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: Nature

Article Title: Saturation Editing of Genomic Regions by Multiplex Homology-Directed Repair

doi: 10.1038/nature13695

Figure Lengend Snippet: (a) Experimental schematic. Cultured cells were co-transfected with a single Cas9-sgRNA construct (CRISPR) and a complex homology-directed repair (HDR) library containing an edited exon that harbors a random hexamer (blue, green, orange) and a fixed selective PCR site (red). CRISPR-induced cutting stimulated homologous recombination with the HDR library, inserting mutant exons into the genomes of many cells. At five days post-transfection, cells were harvested for gDNA and RNA. After reverse transcription, selective PCR was performed followed by sequencing of gDNA and cDNA derived amplicons. Hexamer enrichment scores were calculated by dividing cDNA counts normalized by gDNA counts. (b) Correlation of enrichment scores between biological replicates for hexamers observed in each experiment with positions of previously identified exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs) and stop codons indicated. (c) Rank-ordered plot of enrichment scores with positions of ESEs, ESSs, and stop codons indicated.

Article Snippet: A bicistronic Cas9-sgRNA vector designed to cleave within BRCA1 exon 18 (“pCas9-sgBRCA1x18”) was cloned according to a published protocol by ligating annealed oligonucleotides into a human codon-optimized S. pyogenous Cas9-sgRNA vector from the lab of Feng Zhang (pX330-U6-Chimeric_BB-CBh-hSpCas9; Addgene plasmid #42230).

Techniques: Cell Culture, Transfection, Construct, CRISPR, Random Hexamer Labeling, Homologous Recombination, Mutagenesis, Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

doi: 10.1101/2020.06.16.155101

Figure Lengend Snippet: (A) Schematic of pooled screen. Vero-E6 cells expressing Cas9 were transduced with the genome-wide C. sabaeus library via lentivirus. The transduced cell population then either received a mock treatment or was challenged with SARS-CoV-2 under various culture conditions. Surviving cells from each condition were isolated and the sgRNA sequences were amplified by PCR and sequenced. (B) Volcano plot showing top genes conferring resistance and sensitivity to SARS-CoV-2. The gene-level z-score and −log10(FDR) were both calculated using the mean of the five Cas9-v2 conditions. Non-targeting control sgRNAs were randomly grouped into sets of 4 to serve as “dummy” genes and are shown in green. (C) Performance of individual guide RNAs targeting ACE2, SMARCA4, CTSL, and TMPRSS2. The mean residual across the five Cas9-v2 conditions is plotted for the full library (top) and for the 4 guide RNAs targeting each gene. (D) Heatmaps of the top 25 gene hits for resistance and sensitivity, ranked by mean z-score in the Cas9-v2 conditions.Genes that are included in one of the gene sets labeled in are colored accordingly. Condition a: Cas9v2 D5 2.5e6 Hi-MOI; b: Cas9v2 D5 5e6 Hi-MOI; c: Cas9v2 D2 5e6 Hi-MOI; d: Cas9v2 D10 5e6 Hi-MOI; e: Cas9v2 D5 2.5e6 Lo-MOI.

Article Snippet: Cas9 activity was assessed by transducing parental Vero-E6 or Vero-E6-Cas9 cells with pXPR_047 (Addgene 107645), which expresses eGFP and an sgRNA targeting eGFP ( ).

Techniques: Expressing, Transduction, Genome Wide, Isolation, Amplification, Control, Labeling

Journal: bioRxiv

Article Title: Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

doi: 10.1101/2020.06.16.155101

Figure Lengend Snippet: (A) Correlation matrix depicting the Pearson correlation between the guide-level log-fold change values relative to the plasmid DNA. Cells were cultured in DMEM with 2% FBS (D2), 5% FBS (D5), 10% FBS (D10), plated at 2.5 x 10 6 or 5.0 x 10 6 cells per flask and infected at a MOI 0.1 (Hi) or MOI 0.01 (Lo). (B) Receiver-operator characteristic (ROC) curve for the recovery of guides targeting essential genes in the mock-treated condition of the Cas9-v1 and Cas9-v2 screens. True positives are n = 1,528 essential genes (n = 6,178 guides); true negative genes are n = 622 non-essential genes (n = 2,504 guides). We mapped essential and non-essential genes, which were derived for human cell lines, to the African green monkey genome simply by matching gene symbols. AUC = area under curve. (C) Correlation between gene enrichment in Cas9-v1 and Cas9-v2 screens. Pearson correlation is reported. (D-E) GFP-based Cas9 activity assay in Vero-E6 cells stably expressing either Cas9-v1 (D) or Cas9-v2 (E). The pXPR_047 construct expresses GFP and an sgRNA targeting GFP; therefore, cells without Cas9 activity will express GFP, whereas cells with high Cas9 activity will knock out GFP and resemble parental cells. (F) Approach to calculate residuals from log-fold change data, using ACE2 and the 5%FBS, 5 x 10 6 cells/flask, MOI 0.1 condition as an example. A natural cubic spline with four degrees of freedom is shown in blue, and a residual for each sgRNA is calculated to be the vertical distance from the fit spline.

