirect. Cascade motifs are grouped into same sign of effect (*) and opposing sign of effect (#). ( F ) Scatter plot of FFL (left) and cascade (right) motif target genes displaying logFC response to RUNX1 knockdown and MLL-AF4 knockdown on the x and y axis, respectively. Along each axis is a density plot illustrating the distribution of logFC values. −logFC values are assumed to imply the perturbed factor has a positive regulatory effect on the target, while +logFC values are assumed to imply a negative regulatory effect. Quadrants of scatter plots align with FFL and cascade types shown in ( E ). ( G ) ChIP-seq tracks generated in hg19 for MLL-N, AF4-C, RUNX1 and H3K27ac. ChIP-seq data normalized to 1×10 7 reads. Top locus is for BCL2 , an example of an FFL target, bound by both MLL-AF4 and RUNX1. Bottom locus is CEBPG , an example of a cascade target, bound by RUNX1 but not MLL-AF4. ( H ) Expression of BCL2 (left) and CEBPG (right) from nascent RNA-seq data following knockdowns. Expression normalized as CPM.

Article Snippet: TaqMan probes were used for RUNX1 (Hs00231079_m1); CASP9 (Hs00609647_m1), BCL2 (Hs00608023_m1), CDK6 (Hs01026371_m1), PROM1 (Hs01009257_m1), MYC (Hs0015348_m1), ELF1 (Hs00608023_m1), GNAQ (Hs01586104_m).

Techniques: RNA Sequencing, Knockdown, Quantitative Proteomics, ChIP-sequencing, Generated, Expressing

Journal: bioRxiv

Article Title: Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance

doi: 10.1101/2020.06.30.179796

Figure Lengend Snippet: MLL-AF4 cooperates with RUNX1 in FFL and cascade circuits to regulate downstream targets. ( A ) Volcano plot showing the relationship between −log 10 P value and |logFC| in RUNX1 siRNA knockdown nascent RNA-seq. Orange points represent genes |logFC| > 1 and FDR > 0.05; red represents |logFC| < 1 and FDR < 0.05; green represents |logFC| > 1 and FDR < 0.05. ( B ) MA plot showing the relationship between logFC and mean log 2 CPM in RUNX1 siRNA knockdown nascent RNA-seq. DEGs are highlighted in red. ( C ) Pathway enrichment (Reactome) for overlap between MLL-AF4 and RUNX1 KD DEGs . Size of points represents −log 10 FDR of enrichment, while point color represents fold enrichment over expected number of genes. ( D ) Venn diagram overlaps between RUNX1, MLL-AF4, DOT1L and BRD4 GRNs. ( E ) Statistical significance of overlaps shown in ( D ). Significance calculated using a Fisher’s exact test and displayed as −log 10 P value. ( F ) ChIP-seq tracks generated in hg19 for MLL-N, AF4-C, RUNX1 and H3K27ac. ChIP-seq data normalized to 1×10 7 reads. Displayed loci are for MLL-AF4 FFL circuit targets.

Article Snippet: TaqMan probes were used for RUNX1 (Hs00231079_m1); CASP9 (Hs00609647_m1), BCL2 (Hs00608023_m1), CDK6 (Hs01026371_m1), PROM1 (Hs01009257_m1), MYC (Hs0015348_m1), ELF1 (Hs00608023_m1), GNAQ (Hs01586104_m).

Techniques: Knockdown, RNA Sequencing, ChIP-sequencing, Generated

Journal: bioRxiv

Article Title: Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance

doi: 10.1101/2020.06.30.179796

Figure Lengend Snippet: CASP9 is a key apoptotic gene repressed by MLL-AF4 in a cascade motif. ( A ) Number of apoptosis KEGG pathway (hsa04210) components regulated by TFs within the MLL-AF4 GRN. Top 10 apoptosis regulators are shown. ( B ) Interaction matrix showing a subset of TF regulators of apoptosis pathway (rows) and components of the apoptosis pathway (columns). Red squares indicate a predicted regulatory interaction between the TF and apoptosis component. Full interaction matrix shown in . ( C ) Experimental outline of CRISPR screen performed in combination with 7.4 μM venetoclax treatment of THP-1 cells. Cells harvested for sgRNA counting and analysis at T0 (after transduction and selection) and T18 (18 days treatment with venetoclax). ( D ) Analysis of CRISPR screen using MAGeCK, plotting −log 10 FDR against log 2 gRNA fold change. Left – MAGeCK results, excluding pan-essential genes. Right – MAGeCK results for enriched sgRNA, filtered for MLL-AF4 GRN nodes. ( E ) Top – Western blot for Caspase-9 from CASP9 knockout clones in SEM cells. Clones used for functional analysis are indicated by *. Bottom – CellTiter-Glo viability assay of CASP9 knockout clones after 48 hours treatment with 2.5 μM venetoclax. CTG luminescence displayed as a ratio relative to DMSO control. (n=3, ** < 0.01, *** < 0.001). Error bars represent standard error of the mean. ( F ) Western blot in SEM cells showing RUNX1 protein levels after 48 hours’ MLL-AF4 knockdown. GAPDH used as a loading control. ( G ) qRT-PCR results showing expression of MLL-AF4 , RUNX1 and CASP9 following 48 hours’ MLL-AF4 siRNA knockdown (n=3, * < 0.05, ** < 0.01). Error bars represent standard error of the mean. ( H ) Western blot in SEM cells showing RUNX1 protein levels after 48 hours’ RUNX1 knockdown. GAPDH used as a loading control. ( I ) qRT-PCR results showing expression of RUNX1 and CASP9 following 48 hours’ RUNX1 siRNA knockdown (n=3, ** < 0.01). Error bars represent standard error of the mean. ( J ) Illustration of selected cascade motifs within the MLL-AF4 GRN that explain regulatory connectivity between MLL-AF4 and CASP9. Node color represents logFC response to MLL-AF4 knockdown.

Article Snippet: TaqMan probes were used for RUNX1 (Hs00231079_m1); CASP9 (Hs00609647_m1), BCL2 (Hs00608023_m1), CDK6 (Hs01026371_m1), PROM1 (Hs01009257_m1), MYC (Hs0015348_m1), ELF1 (Hs00608023_m1), GNAQ (Hs01586104_m).

Techniques: CRISPR, Transduction, Selection, Western Blot, Knock-Out, Clone Assay, Functional Assay, Viability Assay, Control, Knockdown, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance

doi: 10.1101/2020.06.30.179796

Figure Lengend Snippet: CRISPR screen in combination with venetoclax treatment to test GRN predicted circuits. ( A ) Interaction matrix showing TF regulators of apoptosis pathway (rows) and components of the apoptosis pathway (columns). Select subset of TFs and pathway components shown. Red squares indicate a predicted regulatory interaction between the TF and apoptosis component. ( B ) Left – Flow cytometry gating for Annexin-V/PI staining of THP-1 cells to assay viability with venetoclax treatment. Right – THP-1 cells were cultured for 48 hours with different concentrations of venetoclax, and assayed for viability. PI: propidium iodide; early/late: early/late apoptotic. Dots show mean; error bars show SD. Lines show unconstrained four parameter sigmoidal least-squares regression best fit; shaded region shows 99% confidence interval. For all curves, R2 > 0.99. (n=5). ( C ) Sequencing validation of amplified sgRNA pool showing sgRNA distribution as a histogram and boxplot. ( D ) Scatter plot of sgRNA counts for biological replicates of T0 and +VEN samples. Correlation between replicates shown as R2, and histograms distributions shown along the axis. ( E ) Analysis of CRISPR screen using MAGeCK, plotting −log 10 FDR against log 2 gRNA fold change. Left – MAGeCK results, including only pan-essential genes. Right – MAGeCK results for depleted sgRNA, filtered for MLL-AF4 GRN nodes. ( F ) Functional validation of CRISPR screen hits by individual gene knockouts using sgRNA from the Brunello library. sgRNA were cloned into lentiCRISPRv2 constructs and transduced into THP-1 cells, followed by 48h treatment with DMSO or 20 μM Venetoclax. CellTiter-Glo (CTG) was used to assay viability, and is normalized as Venetoclax/DMSO viability, relative to AAVS1 silent control . Genes marked * are FDR > 0.1 in the CRISPR screen. Dots show biological replicates (n=3); bars show mean. ( G ) Expression of CASP9 from nascent RNA-seq data following 96 hours MLL-AF4 knockdown. Expression normalized as CPM. ( H ) ChIP-seq tracks generated in hg19 for MLL-N, AF4-C, RUNX1 and H3K27ac at the CASP9 locus. ChIP-seq data normalized to 1×10 7 reads. ( I ) Normalized mean sgRNA counts for key depleted and enriched genes at T0 and T18+Venetoclax (n = 2). Dropout hits are highlighted in blue, enriched in red.