Article Snippet: Cas9 activity was assessed by transducing parental Vero-E6 or Vero-E6-Cas9 cells with pXPR_047 (Addgene 107645), which expresses eGFP and an sgRNA targeting eGFP ( ).

Techniques: Plasmid Preparation, Cell Culture, Infection, Derivative Assay, Activity Assay, Stable Transfection, Expressing, Construct, Knock-Out

Journal: bioRxiv

Article Title: Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

doi: 10.1101/2020.06.16.155101

Figure Lengend Snippet: (A) Performance in the pooled screen of guide RNAs targeting the 25 genes selected for further validation. The mean residual across the five Cas9-v2 conditions is plotted for the full library (top) and for the 3-4 guide RNAs targeting each gene. Genes that scored as resistance hits are shown in red; genes that scored as sensitization hits are shown in blue. The dashed line indicates a residual of 0. (B) 42 unique sgRNAs targeting 25 genes were introduced into VeroE6 cells. SARS-CoV-2 was added at MOI 0.2 and cell viability was measured at 3 dpi. (C). Z-scores from CRISPR screen correlate with cell viability of individually disrupted genes. Genes with multiple sgRNAs from (B) are averaged to generate one point per gene in (C). Data in (B) and (C) is representative of one of three independent experiments each done in quintuplicate.

Article Snippet: Cas9 activity was assessed by transducing parental Vero-E6 or Vero-E6-Cas9 cells with pXPR_047 (Addgene 107645), which expresses eGFP and an sgRNA targeting eGFP ( ).

Techniques: Biomarker Discovery, CRISPR

Journal: bioRxiv

Article Title: Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

doi: 10.1101/2020.06.16.155101

Figure Lengend Snippet: (A) Performance of individual guide RNAs targeting LOC103214541 (HMGB1-like). The mean residual across the five Cas9-v2 conditions is plotted for the full library (top) and for the 3 guide RNAs targeting that gene. (B) Western blot for HMGB1 expression in control and HMGB1 sgRNAs transduced Vero-E6 cells. (C) Control and HMGB1 sgRNAs transduced Vero-E6 cells were infected with SARS-CoV-2 at a MOI of 0.2. Cell viability relative to an uninfected control was measured 3dpi with CellTiter Glo. (D) Vero-E6 cells were infected with SARS-CoV-2 at a MOI of 0.1. Viral production as measured by plaque forming units (PFU/ml) was determined by plaque assay. (E) Vero-E6 cells were infected with VSV-G or SARS-CoV-2-Spike pseudovirus. Luminescence was measured 1dpi. LOD, limit of detection. Data was analyzed by Mann-Whitney test. Shown are means ± SEM. ns, not statistically significant; *P < 0.05; **P < 0.01; ***P < 0.001. Data in (C) are one representative experiment from three independent times each in octuplicate. Data in (D-E) are one representative experiment from two independent experiments each performed in triplicate.

Article Snippet: Cas9 activity was assessed by transducing parental Vero-E6 or Vero-E6-Cas9 cells with pXPR_047 (Addgene 107645), which expresses eGFP and an sgRNA targeting eGFP ( ).