Article Snippet: TaqMan probes were used for RUNX1 (Hs00231079_m1); CASP9 (Hs00609647_m1), BCL2 (Hs00608023_m1), CDK6 (Hs01026371_m1), PROM1 (Hs01009257_m1), MYC (Hs0015348_m1), ELF1 (Hs00608023_m1), GNAQ (Hs01586104_m).

Techniques: CRISPR, Flow Cytometry, Staining, Cell Culture, Sequencing, Biomarker Discovery, Amplification, Functional Assay, Clone Assay, Construct, Control, Expressing, RNA Sequencing, Knockdown, ChIP-sequencing, Generated

Journal: eLife

Article Title: Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function

doi: 10.7554/eLife.84168

Figure Lengend Snippet: ( A ) Violin plot of MTIF3 expression in subcutaneous adipose tissue for rs1885988 from Genotype-Tissue Expression (GTEx) Project eQTL. ( B ) Same as in ( A ), but for rs67785913. ( C ) Representative Sanger sequencing traces of rs67785913 CTCT/CTCT and CT/CT clones obtained after CRISPR/Cas9-mediated allele editing and single-cell cloning. ( D ) Normalized Z -score plot of luciferase reporter assays using vectors carrying different DNA fragments of the MTIF3 gene cloned into pGL4.23 luciferase reporter vector. Hypothesis testing was performed by comparing the transcriptional enhancer activity of each of the 12 vectors (F1–12) to the empty vector (minP). All data were plotted as mean ± standard deviation (SD), n = 4 independent experiments, p values are presented in each graph; ordinary one-way analysis of variance (ANOVA) was used for statistical analysis. ( E ) Relative MTIF3 expression (mRNA) in rs67785913 allele-edited cells 2 days before, at, or 2 days post-differentiation induction (day −2, 0, and 2, respectively). n = 3 clonal populations for CTCT/CTCT genotype, n = 5 clonal populations for CT/CT genotype, error bars show SD. ( F ) as in ( E ), but for GTF3A (mRNA) expression. Two-tailed Student’s t -test was used; p values are presented in each graph.

Article Snippet: Sequence-based reagent , Taqman assay for GTF3A , Thermo Fisher Scientific , Hs00157851_m1 , .

Techniques: Expressing, Sequencing, Clone Assay, CRISPR, Cloning, Luciferase, Plasmid Preparation, Activity Assay, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function

doi: 10.7554/eLife.84168

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , Taqman assay for GTF3A , Thermo Fisher Scientific , Hs00157851_m1 , .

Techniques: Recombinant, Plasmid Preparation, Sequencing, TaqMan Assay, DNA Extraction, Isolation, Sample Prep, Picogreen Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: VEGF-induced vascular permeability is reduced upon CRISPR/Cas9-mediated knockout of Stat3 in zebrafish. (A) VEGF-inducible zebrafish were crossed to Stat3 +/− (heterozygous) zebrafish to generate VEGF-inducible; Stat3 +/− double transgenic fish, which were intercrossed to generate VEGF-inducible; Stat3 −/− (KO) zebrafish. (B) CRISPR/Cas9-generated Stat3 KO zebrafish (bottom) display no overt vascular defects relative to wild-type (WT) zebrafish (top). The vascular system of 3 days post-fertilization (dpf) zebrafish was visualized by microangiography with 2000 kDa FITC-dextran. Representative images of at least three zebrafish per group are shown. Scale bars: 100 μm. (C) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf Stat3 +/+ (negative controls without VEGF induction; left) , VEGF-induced, Stat3 +/+ (middle) and VEGF-induced, Stat3 −/− (right) zebrafish. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at room temperature (RT). Scale bars: 50 μm. (D) Quantitative analysis of vascular permeability upon VEGF stimulation in WT Stat3 +/+ ( n =30) and KO Stat3 −/− ( n =9) zebrafish. Mean±s.e.m., unpaired, two-tailed Student's t -test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, CRISPR, Knock-Out, Transgenic Assay, Generated, Marker, Microscopy, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Endothelial cell-specific STAT3 knockout mice exhibit decreased VEGF-induced permeability. (A) Images of footpads from WT and endothelial cell-specific STAT3 knockout (STAT3 ECKO ) mice following tail vein injection with 1% Evans Blue dye and human recombinant VEGF-165 protein (2.5 µg/ml; left footpads) or PBS vehicle (right footpads) being injected into the root of the footpad. (B,C) Quantitation of Evans Blue leakage in Tie2-Cre negative; STAT3 flox/flox (WT) and Tie2-Cre positive; STAT3 flox/flox (STAT3 ECKO ) mice. n =7 mice in WT group and n =6 mice in STAT3 ECKO group. Each mouse was injected with PBS on the right anterior and posterior footpads and VEGF on the left anterior and posterior footpads. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. A.U., arbitrary units.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Knock-Out, Permeability, Injection, Recombinant, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Pharmacological inhibition of STAT3 stabilizes endothelial barrier integrity following VEGF stimulation in human endothelial cells. (A) Serum-starved human umbilical vein endothelial cells (HUVECs) were pretreated with DMSO (vehicle control) for 1 h, 30 µM AQ for 4 h, or 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 2 or 5 min. Lysates were immunoblotted. Densitometry was performed, and the values below the rows of bands represent the ratio of phosphorylated protein to respective total protein. (B) Human VEGF-165 recombinant protein (VEGF; 25 ng/ml) stimulation of HUVECs promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows; left column; DMSO vehicle control pretreatment for 1 h prior to VEGF stimulation). ZO-1 organization is maintained upon pretreatment with 30 μM AQ for 4 h (magenta arrows; middle column) or 10 μM PYR for 1 h (magenta arrows; right column) prior to VEGF stimulation. Nuclei were stained with DAPI (blue). (C) Serum-starved human pulmonary artery endothelial cells (HPAECs) were pretreated with 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 5 or 30 min. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained when HPAECs were pretreated with PYR (magenta arrows). Nuclei were stained with DAPI (blue). (D) VEGF (25 ng/ml) stimulation of human lung microvascular endothelial cells (HMVEC-Ls) promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained upon pretreatment with 20 μM PYR for 6 h prior to VEGF stimulation (magenta arrows). Nuclei were stained with DAPI (blue). At least two biological replicates were performed for each experiment depicted in A-D. Scale bars: 20 µm.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Inhibition, Recombinant, Staining