Techniques: Western Blot, Expressing, Control, Infection, Plaque Assay, MANN-WHITNEY

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Specific commercial products and services available to the researchers to implement CRISPR technology.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Genome Wide, Clone Assay, Stable Transfection, Selection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Negative Control, Positive Control, Construct, Knock-In, Multiplex Assay, Amplification, Sequencing

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet: ( a ) Representative immunofluorescence images of HEK293T, HEK293 cell lines and HEK293 cells transiently expressing SV40 large T (LT) antigen. Cells were fixed, stained with SMC6 (green), LT (red) antibodies and DAPI (blue). Scale bar 10 μm. ( b ) Schematic of SMC5/6 complex subunits showing the SLF1/SLF2 cofactors recruiting the complex to DNA lesions and depicting the hypothesis of SMC5/6 cofactor involved in SV40 virus lifecycle. ( c ) Experimental design for human SMC5 BioID with SILAC. ( d ) The top SMC5 interacting proteins identified in BioID screen are listed with their ratio of heavy ( H ) to light ( L ) enrichment. Where known, fission and budding yeast orthologues are listed. The green rows contain known SMC5/6 subunits, grey ones are possible novel interactors of human SMC5. BioID datasets are available in the PRIDE database ( https://www.ebi.ac.uk/pride/archive ) under the accession code PRIDE PXD033923 and in .

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Immunofluorescence, Expressing, Staining, Virus, Multiplex sample analysis

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet: ( a ) Schematic representation of SIMC1 and SLF2. SIM1/2, SUMO interaction motifs; IDR, intrinsically disordered regions; APIM, AlkB homologue 2 PCNA-interacting motif. ( b ) Representative immunofluorescence images of U2OS cells overexpressing mCherry-SIMC1 and GFP-SLF2 that were fixed and stained with DAPI. Dotted outline marks nucleus. Scale bar 10 µm. ( c ) Western blot of GFP-Trap immunoprecipitation from HEK293 cells transfected with plasmids expressing the indicated combinations of proteins. Truncation constructs of SIMC1 and SLF2 are represented schematically including the status of SIMC1-SLF2 interaction that is, +++ > +, – undetectable. Full and unedited blots provided in . Figure 2—source data 1. Full and unedited blots corresponding to panel ( c ).

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Immunofluorescence, Staining, Western Blot, Immunoprecipitation, Transfection, Expressing, Construct

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet: Western blot of GFP-Trap immunoprecipitation from HEK293 cells transfected with plasmids expressing the indicated combinations of proteins. Full and unedited blots provided in . Figure 2—figure supplement 2—source data 1. Full and unedited blots.

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Western Blot, Immunoprecipitation, Transfection, Expressing

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet: ( a, b ) A subset of SIMC1 interacting proteins identified by BioID screen carried out under native ( a ) or denaturing ( b ) lysis conditions, respectively. Pink circle illustrates the biotinylation radius of BirA*. BioID datasets are available in the PRIDE database ( https://www.ebi.ac.uk/pride/archive ) under the accession code PRIDE PXD033923 and in . ( c ) Representative immunofluorescence images of U2OS cells overexpressing mCherry-SIMC1 or mCherry-SIMC1 SIM mut (mutations: FIDL to AADA (26–29 aa) and VIDL to AADA (45–48 aa)) that were stained for PML (left three panels); or were co-expressing GFP-ZNF451 (right three panels). The cells were stained with DAPI to mark nucleus (dotted lines). Scale bar 10 µm. ( d ) HEK293 cells overexpressing ZNF451, BirA*-SIMC1, SLF2, BirA* control in indicated combination were cultured in the presence of 50 μM supplemental biotin. The total cell extracts and streptavidin pulldown were analyzed by western blot using anti-GFP or Myc antibodies. Full and unedited blots provided in . Figure 3—source data 1. Full and unedited blots corresponding to panel ( d ).

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Lysis, Immunofluorescence, Staining, Expressing, Control, Cell Culture, Western Blot