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Suppression of STAT3 activity by pyrimethamine (PYR) inhibits VEGF-induced vascular permeability in zebrafish and mice. (A) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf zebrafish without induced VEGF pretreated with DMSO ( n =6) or 25 μM PYR ( n =5) or 3 dpf zebrafish with induced VEGF pretreated with DMSO ( n =4) or 25 μM PYR ( n =9) for 3 days. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at RT. Scale bars: 50 μm. (B) The quantitative analysis of vascular permeability without VEGF stimulation or upon VEGF stimulation in zebrafish pretreated with DMSO or PYR. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. (C) Representative images of footpads from mice treated with vehicle or PYR following tail vein injection with 1% Evans Blue and footpad injection of VEGF (2.5 μg/ml) or PBS vehicle. (D) Quantitation of Evans Blue dye leakage in C57BL/6 WT mice treated with vehicle or PYR. n =9 mice in the vehicle group and n =7 mice in the PYR group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Activity Assay, Permeability, Marker, Microscopy, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: JAK2 phosphorylates STAT3 to transduce VEGF/VEGFR-2 signaling and promote vascular permeability. (A) To perform a STAT3 GST pull-down of VEGFR-2 and JAK2, lysates of HUVECs stimulated with serum for 30 min were used as prey. GST fusion protein STAT3 expressed in 293F cells was used as bait. GST alone served as a negative control. Binding experiments were analyzed by SDS-PAGE and visualized by immunoblotting. GST-STAT3 and GST were each detected using an anti-GST antibody. Three biological replicates were performed and depicted findings are representative. (B) JAK2 phosphorylates STAT3 in vitro . In vitro kinase assays were performed using purified human STAT3 protein and kinase active JAK2 protein. The results shown here are representative of two independent experiments. (C) Representative images of footpads from C57BL/6 WT mice treated with vehicle or JAK2 inhibitor AG490. Following tail vein injection with 1% Evans Blue dye, human VEGF-165 protein (2.5 μg/ml) or PBS vehicle was injected into the root of the footpad. After 30 min, the mice were euthanized and the footpads were excised. (D) Quantitation of Evans Blue dye leakage in C57BL/6 mice treated with vehicle or AG490. n =4 mice per group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Two biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Transduction, Permeability, Negative Control, Binding Assay, SDS Page, Western Blot, In Vitro, Purification, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: STAT3 transcriptionally activates ICAM-1, a cell adhesion molecule that promotes vascular permeability. (A) Top: the pGL3-ICAM1-WT plasmid containing the human ICAM-1 promoter with a STAT3 binding site located at −115 to −107 bp. Bottom: the pGL3-ICAM1-SDM plasmid with a site-directed mutation (SDM) in the STAT3 binding site as indicated. (B) Dual luciferase assays were performed in HUVECs that were transfected with pGL3-ICAM1-WT or pGL3-ICAM1-SDM and empty vector or constitutively active STAT3. Firefly and Renilla luminescence was measured and plotted as a ratio. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. n =9 technical replicates. Depicted findings are representative of three independent experiments. (C) HUVECs that had been stably transduced with lentivirus encoding STAT3-specific shRNA or control shRNA were stimulated with human VEGF-165 protein (25 ng/ml) and the lysates were immunoblotted for ICAM1, p-STAT3 (Y705) and total STAT3. Depicted data are representative of three biological replicates. (D) RNA was harvested from VEGF; Stat3 +/+ or VEGF; Stat3 −/− 3 dpf embryos for quantitative PCR. stat3 transcripts are reduced in VEGF; Stat3 −/− ( n =5) compared to VEGF; Stat3 +/+ zebrafish ( n =7). Mean±s.e.m., unpaired, two-tailed Student's t -test. (E) The expression of icam-1 was assessed by real-time quantitative PCR using RNA derived from each zebrafish embryo in the absence of VEGF induction (Stat3 +/+ , n =3; Stat3 −/− , n =2) or 8 h following VEGF induction (Stat3 +/+ , n =4; Stat3 −/− , n =3) in the heat-inducible VEGF; Stat3 mutant zebrafish. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, Plasmid Preparation, Binding Assay, Mutagenesis, Luciferase, Transfection, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, Two Tailed Test, Expressing, Derivative Assay

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 3. Enhancer activity compacts SOX9 promoter-enhancer hub in individual TNBC cells. (A and B) SOX9 promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 cells. Left: Allele percentages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both (A) and SOX9.EC2, SOX9. EC3, or both (B) in MB157 (n = alleles). Right-top: SOX9 locus schematic, three-color DNA FISH 50-kb probes at SOX9 promoter (green), SOX9.EC3 (magenta), and SOX9.EC1 (A, red) or SOX9.EC2 (B, yellow). Locations per fig. S3A. Right-bottom: Representative cells. Blue: 4′,6-Diamidino-2-phenylindole (DAPI). (C and F) SOX9 enhancers inactiva- tion expands SOX9-EC1-EC3 and SOX9-EC2-EC3 hubs in individual TNBC MB157 cells. Cumulative distribution functions (CDFs) of SOX9-EC1-EC3 (C) and SOX9-EC2-EC3 (F) spatial perimeters in each MB157-dCas9-KRAB expressing control (CTRL), SOX9.EC1, SOX9.EC2, or SOX9.EC3 sgRNA [Kolmogorov-Smirnov (KS) test, n = cells]. Mean (±SD) perimeters (micrometers): (C) Left: CTRL/SOX9.EC1 sgRNA: 3.78 (±2.63)/4.36 (±2.63); middle: CTRL/SOX9.EC2 sgRNA: 3.78 (±2.63)/4.47 (±2.64); right: CTRL/SOX9.EC3 sgRNA: 3.39 (±2.56)/4.28 (±2.65). (G) Left: CTRL/SOX9.EC1 sgRNA: 3.83 (±2.71)/4.45 (±2.72); middle: CTRL/SOX9.EC2 sgRNA: 3.19 (±2.44)/4.26 (±2.61); right: CTRL/SOX9.EC3 sgRNA: 3.83 (±2.71)/4.22 (±2.56). (D and G) Allele percentages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both (D) and SOX9.EC1, SOX9. EC3, or both (G) in MB157-dCas9-KRAB expressing CTRL, SOX9.EC1, SOX9.EC2, or SOX9.EC3 sgRNA (n = alleles). (E and H) Representative cells of 3C and 3D (E) or 3F and 3G (H). Blue: DAPI. (I) SOX9 promoter inactivation decreases SOX9-EC1-EC3 three-way interaction frequency across individual alleles in TNBC MB157. Top-left: Allele percent- ages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both in MB157-dCas9-KRAB expressing CTRL or SOX9 promoter sgRNA (SOX9.P sgRNA) (n = alleles). Bottom-left: CDFs of SOX9-EC1-EC3 spatial perimeter in each MB157-dCas9-KRAB cell (KS test, n = cells). CTRL/SOX9.P sgRNA mean (±SD) perimeter: 3.90 (±2.62)/4.44 (±2.66) μm. Right: Representative cells. Blue: DAPI. Scale bars, 3 μm for nuclei and 0.5 μm for alleles.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Activity Assay, Expressing, Control

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 6. SOX9 regulates oncogene MYC by positioning its enhancers. (A) Genome tracks showing enrichment of pairwise MYC enhancer-enhancer and promoter- enhancer interactions in a population of MB157 cells. From top to bottom: Colored circles marking location of Oligopaint DNA FISH probes labeling 50-kb regions at MYC promoter (green), MYC.EC1 (magenta), MYC.EC2 (red), MYC.EC3 (yellow), and T-ALL-restricted enhancer (black), H3K27ac and SOX9 levels as measured by ChIP-seq, and normalized interaction frequency as measured by SMC1 HiChIP at the MYC locus in MB157. MYC enhancer clusters are marked by grey boxes. (B) A total of 80% of differ- entially expressed genes with SOX9-bound promoter and distal enhancer participate in ensemble hyper-interacting hubs. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. Hyper-interacting ensemble promoter-enhancer hubs containing genes that are significantly down-regulated in MB157-Cas9 cells transfected with SOX9 targeting sgRNA versus control sgRNA for 4 days and have SOX9-bound promoter and distal enhancer are marked in orange. (C to E) SOX9 loss significantly in- creases 3D distances between the MYC promoter and SOX9-bound MYC.EC2 (C) or MYC.EC3 (D) and SOX9-unbound MYC.EC1 (E) in individual cells. CDFs (left) and box and whiskers (middle) of the distances between the MYC promoter and SOX9-bound MYC.EC2 (C) and MYC.EC3 (D) and SOX9-unbound MYC.EC1 (E) in each MB157-Cas9 6 days after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA (SOX9 KO) (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) distance between MYC promoter and MYC.EC2: 0.389 (±0.358)/0.749 (±0.666) μm; MYC.EC3: 0.447 (±0.457)/0.591 (±0.551) μm; MYC.EC1: 0.494 (±0.447)/0.651 (±0.556) μm. Right: Represen- tative cells. Scale bar per 3A. Blue: DAPI.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Labeling, ChIP-sequencing, HiChIP, Transfection, Control, Transduction