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet: ( a ) Representative immunofluorescence images of SV40 LT (green), SMC6 (red) and PML (light blue) in HEK293 and HEK293 SIMC1 -/- cells fixed 48 h after SV40 transfection. Scale bar 10 μm. The bar graph shows relative quantification of the number of cells containing SMC6 foci. A minimum of 175 cells with at least four SV40 LT foci were counted for each cell line. Primary data for graph in panel ( a ) provided in . ( b ) Representative immunofluorescence images of HEK293 SIMC1 -/- cells with integrated empty vector or vectors expressing FLAG-SIMC1 or FLAG-SIMC1 SIM mut, respectively, 48 hr after SV40 transfection. SV40 LT (green), SMC6 (red), FLAG (light blue); Scale bar 10 μm. ( c ) Relative quantification of the number of cells containing SMC6 and FLAG foci (representative images shown in panel b ). A minimum of 225 cells with at least four SV40 LT foci were counted for each cell line. Primary data provided in . ( d ) Immunoblot from HEK293 SIMC1 -/- cells with integrated empty vector or vector expressing FLAG-SIMC1 or SIMC1 SIM mut, respectively, and transfected with a plasmid carrying SV40 genome. Cells were harvested 48 hr after transfection. PSTAIR serves as a loading control. Full and unedited blots provided in . Statistics in ( a, c ): Means and error bars (s.d.) were derived from a minimum of three independent SV40 transfections representing biological replicates. (∗) p<0.05; (∗∗) p<0.005; (∗∗∗) p<0.0005; (n.s.) p>0.05 (two-tailed unpaired t-test). Figure 4—source data 1. Primary data for graphs in panel ( a ). Figure 4—source data 2. Primary data for graphs in panel ( c ). Figure 4—source data 3. Full and unedited blots corresponding to panel ( d ).

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Immunofluorescence, Transfection, Quantitative Proteomics, Plasmid Preparation, Expressing, Western Blot, Control, Derivative Assay, Two Tailed Test

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet: ( a ) Positions of the mutations on SIMC1 and SLF2. SIMC1 set A (Y528D, M529D, K533D, L537D), set B (S778D, F775E), and set C (L847E, L850A, L851E, Y854D) mutations and SLF2 set A (L775E, F786E, F792E), set B (C820W, I821W), set C (M831R, I835R), and set D (P957D, V959D, W1006A) mutations are indicated on the SIMC1 interface (left), the side view of the complex (center), and the SLF2 interface (right). SIMC1’s N-terminal loop (425–467 aa) is shown with surface. ( b, c ) Western blot of GFP-Trap immunoprecipitation from HEK293 cells transiently transfected with plasmids expressing GFP-SIMC1 variants and FLAG-SLF2 CTD ( b ) or GFP-SIMC1 and Myc-SLF2 CTD variants ( c ). Full and unedited blots of ( b ) and ( c ) are provided in , , respectively. Figure 5—figure supplement 14—source data 1. Full and unedited blots corresponding to ( b ). Figure 5—figure supplement 14—source data 2. Full and unedited blots corresponding to ( c ).

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Western Blot, Immunoprecipitation, Transfection, Expressing

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet: ( a ) Conservation mapping on the surface of the SIMC1-SLF2 complex. The conservation score obtained from Consurf server are shown by the color graduation as indicated. The boundary of SIMC1’s surface is indicated by yellow lines for clarity. Mutated amino acids (R473D/N477A/E480K/E481K) in the α1 of SIMC1 α-solenoid are labeled. ( b ) Western blot of GFP-trap immunoprecipitation from HEK293 cells transfected with either GFP-SIMC1 or GFP-SIMC1 combo mutant in combination with FLAG-SLF2 CTD and Myc-SMC6. Full and unedited blots provided in ( c ) Representative immunofluorescence images of HEK293 SIMC1 -/- cells with integrated vectors expressing FLAG-SIMC1 or FLAG-SIMC1 combo mut, respectively. Cells were fixed 48 hr after SV40 transfection and stained with SV40 LT (green), SMC6 (red), FLAG (light blue) antibodies. Scale bar 10 μm. ( d ) Relative quantification of the number of cells containing SMC6 and FLAG foci (representative images shown in panel c ). A minimum of 210 cells with at least four SV40 LT foci were counted for each cell line. Means and error bars (s.d.) were derived from three independent SV40 transfections representing biological replicates. (∗) p<0.05; (∗∗) p<0.005; (∗∗∗) p<0.0005; (n.s.) p>0.05 (two-tailed unpaired t-test). Primary data provided in . Figure 6—source data 1. Full and unedited blots corresponding to panel ( b ). Figure 6—source data 2. Primary data for graphs in panel ( d ).

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Labeling, Western Blot, Immunoprecipitation, Transfection, Mutagenesis, Immunofluorescence, Expressing, Staining, Quantitative Proteomics, Derivative Assay, Two Tailed Test

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet: ( a ) Schematic of SLF1 domain boundaries on the AlphaFold model. ( b ) Superposition of the α-solenoid domains of SLF1 and SIMC1. ( c ) The ColabFold-predicted structural model of the SLF1 410 -SLF2 CTD complex. ( d ) Western blot of GFP-Trap immunoprecipitation from HEK293 cells transiently transfected with plasmids expressing the respective protein combination. Schematic on the right represents domains in SLF1 truncated variants. Full and unedited blots provided in ( e ) Western blot of Myc-Trap or GFP-Trap immunoprecipitation from HEK293 cells transfected with FLAG-SIMC1, Myc-SLF2 CTD and GFP-SLF1 410 or GFP-SLF1 410_ΔARD (linker) , respectively. Domains of SLF1 constructs are shown in the scheme on the right side. Full and unedited blots provided in . Figure 7—source data 1. Full and unedited blots corresponding to panel ( d ). Figure 7—source data 2. Full and unedited blots corresponding to panel ( e ).

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Western Blot, Immunoprecipitation, Transfection, Expressing, Construct

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet: ( a ) Positions of the mutations are indicated on SLF1 336 (left), the SLF1 336 -SLF2 CTD complex (center), and SLF2 CTD (right). The ARD domain (726–934 aa) and the N-terminal disordered region (336–350 aa) are omitted for clarity. SLF1 mutations are set A (G464D, H468E, A472D, L476E), set B (F957E, L964E), and set C (A1042D, I1045E, M1049E). SLF2 mutations are same as shown in . ( b ) Western blot of Myc-Trap immunoprecipitation from HEK293 cells transiently transfected with plasmids expressing Myc-SLF2 CTD and GFP-SLF1 variants. ( c, d ) Western blot of GFP-Trap immunoprecipitation from HEK293 cells transiently transfected with plasmids expressing GFP-SLF1 336 variants and FLAG-SLF2 CTD ( c ) or GFP-SLF1 336 and Myc-SLF2 CTD variants ( d ). Full and unedited blots of ( b–d ) are provided in , , , respectively. Figure 7—figure supplement 8—source data 1. Full and unedited blots corresponding to ( b ). Figure 7—figure supplement 8—source data 2. Full and unedited blots corresponding to ( c ). Figure 7—figure supplement 8—source data 3. Full and unedited blots corresponding to ( d ).

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Western Blot, Immunoprecipitation, Transfection, Expressing

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet: ( a ) U2OS cells stably expressing either FLAG-SLF1 or FLAG-SIMC1 were exposed to laser microirradiation. Cells were fixed 1 hr later and stained with gamma-H2A.X (phosphorylation of histone H2A.X Ser139) and FLAG antibodies. Scale bar 10 μm. ( b ) Localization of SLF1 to LT foci depends on SIMC1. Representative immunofluorescence images of HEK293 WT and SIMC1-/- cells with integrated vector expressing FLAG-SLF1. Cells were fixed 48 hr after SV40 transfection and stained with SV40 LT (green), FLAG (red), PML (light blue) antibodies. Scale bar 10 μm. ( c ) Relative quantification of the number of cells containing FLAG and PML foci representative images shown in panel ( b ). A minimum of 267 cells with at least four SV40 LT foci were counted for each cell line. Means and error bars (s.d.) were derived from three independent SV40 transfections representing biological replicates. (∗) p<0.05; (∗∗) p<0.005; (∗∗∗) p<0.0005; (n.s.) p>0.05 (two-tailed unpaired t-test). Primary data provided in . ( d ) Comparison of localization of FLAG-SIMC1 and FLAG-SLF1 (red), stably expressed from integrated vectors, and LT foci (green) in HEK293 cells. Cells fixed 48 hr after SV40 transfection. Scale bar 10 μm. Figure 8—source data 1. Primary data for graph in ( c ).

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Stable Transfection, Expressing, Staining, Phospho-proteomics, Immunofluorescence, Plasmid Preparation, Transfection, Quantitative Proteomics, Derivative Assay, Two Tailed Test, Comparison

Journal: eLife

Article Title: The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers

doi: 10.7554/eLife.79676

Figure Lengend Snippet:

Article Snippet: HEK293 and U2OS cell lines were authenticated by STR profiling (ATCC).