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 7. SOX9 loss decompacts MYC promoter-enhancer hubs. (A) MYC promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 and MDA-MB-468 but not ER+ MCF7. Left: Percentage of alleles with MYC promoter interacting (<350 nm) with SOX9-unbound MYC.EC1, SOX9-bound MYC.EC3, or both MYC.EC1 and MYC.EC3 in MB157, MDA-MB-468, and MCF7 as measured by three-color Oligopaint DNA FISH with probes marked in Fig. 6A top genome track (n = alleles). Right: Representative MB157, MDA-MB-468, and MCF7 nuclei and two magnified alleles from three-color DNA FISH. Scale bar per 3A. Blue: DAPI. (B and C) SOX9 loss expands MYC-EC1-EC2 (B) and MYC-EC1-EC3 (C) promoter-enhancer hubs in individual MB157 and decreases three-way interaction frequency across individual alleles. Left: CDFs of MYC-EC1-EC2 (B) and MYC-EC1-EC3 (C) spatial perimeters in each MB157-Cas9 cell expressing CTRL or SOX9 KO sgRNA (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) perimeters MYC-EC1-EC2 (B): 3.84 (±2.70)/4.53 (±2.67) μm; MYC-EC1-EC3 (C): 3.10 (±2.58)/3.90 (±2.64) μm (n = cells). Middle: Allele per- centages with MYC promoter interacting (<350 nm) with MYC.EC1, MYC.EC2, or both MYC.EC1 and MYC.EC2 (B) and MYC.EC1, MYC.EC3, or both MYC.EC1 and MYC.EC3 (C) in CTRL and SOX9 KO MB157-Cas9. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Expressing

Journal: Science Advances

Article Title: ACTR5 controls CDKN2A and tumor progression in an INO80-independent manner

doi: 10.1126/sciadv.adc8911

Figure Lengend Snippet: ( A ) RNA-seq and GSEA analyses showing changes in expression of the E2F_Pathway gene set in sgiNTC- versus sgiACTR5-transduced HepG2-dCas9-Krab cells. (Right) Each dot indicates one gene set from the GSEA HALLMARK Database. NES, normalized enrichment score. ( B ) Venn diagram revealed 13 ACTR5-bound target genes within the E2F_Pathway gene set (green). ( C ) RNA-seq expression change of the ACTR5-regulated E2F-Pathway genes (green dots) induced by sgiACTR5 in dCas9-Krab–expressing HepG2 ( y axis) versus U87 ( x axis) cells. ( D ) Western blot of ACTR5, CDKN2A, and β-actin in dCas9-Krab–expressing HepG2 and U87 cells transduced with sgiNTC and sgiACTR5. ( E ) TST-mediated ChIP-seq and ChIP-qPCR of ACTR5 at the CDKN2A locus in HepG2 cells. ( F ) Level of H3K9me2 and ( G ) H3K27me3 at the CDKN2A and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) loci detected by ChIP-qPCR. Data are presented as means ± SEM. * P < 0.01 by two-sided Student’s t test. n.s., not significant.

Article Snippet: Membranes were immersed in 5% nonfat milk then probed with rabbit antibodies against ACTR5 (sc-376364, Santa Cruz Biotechnology; 1:1000), IES6 (PA5-61869, Thermo Fisher Scientific; 1:1000), CDKN2A (ab108349, Abcam; 1:1000), CDK6 (ab124821, Abcam; 1:1000), E2F1 (3742S, Cell Signaling Technology; 1:1000), Rb (ab181616, Abcam; 1:1000), phospho-S780 Rb (ab173289, Abcam; 1:1000), and β-actin (4970S, Cell Signaling Technology; 1:1000) at 4°C overnight.

Techniques: RNA Sequencing, Expressing, Western Blot, Transduction, ChIP-sequencing, ChIP-qPCR

Journal: Science Advances

Article Title: ACTR5 controls CDKN2A and tumor progression in an INO80-independent manner

doi: 10.1126/sciadv.adc8911

Figure Lengend Snippet: ( A and C ) Western blot of ACTR5, CDKN2A, CDK6, Rb, p-Rb, E2F1, and β-actin in (A) dCas9-Krab–expressing HCC (HepG2 and SNU475) and glioblastoma (U251 and U87) cells, and (C) vector versus ACTR5-TST–expressing HepG2-dCas9-Krab cells transduced with sgiNTC and sgiACTR5. ( B ) Cell cycle monitored by 5-Ethynyl-2′-deoxyuridine incorporation in HepG2-dCas9-Krab cells transduced with sgiNTC and sgiACTR5 ( n = 3). ( D ) Growth competition assay of vector versus ACTR5-TST–expressing HepG2-dCas9-Krab cells transduced with RFP-labeled sgiNTC and sgiACTR5 ( n = 3 each group). ( E ) Effect of targeting ACTR5 on CDKN2A- and CDK6-triggered cell cycle signaling. Data are presented as means ± SEM. * P < 0.01 by two-sided Student’s t test.

Article Snippet: Membranes were immersed in 5% nonfat milk then probed with rabbit antibodies against ACTR5 (sc-376364, Santa Cruz Biotechnology; 1:1000), IES6 (PA5-61869, Thermo Fisher Scientific; 1:1000), CDKN2A (ab108349, Abcam; 1:1000), CDK6 (ab124821, Abcam; 1:1000), E2F1 (3742S, Cell Signaling Technology; 1:1000), Rb (ab181616, Abcam; 1:1000), phospho-S780 Rb (ab173289, Abcam; 1:1000), and β-actin (4970S, Cell Signaling Technology; 1:1000) at 4°C overnight.

Techniques: Western Blot, Expressing, Plasmid Preparation, Transduction, Competitive Binding Assay, Labeling

Journal: Science Advances

Article Title: ACTR5 controls CDKN2A and tumor progression in an INO80-independent manner

doi: 10.1126/sciadv.adc8911

Figure Lengend Snippet: ( A ) Growth competition assay of HepG2-dCas9-Krab cells transduced with RFP-labeled nontargeting control (gray lines; n = 2 independent sgiNTC sequences) and IES6 -targeting sgiRNAs (blue lines; n = 4 independent sgiIES6 sequences). ( B ) Two-dimensional annotation of CRISPR gene tiling scans for IES6 in HepG2 (red) and U87 (blue) cells. The solid lines indicate the smoothened model of the CRISPR scan score derived from individual sgRNAs (dots). The median CRISPR scan scores of the positive control (dotted line; defined as −1.0) and negative control (defined as 0.0) sgRNAs are designated. ( C ) Three-dimensional annotation of IES6 CRISPR scan score relative to the AlphaFold structural model of IES6 (ID, Q6PI98). ( D ) Western blot of IES6 and ACTR5 in the TST-purified WT-, ΔI1-, and ΔI2-IES6 protein complexes. ( E ) Effect of WT- and ΔI2-IES6 expression on the growth competition assay of HepG2-dCas9-Krab cells transduced with sgiIES6 ( n = 3 each group). ( F ) Western blot of IES6, ACTR5, CDKN2A, CDK6, Rb, p-Rb, E2F1, and β-actin in HepG2-dCas9-Krab cells transduced with sgiNTC and sgiIES6. ( G and H ) CellTiter-Glo analysis of the (G) sgiACTR5- and (H) sgiIES6-transduced HepG2-dCas9-Krab cells incubated with dimethyl sulfoxide (DMSO) or ribociclib ( n = 4 each group). ( I ) Model of the ACTR5/IES6 complex supporting CDK6-driven cell proliferation. Data are presented as means ± SEM. * P < 0.01 by two-sided Student’s t test.

Article Snippet: Membranes were immersed in 5% nonfat milk then probed with rabbit antibodies against ACTR5 (sc-376364, Santa Cruz Biotechnology; 1:1000), IES6 (PA5-61869, Thermo Fisher Scientific; 1:1000), CDKN2A (ab108349, Abcam; 1:1000), CDK6 (ab124821, Abcam; 1:1000), E2F1 (3742S, Cell Signaling Technology; 1:1000), Rb (ab181616, Abcam; 1:1000), phospho-S780 Rb (ab173289, Abcam; 1:1000), and β-actin (4970S, Cell Signaling Technology; 1:1000) at 4°C overnight.

Techniques: Competitive Binding Assay, Transduction, Labeling, Control, CRISPR, Derivative Assay, Positive Control, Negative Control, Western Blot, Purification, Expressing, Incubation

Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet: (A) Immunoblot analysis of CD40, poly-ubiquitin (poly-Ub), or control GAPDH levels in WCE from Cas9 + Daudi B cells that expressed the indicated sgRNA, treated with DMSO or the proteasome inhibitor bortezomib (200 nM) for 16 h. Increased poly-Ub signal indicates on-target bortezomib activity. (B) qRT-PCR analysis of CD40 mRNA abundances relative to 18S control levels in Cas9 + Daudi B cells expressing the indicated sgRNA. (C) FACS analysis of PM CD40 abundances in Cas9 + Daudi B cells expressing the indicated control or FBXO11 targeting sgRNA as well as the indicated control or CD40 cDNA construct. (D) FACS analysis of Daudi B cell PM CD40 MFI as in (C) from n = 3 replicates. (E) FACS analysis of PM Fas abundances in Cas9 + Daudi B cells expressing the indicated control or FBXO11 targeting sgRNA as well as the indicated control or CD40 cDNA rescue construct, stimulated by Mega-CD40L (50 ng/mL for 48 h), as indicated. (F) Mean + SD Log2-normlized CTBP1 sgRNA abundances from both pre-FACS sort input libraries and from all four screen replicates are shown. (G) Immunoblot analysis of CTBP1, FBXO11, and GAPDH control abundances in WCE from Cas9 + Daudi B cells expressing the indicated control, FBXO11 , or either of two independent CTBP1 targeting sgRNAs. (H) PM CD40 abundances in Cas9 + Daudi B cells expressing the indicated two sgRNAs. Mean + SD levels from at least n = 3 experiments and shown in (B), (D), and (H). *p < 0.05, **p < 0.01, ***p < 0.001, ns, non-significant. Immunoblot results were representative of n = 3 experiments. See also .

Article Snippet: Mouse Ubiquitin (P4D1) monoclonal antibody , Cell Signaling Technology , Cat# 3936S; RRID:AB_331292.

Techniques: Western Blot, Ubiquitin Proteomics, Control, Activity Assay, Quantitative RT-PCR, Expressing, Construct

Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet:

Article Snippet: Mouse Ubiquitin (P4D1) monoclonal antibody , Cell Signaling Technology , Cat# 3936S; RRID:AB_331292.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Protease Inhibitor, SYBR Green Assay, Purification, Gel Extraction, Reverse Transcription, Quantitative RT-PCR, Plasmid Preparation, Isolation, Cell Culture, Immunoprecipitation, Gene Expression, CRISPR, Software, Sequencing, Modification

Journal: Nature Communications

Article Title: A single genetic locus controls both expression of DPEP1/CHMP1A and kidney disease development via ferroptosis

doi: 10.1038/s41467-021-25377-x

Figure Lengend Snippet: a LocusZoom plots of eGFR GWAS, human kidney mQTL analysis (genotype-methylation, n = 188), eQTLs (genotype-expression of DPEP1 ) in kidney compartments (tubule n = 121 or glomerulus n = 119). b LocusZoom plots of eGFR GWAS, human kidney mQTL analysis (genotype-methylation, n = 188), eQTLs (genotype-expression of CHMP1A ) in kidney compartments (tubule n = 121 or glomerulus n = 119). The x-axis indicates the genomic location on chromosome 16. The arrow indicates the transcriptional direction for specific genes. Each dot represent one SNP. The dots are colored according to their correlation to the index SNP (rs164748). The red dots indicates strong correlation ( r 2 > 0.8) (LD) with the index SNP. The left y-axis indicates −log 10 ( P value). The right y-axis indicates recombination rate (cM/Mb). c Genotype (rs164748) and gene expression ( DPEP1 and CHMP1A ) association in human tubules ( n = 121) and glomeruli ( n = 119) in the Susztak lab database . The effect size estimate (Beta) and standard error (SE) are as below: DPEP1 tubule Beta = 0.811 and SE = 0.11; DPEP1 glom Beta = 0.889 and SE = 0.11; CHMP1A tubule Beta = −0.766 and SE = 0.113; CHMP1A glom Beta = −0.587 and SE = 0.127. Centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the 5th and 95th percentiles. P value was calculated as previously published .

Article Snippet: Kidney tissue or cultured cell lysates were prepared with ice-cold lysis buffer (CST # 9806) containing protease inhibitor cocktail (cOmplete Mini, Roche #11836153001) and phosphatase inhibitor (PhosSTOP, Roche #4906837001), resolved on 8–12% gradient gels, transferred on to polyvinylidene difluoride membranes, and probed with the following antibodies: DPEP1 (Proteintech #12222-1-AP 1:500), CHMP1A (Proteintech #15761-1-AP 1:500), RIPK3 (Sigma #PRS2283 1:1000), Cleaved Caspase 1 (Santa cruz #sc-56036 1:500), Collagen III (Abcam #ab7778 1:1000), Fibronectin (Abcam #ab2413 1:1000), aSMA (Sigma #A5228 1:1000), ACSL4 (Abcam #ab155282 1:1000), CD63 (Abcam #ab193349 1:1000), GPX4 (Abcam #ab125066 1:1000), Actin (Sigma #A3854 1:20000), GAPDH (Proteintech #60004-1-Ig 1:1000), and Tubulin (BioLegend #801202 1:1000).

Techniques: Methylation, Expressing, Gene Expression

Journal: Nature Communications

Article Title: A single genetic locus controls both expression of DPEP1/CHMP1A and kidney disease development via ferroptosis

doi: 10.1038/s41467-021-25377-x

Figure Lengend Snippet: a From top to bottom: locuszoom plots of eGFR GWAS; Gene browser view of the single nucleotide polymorphisms within the regions; genome browser view of chromatin accessibility for proximal tubules (PT), loop of Henle (LOH), distal convoluted tubule (DCT), collecting duct principal cell types (PC), collecting duct intercalated cells (IC), podocytes (Podo), endothelial cells (Endo), immune cells (Immune); genome browser view of whole kidney H3K27ac, H3K4me1, and H3K4me3 histone ChIP-seq; ChomHMM annotation human adult and fetal kidneys. Proximal tubule-specific open chromatin region across this region was circled and numbered. b Schematic of CRISPR/Cas9 mediated open chromatin region deletion. c Relative transcript levels of DPEP1 and CHMP1A following open chromatin region deletion ( n = 4). All data are represented as mean ± SEM. P value was calculated by one-way ANOVA with post hoc Tukey test. P < 0.05 is statistically significant. A Source Data file is available for this figure.

Article Snippet: Kidney tissue or cultured cell lysates were prepared with ice-cold lysis buffer (CST # 9806) containing protease inhibitor cocktail (cOmplete Mini, Roche #11836153001) and phosphatase inhibitor (PhosSTOP, Roche #4906837001), resolved on 8–12% gradient gels, transferred on to polyvinylidene difluoride membranes, and probed with the following antibodies: DPEP1 (Proteintech #12222-1-AP 1:500), CHMP1A (Proteintech #15761-1-AP 1:500), RIPK3 (Sigma #PRS2283 1:1000), Cleaved Caspase 1 (Santa cruz #sc-56036 1:500), Collagen III (Abcam #ab7778 1:1000), Fibronectin (Abcam #ab2413 1:1000), aSMA (Sigma #A5228 1:1000), ACSL4 (Abcam #ab155282 1:1000), CD63 (Abcam #ab193349 1:1000), GPX4 (Abcam #ab125066 1:1000), Actin (Sigma #A3854 1:20000), GAPDH (Proteintech #60004-1-Ig 1:1000), and Tubulin (BioLegend #801202 1:1000).