Techniques: Derivative Assay, CRISPR, Expressing, Transfection, Construct, Plasmid Preparation, Retroviral, Stable Transfection, Recombinant, Sequencing, Protease Inhibitor, Multiplex sample analysis, Cloning, Software

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: TMEM106B is required for a post-endocytic stage of virus entry (A) Wild-type (WT) or monoclonal TMEM106B KO NCI-H1975 cells, untransduced (control) or transduced with ACE2 cDNA and infected with SARS-CoV-2 Belgium/GHB-03021/2020 at MOI 0.03. Viral RNA in cells was measured by qPCR (n = 8 wells from two experiments). (B) Monoclonal NCI-H1975 ACE2 KO cells or TMEM106B KO cells overexpressing ACE2 , infected with SARS-CoV-2 Belgium/GHB-03021/2020 or HCoV-229E in the presence of anti-TMEM106B (Ab09). After 6 h (SARS-CoV-2) or 24 h (HCoV-229E), cells were stained for dsRNA (n = 8 wells from three experiments; untreated, n = 36). (C) Monoclonal NCI-H1975 ACE2 KO cells or TMEM106B KO cells overexpressing ACE2 , infected with SARS-CoV-2 Belgium/GHB-03021/2020 or HCoV-229E pretreated with different concentrations of heparin or heparan sulfate. After 6 h (SARS-CoV-2) or 24 h (HCoV-229E), cells were stained for dsRNA (n = 6 wells from two experiments; untreated, n = 28). (D) Monoclonal NCI-H1975 ACE2 KO cells or TMEM106B KO cells overexpressing ACE2 , with or without an sgRNA targeting EXT1 ( EXT1 KO ), infected with SARS-CoV-2 Belgium/GHB-03021/2020. After 6 h, cells were stained for dsRNA (n = 8 wells from two experiments). (E) WT NCI-H1975 cells, ACE2 KO (monoclonal), TMEM106B KO (monoclonal), ACE2/EXT1 KO (monoclonal), or ACE2/EXT1/TMEM106B KO (polyclonal) cells, incubated with SARS-CoV-2 Belgium/GHB-03021/2020 on ice. Viral RNA bound on cells was measured by qPCR (n = 12 wells from two experiments). Data were analyzed using one-way ANOVA with Dunnett’s multiple comparison test, comparing each condition with WT cells. (F) NCI-H1975 cells, untransduced (control) or transduced with ACE2 cDNA, treated with E64d or camostat, infected with SARS-CoV-2 Belgium/GHB-03021/2020, and stained for nucleocapsid after 6 h (n = 6 wells from two experiments). (G) NCI-H1975 WT or monoclonal TMEM106B KO cells, incubated with SARS-CoV-2 at MOI 8 on ice, followed by virus internalization at 35°C for 0 or 2 h in the presence of 20 μg/mL cycloheximide to block translation. Cells were stained for nucleocapsid before permeabilization (green) and after permeabilization (red) and nuclei (blue). Shown are representative images, scale bars, 10 μm. A magnification of the area in the square is shown in each upper right corner. (H) Quantified results from (G) (n = 12 wells from two experiments.). (B–D, F, and H) Upper dotted line: untreated level. Lower dotted line: detection limit. Data were log-transformed (B, C, and F) and analyzed using two-way ANOVA with Tukey’s (H), Šidák’s (B and D), or Dunnett’s (C and F) multiple comparison test, comparing each condition with the untreated control. (I) HEK293T cells co-transfected with three plasmids, encoding (1) SARS-CoV-2 Belgium/GHB-03021/2020 spike and mNeonGreen, (2) TMPRSS2, and (3) a receptor (ACE2 or TMEM106B) or control protein (Luc). Left: representative images, scale bars, 100 μm. Right: quantified syncytium area (n = 2 wells from one of two experiments with similar results). The area under the curve was calculated, followed by one-way ANOVA with Dunnett’s multiple comparison test, comparing each condition with Luc. (C, F, and I) Data are mean ± SEM. ∗∗∗∗ p < 0.0001; ∗∗∗ 0.0001 < p < 0.001; ∗∗ 0.001 < p < 0.01; ∗ 0.01 < p < 0.05; ns, not significant. See also Figure S5 .

Article Snippet: HEK293T (obtained from Jason Moffat, University of Toronto), Vero E6 (ATCC- CRL-1586), HEp-2 (ATCC CCL-23), U-87 MG (ATCC HTB-14) and Huh-7 (CLS - 300156; human hepatoblastoma) were grown in Dulbecco’s Modified Eagle Medium (DMEM).

Techniques: Virus, Control, Transduction, Infection, Staining, Incubation, Comparison, Blocking Assay, Transformation Assay, Transfection

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet: Analysis of TMEM106B cell surface expression, SARS-CoV-2 uptake into TMEM106B KO cells, and ACE2 expression in various cell lines, related to Figures 4 and (A) Confirmation of EXT1 knockout in monoclonal NCI-H1975 cell lines generated by CRISPR-Cas9. The cut site within the sgRNA is indicated by an arrowhead, and the protospacer adjacent motif (PAM) is underlined. Sequences of wild-type and EXT1 knockout cells were determined by Sanger sequencing and are shown as chromatograms. Inserted nucleotides are shown in red. (B and C) Wild-type (WT) or TMEM106B KO NCI-H1975 cells stained for TMEM106B (Ab09; green), membranes (CellBrite Fix 640; red), and nuclei (blue). Shown are representative images from one out of two independent experiments with similar results. Cells were either permeabilized before staining (B) or not permeabilized (C) to visualize only TMEM106B expressed on the cell surface. Scale bars, 10 μm. (D) WT or TMEM106B KO NCI-H1975 cells incubated with SARS-CoV-2 at MOI 1 for 24 h and stained for SARS-CoV-2 N (red), LAMP-1 (green), and nuclei (blue). Shown are representative images from one out of three independent experiments with similar results. Scale bars, 10 μm. Note that WT cells show more widespread N staining due to the translation of new N protein during productive infection. (E) Analysis of ACE2 expression levels in different cell lines. Lysates of the indicated wild-type cell lines or HEK293T cells transduced with an ACE2 overexpression construct were analyzed using a ProteinSimple Wes system, with antibodies specific for ACE2 and the endogenous controls vinculin and GAPDH.

Article Snippet: HEK293T (obtained from Jason Moffat, University of Toronto), Vero E6 (ATCC- CRL-1586), HEp-2 (ATCC CCL-23), U-87 MG (ATCC HTB-14) and Huh-7 (CLS - 300156; human hepatoblastoma) were grown in Dulbecco’s Modified Eagle Medium (DMEM).

Techniques: Expressing, Knock-Out, Generated, CRISPR, Sequencing, Staining, Incubation, Infection, Transduction, Over Expression, Construct

Journal: Cell

Article Title: TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet:

Article Snippet: HEK293T (obtained from Jason Moffat, University of Toronto), Vero E6 (ATCC- CRL-1586), HEp-2 (ATCC CCL-23), U-87 MG (ATCC HTB-14) and Huh-7 (CLS - 300156; human hepatoblastoma) were grown in Dulbecco’s Modified Eagle Medium (DMEM).

Techniques: Virus, Recombinant, Transfection, Staining, Lysis, Proliferation Assay, Gene Expression, Expressing, Knock-Out, Plasmid Preparation, Construct, Software, Cell Analysis, Simple Western

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of ADAMTS17 shows location and targeting of exon 3 by CRISPR/Cas9 gRNA to induce nonhomologous end joining. The nucleotide and amino acid sequence of the ADAMTS17 WT allele (green) and after AT insertion (red) are indicated. The dinucleotide insertion induced a frameshift, which resulted in a premature stop codon after 12 amino acids. (B) Sanger sequencing traces of a PCR product generated with primers flanking exon 3 showing the AT insertion (underlined) in the Adamts17 KO. (C) Quantitative real-time PCR using cDNA prepared from WT and Adamts17 KO lung tissue as a template shows significant reduction of ADAMTS17 mRNA in the KO (n = 3). (D) Micrographs of ADAMTS17 immunostaining of sections through WT and Adamts17 KO skin (left), DKO growth plates (middle), and of primary DKO mouse skin fibroblasts (right). The signal in the dermis around hair follicles, in growth plate chondrocytes, and in fibroblasts and their ECM originating from the monoclonal ADAMTS17 antibody was strongly reduced in KO and DKO tissues and cells, indicating lack of ADAMTS17 protein in Adamts17 KO mice. (E) Pie chart showing Mendelian distribution of genotypes recovered from Adamts17 Het intercrosses at the time of genotyping (P7–P10) (n = 94 mice). (F) Breeding scheme to generate WT, Adamts10 KO (10KO), Adamts17 KO (17KO), and DKO mice. (G) Pie chart showing distribution of genotypes recovered from Adamts10 Het; Adamts17 Het intercrosses at P7–P10 (n = 180 mice). Statistical analysis was performed using Chi square calculation. (H) Kaplan–Meier survival analysis of DKO mice. The numbers of observed dead/total mice for the individual genotypes are indicated in brackets. Statistical significance was determined using a log-rank test. (I) Whole mount images of WT, 10KO, 10KO;17Het mice at 4 wk of age show progressive reduction in body size. (J) Bar graphs showing body weights of 4-wk-old mice of the indicated genotypes. The number of mice is indicated below the genotypes. (I, K) Bar graphs showing body weight normalized to average femur length for the genotypes that were significantly different in (I). Bars in (C) indicate mean values and whiskers the SD. In (J, K) floating bars indicate the 25th–75th percentile range, lines the mean value, and whiskers the SD. (C, J, K) Statistical differences in (C) were determined using a two-sided t test and (J, K) were using a one-way ANOVA with post hoc Tukey test. a, P < 0.05 compared with WT.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: CRISPR, Sequencing, Generated, Real-time Polymerase Chain Reaction, Immunostaining