Techniques: ChIP-sequencing, CRISPR

Journal: Nature Communications

Article Title: A single genetic locus controls both expression of DPEP1/CHMP1A and kidney disease development via ferroptosis

doi: 10.1038/s41467-021-25377-x

Figure Lengend Snippet: a Serum blood urea nitrogen (BUN) and creatinine measurement of control and Dpep1 +/− mice following sham or cisplatin injection. Sham-treated group: WT ( n = 4), Dpep1 +/− ( n = 4); cisplatin-treated group: WT ( n = 7), Dpep1 +/− ( n = 6). b Representative images of HE-stained kidney sections from control and Dpep1 +/− mice following sham or cisplatin injection. Scale bar: 20 μm. c Relative mRNA level of injury markers Kim1 and Lcn2 in the kidneys of control and Dpep1 +/− mice following sham or cisplatin injection. Sham-treated group: WT ( n = 4), Dpep1 +/− ( n = 4); cisplatin-treated group: WT ( n = 7), Dpep1 +/− ( n = 6). d Serum BUN and creatinine measurement of control and Dpep1 +/− mice following sham or folic acid (FA) injection. Sham-treated group: WT ( n = 3), Dpep1 +/− ( n = 3); cisplatin-treated group: WT ( n = 5), Dpep1 +/− ( n = 7). e Representative images of HE- and Sirius Red-stained kidney sections from control and Dpep1 +/− mice following sham or FA injection. Scale bar: 20 μm. f Western blots of fibrosis markers aSMA, Collagen3, and Fibronectin in kidneys of control and Dpep1 +/− mice following sham or FA injection. g Serum BUN and creatinine measurement of control and Chmp1a +/− mice following sham or cisplatin injection. Sham-treated group: WT ( n = 3), Dpep1 +/− ( n = 3); cisplatin-treated group: WT ( n = 4), Dpep1 +/− ( n = 5). h Representative images of HE-stained kidney sections from control and Chmp1a +/− mice following sham or cisplatin injection. Scale bar: 20 μm. i Relative transcript level of injury markers Kim1 and Lcn2 in control and Chmp1a +/− mice following sham or cisplatin injection. Sham-treated group: WT ( n = 3), Dpep1 +/− ( n = 3); cisplatin-treated group: WT ( n = 4), Dpep1 +/− ( n = 5). j Serum BUN and creatinine levels of control and Chmp1a +/− mice following sham or FA injection ( n = 3 per group). k Representative images of HE- and Sirius Red-stained kidney sections from control and Chmp1a +/− mice following sham or FA injection. Scale bar: 20 μm. l Western blots of fibrosis markers aSMA, Collagen3, and Fibronectin in control and Chmp1a +/− mice following sham or FA injection. All data are represented as mean ± SEM. P value was calculated by two-way ANOVA with post hoc Tukey test. P < 0.05 is statistically significant. A Source Data file is available for this figure.

Article Snippet: Kidney tissue or cultured cell lysates were prepared with ice-cold lysis buffer (CST # 9806) containing protease inhibitor cocktail (cOmplete Mini, Roche #11836153001) and phosphatase inhibitor (PhosSTOP, Roche #4906837001), resolved on 8–12% gradient gels, transferred on to polyvinylidene difluoride membranes, and probed with the following antibodies: DPEP1 (Proteintech #12222-1-AP 1:500), CHMP1A (Proteintech #15761-1-AP 1:500), RIPK3 (Sigma #PRS2283 1:1000), Cleaved Caspase 1 (Santa cruz #sc-56036 1:500), Collagen III (Abcam #ab7778 1:1000), Fibronectin (Abcam #ab2413 1:1000), aSMA (Sigma #A5228 1:1000), ACSL4 (Abcam #ab155282 1:1000), CD63 (Abcam #ab193349 1:1000), GPX4 (Abcam #ab125066 1:1000), Actin (Sigma #A3854 1:20000), GAPDH (Proteintech #60004-1-Ig 1:1000), and Tubulin (BioLegend #801202 1:1000).

Techniques: Control, Injection, Staining, Western Blot

Journal: Nature Communications

Article Title: A single genetic locus controls both expression of DPEP1/CHMP1A and kidney disease development via ferroptosis

doi: 10.1038/s41467-021-25377-x

Figure Lengend Snippet: a Relative mRNA level of Dpep1 in scramble siRNA (siControl) and Dpep1 siRNA (siDpep1) transfected tubule cell ( n = 3). b Western blots of DPEP1 and CHMP1A in scramble and Dpep1 siRNA transfected tubule cell. c LDH level of NRK52E cell treated with varying dose of cisplatin for varying degree of time ( n = 3). d The percentage of viable cells following siControl and siDpep1 transfection and in the presence and absence of cisplatin treatment ( n = 3). e LDH level of following siControl and siDpep1 transfection and sham or cisplatin treatment ( n = 3). f The ratio of cell-impermeable peptide substrate bis-AAF-R110 (dead cell indicator) to cell-permeable GF-AFC substrate (live cell indicator) from siControl and siDpep1 transfected cell following sham or cisplatin treatment ( n = 3). g Relative transcript level of Ripk1 and Mlkl of siControl and siDpep1 transfected cell following sham or cisplatin treatment ( n = 3). h Relative transcript level of Ripk1 in kidneys of folic acid and cisplatin-treated wild-type and Dpep1 +/− mice. Sham-treated group: WT ( n = 3), Dpep1 +/− ( n = 3); FA-treated group: WT ( n = 5), Dpep1 +/− ( n = 7); cisplatin-treated group: WT ( n = 7), Dpep1 +/− ( n = 6). i Relative transcript level of Mlkl in kidneys of folic acid and cisplatin-treated wild-type and Dpep1 +/− mice. Sham-treated group: WT ( n = 3), Dpep1 +/− ( n = 3); FA-treated group: WT ( n = 5), Dpep1 +/− ( n = 7); cisplatin-treated group: WT ( n = 7), Dpep1 +/− ( n = 6). j Relative transcript level of Nlrp3 and Il1beta from siControl and siDpep1 transfected cell following sham or cisplatin treatment ( n = 3). k Relative transcript level of Nlrp3 and Il1beta in kidneys of folic acid and cisplatin-treated wild-type and Dpep1 +/− mice. Sham-treated group: WT ( n = 3), Dpep1 +/− ( n = 3); FA-treated group: WT ( n = 5), Dpep1 +/− ( n = 7); cisplatin-treated group: WT ( n = 7), Dpep1 +/− ( n = 6). l Western blots of RIPK3 and cleaved caspase 1 in kidneys of wild-type and Dpep1 +/− mice following sham or cisplatin treatment. m LDH level of siControl and siDpep1 transfected cell following sham or Nigericin treatment ( n = 3). n Relative mRNA level of Ly6G in kidneys of control, folic acid and cisplatin-treated wild-type and Dpep1 +/− mice. Sham-treated group: WT ( n = 3), Dpep1 +/− ( n = 3); FA-treated group: WT ( n = 5), Dpep1 +/− ( n = 7); cisplatin-treated group: WT ( n = 7), Dpep1 +/− ( n = 6). All data are represented as mean ± SEM. P value was calculated by two-way ANOVA with post hoc Tukey test for c – n . P value was calculated by two-tailed t -test for a . P < 0.05 is statistically significant. A Source Data file is available for this figure.

Article Snippet: Kidney tissue or cultured cell lysates were prepared with ice-cold lysis buffer (CST # 9806) containing protease inhibitor cocktail (cOmplete Mini, Roche #11836153001) and phosphatase inhibitor (PhosSTOP, Roche #4906837001), resolved on 8–12% gradient gels, transferred on to polyvinylidene difluoride membranes, and probed with the following antibodies: DPEP1 (Proteintech #12222-1-AP 1:500), CHMP1A (Proteintech #15761-1-AP 1:500), RIPK3 (Sigma #PRS2283 1:1000), Cleaved Caspase 1 (Santa cruz #sc-56036 1:500), Collagen III (Abcam #ab7778 1:1000), Fibronectin (Abcam #ab2413 1:1000), aSMA (Sigma #A5228 1:1000), ACSL4 (Abcam #ab155282 1:1000), CD63 (Abcam #ab193349 1:1000), GPX4 (Abcam #ab125066 1:1000), Actin (Sigma #A3854 1:20000), GAPDH (Proteintech #60004-1-Ig 1:1000), and Tubulin (BioLegend #801202 1:1000).

Techniques: Transfection, Western Blot, Control, Two Tailed Test

Journal: Nature Communications

Article Title: A single genetic locus controls both expression of DPEP1/CHMP1A and kidney disease development via ferroptosis

doi: 10.1038/s41467-021-25377-x

Figure Lengend Snippet: a Relative mRNA level of Chmp1a in scramble siRNA (siControl) and Chmp1a siRNA (siChmp1a) transfected kidney tubule cell ( n = 3). b Western blots of CHMP1A and DPEP1 from siControl and siChmp1a transfected cells. c Viability of siControl and siChmp1a transfected tubule cell following sham or cisplatin treatment ( n = 3). d LDH level of siControl and siChmp1 transfected tubule cell following sham or cisplatin treatment ( n = 3). e The ratio of cell-impermeable peptide substrate bis-AAF-R110 (dead cell indicator) to cell-permeable GF-AFC substrate (live cell indicator) from siControl siChmp1a transfected cell following sham or cisplatin treatment ( n = 3). f Relative mRNA level of Ripk1 and Mlkl of siControl and siChmp1a transfected cell following sham or cisplatin treatment ( n = 3). g Relative mRNA level of Ripk1 and Mlkl in kidneys of folic acid and cisplatin-treated wild-type and Chmp1a +/− mice. Sham-treated group: WT ( n = 3), Chmp1a +/− ( n = 3); FA-treated group: WT ( n = 5), Chmp1a +/− ( n = 5); cisplatin-treated group: WT ( n = 4), Chmp1a +/− ( n = 5). h Relative mRNA level of Nlrp3 and Il1beta of siControl and siChmp1a transfected cell following sham or cisplatin treatment ( n = 3). i Relative mRNA level of Nlrp3 and Il1beta in kidneys of folic acid and cisplatin-treated wild-type and Chmp1a +/− mice. Sham-treated group: WT ( n = 3), Chmp1a +/− ( n = 3); FA-treated group: WT ( n = 5), Chmp1a +/− ( n = 5); cisplatin-treated group: WT ( n = 4), Chmp1a +/− ( n = 5). j (left) Representative cleaved Caspase 3 staining of siControl and siChmp1a transfected cell following sham or cisplatin treatment. (right) Quantification of cleaved Caspase 3 positive cell ( n = 5). Scale bar: 20 μm. k Relative mRNA transcript of Bak and Bax in kidneys of folic acid and cisplatin-treated wild-type and Chmp1a +/− mice. Sham-treated group: WT ( n = 3), Chmp1a +/− ( n = 3); FA-treated group: WT ( n = 5), Chmp1a +/− ( n = 5); cisplatin-treated group: WT ( n = 4), Chmp1a +/− ( n = 5). l LDH level of siControl and siChmp1a transfected cell with or without cisplatin and necroptosis (Nec1), pyroptosis (Vx765), and apoptosis (Z-VAD-FMK) inhibitors ( n = 3). All data are represented as mean ± SEM. P value was calculated by two-way ANOVA with post hoc Tukey test for c – l . P value was calculated by two-tailed t-test for a . P < 0.05 is statistically significant. A Source Data file is available for this figure.

Article Snippet: Kidney tissue or cultured cell lysates were prepared with ice-cold lysis buffer (CST # 9806) containing protease inhibitor cocktail (cOmplete Mini, Roche #11836153001) and phosphatase inhibitor (PhosSTOP, Roche #4906837001), resolved on 8–12% gradient gels, transferred on to polyvinylidene difluoride membranes, and probed with the following antibodies: DPEP1 (Proteintech #12222-1-AP 1:500), CHMP1A (Proteintech #15761-1-AP 1:500), RIPK3 (Sigma #PRS2283 1:1000), Cleaved Caspase 1 (Santa cruz #sc-56036 1:500), Collagen III (Abcam #ab7778 1:1000), Fibronectin (Abcam #ab2413 1:1000), aSMA (Sigma #A5228 1:1000), ACSL4 (Abcam #ab155282 1:1000), CD63 (Abcam #ab193349 1:1000), GPX4 (Abcam #ab125066 1:1000), Actin (Sigma #A3854 1:20000), GAPDH (Proteintech #60004-1-Ig 1:1000), and Tubulin (BioLegend #801202 1:1000).

Techniques: Transfection, Western Blot, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: A single genetic locus controls both expression of DPEP1/CHMP1A and kidney disease development via ferroptosis

doi: 10.1038/s41467-021-25377-x

Figure Lengend Snippet: a (Left) Representative cleaved caspase 3 staining of siControl and siDpep1 cell following sham or cisplatin treatment. (Right) Quantification of positive cleaved caspase 3 cell ( n = 7). Scale bar: 20 μm. b Relative mRNA transcript of Bax and Bak in kidneys of folic acid and cisplatin-treated wild-type and Dpep1 +/− mice. Sham-treated g group: WT ( n = 3), Dpep1 +/− ( n = 3); FA-treated group: WT ( n = 5), Dpep1 +/− ( n = 7); cisplatin-treated group: WT ( n = 7), Dpep1 +/− ( n = 6). c (Left) LDH level of siControl and siDpep1 transfected cell with or without apoptosis activator camptothecin (CPT) treatment. (Right) The ratio of cell-impermeable peptide substrate bis-AAF-R110 (dead cell indicator) to cell-permeable GF-AFC substrate (live cell indicator) of siControl and siDpep1 transfected cell with or without CPT treatment ( n = 3). d (left) Representative BODIPY 581/591 C11 fluorescence of siControl and siDpep1 transfected cell following sham or cisplatin treatment. (right) Quantification of the oxidized vs. reduced probe ( n = 5). Scale bar: 50 μm. e Ferrous iron concentration (normalized to kidney weight) in kidneys of cisplatin-treated wild-type and Dpep1 +/− mice. Sham-treated group: WT ( n = 3), Dpep1 +/− ( n = 3); cisplatin-treated group: WT ( n = 7), Dpep1 +/− ( n = 6). f Relative mRNA level of Acsl4 in kidneys of folic acid and cisplatin-treated wild-type and Dpep1 +/− mice. Sham-treated group: WT ( n = 3), Dpep1 +/ − ( n = 3); FA-treated group: WT ( n = 5), Dpep1 +/− ( n = 7); cisplatin-treated group: WT ( n = 7), Dpep1 +/− ( n = 6). g Representative images of ACSL4 immunostaining in kidney sections of folic acid-treated wild-type and Dpep1 +/− mice. Scale bar: 10 μm. h Western blots of ACSL4 kidneys of folic acid-treated wild-type and Dpep1 +/− mice. i LDH level of scramble siControl and siDpep1 transfected cell following sham or ferroptosis activator Erastin, FIN56, FINO2, and RSL3 treatment ( n = 3). j (Left) Representative images of transferrin uptake of siControl and siDpep1 transfected cells. (Right) Quantification of arbitrary fluorescence unit of transferrin in siControl and siDpep1 transfected cell ( n = 10). Scale bar: 50 μm. All data are represented as mean ± SEM. P value was calculated by two-way ANOVA with post hoc Tukey test for a – i and was calculated by two-tailed t -test for j . P < 0.05 is statistically significant. A Source Data file is available for this figure.

Article Snippet: Kidney tissue or cultured cell lysates were prepared with ice-cold lysis buffer (CST # 9806) containing protease inhibitor cocktail (cOmplete Mini, Roche #11836153001) and phosphatase inhibitor (PhosSTOP, Roche #4906837001), resolved on 8–12% gradient gels, transferred on to polyvinylidene difluoride membranes, and probed with the following antibodies: DPEP1 (Proteintech #12222-1-AP 1:500), CHMP1A (Proteintech #15761-1-AP 1:500), RIPK3 (Sigma #PRS2283 1:1000), Cleaved Caspase 1 (Santa cruz #sc-56036 1:500), Collagen III (Abcam #ab7778 1:1000), Fibronectin (Abcam #ab2413 1:1000), aSMA (Sigma #A5228 1:1000), ACSL4 (Abcam #ab155282 1:1000), CD63 (Abcam #ab193349 1:1000), GPX4 (Abcam #ab125066 1:1000), Actin (Sigma #A3854 1:20000), GAPDH (Proteintech #60004-1-Ig 1:1000), and Tubulin (BioLegend #801202 1:1000).

Techniques: Staining, Transfection, Fluorescence, Concentration Assay, Immunostaining, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: A single genetic locus controls both expression of DPEP1/CHMP1A and kidney disease development via ferroptosis

doi: 10.1038/s41467-021-25377-x

Figure Lengend Snippet: a Relative expression of DPEP1 (y-axis) and CHMP1A (x-axis) in 432 microdissected human kidney tubule samples. Pearson correlation is shown. Student t -test based on the Pearson correlation coefficient was used to calculate the statistical significance of the association. b Genes showing significant correlation with DPEP1 and CHMP1A in microdissected human kidney tubule samples. y - axis represents the Pearson correlation P value. c Relative transcript levels of ACSL4 , ACSL3 , and SLC3A2 (FPKM, y-axis), and kidney function (eGFR, x-axis) or kidney fibrosis (x-axis) as analyzed in 432 microdissected human kidney samples. Pearson correlation is shown. Student’s t -test based on the Pearson correlation coefficient was used to calculate the statistical significance of the association. d Representative images of ACSL4 immunostaining in healthy control and CKD kidney samples. Scale bar: 20 μm. e Western blots of ACSL4 in healthy control and CKD kidney samples.

Article Snippet: Kidney tissue or cultured cell lysates were prepared with ice-cold lysis buffer (CST # 9806) containing protease inhibitor cocktail (cOmplete Mini, Roche #11836153001) and phosphatase inhibitor (PhosSTOP, Roche #4906837001), resolved on 8–12% gradient gels, transferred on to polyvinylidene difluoride membranes, and probed with the following antibodies: DPEP1 (Proteintech #12222-1-AP 1:500), CHMP1A (Proteintech #15761-1-AP 1:500), RIPK3 (Sigma #PRS2283 1:1000), Cleaved Caspase 1 (Santa cruz #sc-56036 1:500), Collagen III (Abcam #ab7778 1:1000), Fibronectin (Abcam #ab2413 1:1000), aSMA (Sigma #A5228 1:1000), ACSL4 (Abcam #ab155282 1:1000), CD63 (Abcam #ab193349 1:1000), GPX4 (Abcam #ab125066 1:1000), Actin (Sigma #A3854 1:20000), GAPDH (Proteintech #60004-1-Ig 1:1000), and Tubulin (BioLegend #801202 1:1000).

Techniques: Expressing, Immunostaining, Control, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Novel Mouse Model of Granular Corneal Dystrophy Type II Reveals Impaired Autophagy and Recapitulates Human Pathogenesis

doi: 10.1167/iovs.66.12.7

Figure Lengend Snippet: Generation and genotyping of TGFBI –R124H knock-in mice. ( A ) Schematic representation of the CRISPR/Cas9-mediated editing process used to introduce the R124H mutation in the TGFBI gene of mice. ( B ) Genotyping of TGFBI –R124H mice by restriction enzyme digestion and PCR. ( C ) DNA sequencing confirmed the presence of the R124H mutation in heterozygous mice.

Article Snippet: Immunofluorescence staining evaluated TGFBI (10188-1-AP, 1:1000; Proteintech, Wuhan, China) expression in the corneas of WT, HE, and HO mice.

Techniques: Knock-In, CRISPR, Introduce, Mutagenesis, DNA Sequencing

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Novel Mouse Model of Granular Corneal Dystrophy Type II Reveals Impaired Autophagy and Recapitulates Human Pathogenesis

doi: 10.1167/iovs.66.12.7

Figure Lengend Snippet: Corneal opacity in WT and TGFBI –R124H mutant mice. ( A ) Representative slit-lamp microscopy images of corneas from WT, HE, and HO TGFBI –R124H mice at 15 months of age. ( B ) AS-OCT images of the corneas in WT, HE, and HO mice. ( C ) Frequency of corneal opacity by genotype and observation time (total numbers: WT = 31, HE = 45, HO = 26).

Article Snippet: Immunofluorescence staining evaluated TGFBI (10188-1-AP, 1:1000; Proteintech, Wuhan, China) expression in the corneas of WT, HE, and HO mice.

Techniques: Mutagenesis, Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Novel Mouse Model of Granular Corneal Dystrophy Type II Reveals Impaired Autophagy and Recapitulates Human Pathogenesis

doi: 10.1167/iovs.66.12.7

Figure Lengend Snippet: Microscopic examination of the cornea in WT and TGFBI –R124H mutant mice. ( A , B ) H&E staining ( A ) and Masson's trichrome staining ( B ) of corneal sections. ( C , D ) SEM and TEM images of corneal tissues showing irregular, amorphous deposits ( yellow arrowheads ) and abnormal collagen arrangement in the corneal stroma. ( E , F ) Immunofluorescence staining of corneal sections revealed increased TGFBIp deposition in HE and HO mice compared to WT controls ( n = 3 per group). ( G ) Western blot analysis showed significantly elevated TGFBIp levels in both HE and HO mice ( n = 3 per group). ( H ) qRT-PCR analysis demonstrated significantly higher TGFBI mRNA expression in HO mice ( n = 3 per group). *** P < 0.001.

Article Snippet: Immunofluorescence staining evaluated TGFBI (10188-1-AP, 1:1000; Proteintech, Wuhan, China) expression in the corneas of WT, HE, and HO mice.

Techniques: Mutagenesis, Staining, Immunofluorescence, Western Blot, Quantitative RT-PCR, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Novel Mouse Model of Granular Corneal Dystrophy Type II Reveals Impaired Autophagy and Recapitulates Human Pathogenesis

doi: 10.1167/iovs.66.12.7

Figure Lengend Snippet: Gene module detection by WGCNA from TGFBI –R124H mutant mice. ( A ) Scale independence and mean connectivity calculated by the WGCNA package. ( B ) Module discovery was performed by clustering genes based on topological overlap matrix dissimilarity ( y -axis is height). Similar clusters were merged using a dissimilarity threshold of 0.25 (merged dynamic). ( C ) Quantified traits were correlated to the eigengene expression of each module to prioritize modules of interest. Benjamini–Hochberg adjusted P values were reported in each cell of the heatmap (colored by Pearson's r ). Mutation (Mut) is defined as a binary trait (1, HO or HE; 0, WT). HO is defined as a binary trait (1, HO; 0, HE or WT). The sample size for each group was 10.

Article Snippet: Immunofluorescence staining evaluated TGFBI (10188-1-AP, 1:1000; Proteintech, Wuhan, China) expression in the corneas of WT, HE, and HO mice.

Techniques: Mutagenesis, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Novel Mouse Model of Granular Corneal Dystrophy Type II Reveals Impaired Autophagy and Recapitulates Human Pathogenesis

doi: 10.1167/iovs.66.12.7

Figure Lengend Snippet: Impaired autophagy flux in TGFBI –R124H mutant mice. ( A , B ) GO and KEGG enrichment analysis of genes in the TGFBI –R124H mutation related modules (MEgrey60 and MEcyan). ( C ) Heatmap based on GSVA analysis of macroautophagy, protein–macromolecule adaptor activity, phosphoprotein binding, endocytosis, ECM–receptor interaction–related gene sets. ( D ) Western blot analysis showing elevated levels of LC3 and SQSTM1 in the corneas of TGFBI –R124H mutant mice ( n = 3 per group). ( E ) TEM showed autophagolysosomes were more abundant in HE and HO corneal fibroblasts. The yellow arrow points to the autophagolysosomes. P values were calculated by comparing HE and HO with WT. * P < 0.05, ** P < 0.01.

Article Snippet: Immunofluorescence staining evaluated TGFBI (10188-1-AP, 1:1000; Proteintech, Wuhan, China) expression in the corneas of WT, HE, and HO mice.

Techniques: Mutagenesis, Activity Assay, Binding Assay, Western Blot