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Micrographs of Masson’s trichrome–stained cross sections through dorsal skin from 4-wk-old WT, Adamts10 KO (10KO), Adamts17 KO (17KO), and DKO mice. ED, epidermis; D, dermis; HD, hypodermis; PC, panniculus carnosus. (B, C, D) Bar graphs showing quantification of overall skin thickness (B) and the thicknesses of the epidermis, dermis, hypodermis (C), and panniculus carnosus (p. carnosus, (D)). Individual data points represent multiple measurements along the different skin layers from n = 3 mice/genotype. (E) Stacked bar graphs showing the relative proportions of individual skin layers. The percentage values are indicated. (F) Bar graphs show the quantification of hair follicle numbers in the skin for each genotype. (G, H) Bar graphs showing normalized gene expression in fragments per kilobase of transcript per million mapped reads (FPKM) for Adamts10 and Adamts17 in individual skin cell types at E14.5 (G) and P5 (H). Data were extracted from the Hair-GEL database ( ; ). (I, J, K) Micrographs showing the localization of ADAMTS17 mRNA (red/dark purple) in WT skin cross sections at E13.5 (I), E16.5 (J), and P0 (K) detected by RNAscope in situ hybridization with a probe specific for ADAMTS17 mRNA. Sections were counterstained with hematoxylin. (L) Micrograph of ADAMTS17 immunostaining (green) of cross sections through WT skin. Nuclei were stained with DAPI (blue). (M) Micrographs of primary mouse skin fibroblasts after immunostaining for fibrillin-1 (red) and fibronectin (green). Nuclei were counterstained with DAPI (blue). (M, N) Quantification of mean fluorescence intensity from (M) (n = 4 biological replicates). In (B, C, D, F), floating bars indicate 25th–75th percentile range, lines the mean value and whiskers the SD. In (N), the bars represent the mean value and the whiskers the SD. Statistical differences in (B, C, D, F, N) were determined using a one-way ANOVA with post hoc Tukey test. a, P < 0.05 compared with WT; b, P < 0.05 compared with Adamts10 KO; P < 0.05 compared with Adamts17 KO.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Staining, Gene Expression, RNAscope, In Situ Hybridization, Immunostaining, Fluorescence

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of ADAMTS10 and ADAMTS17, which is identical. The degenerate (ADAMTS10) and canonical (ADAMTS17) furin-processing sites and the localization of the catalytic residue Glu-390 in ADAMTS17 (17) that was mutated into Ala to generate proteolytically inactive ADAMTS17-EA (17-EA) are indicated. The domain organization of the catalytic (17-PCD) and ancillary (17-AD) domain constructs is indicated. (B) Schematic representation of experimental design for coculture of human dermal fibroblasts (HDF) with HEK293 cells stably expressing 17- or 17-EA (left) or co-transfection of 17- or 17-EA–encoding plasmids with FN1 or COL6A2-encoding plasmids in HEK293 cells (right). (C) Volcano plot showing N-terminally labeled peptides identified by N-terminomics method TAILS in conditioned medium from ADAMTS17-expressing HEK293 cells cocultured with HDFs. Peptides present only in samples from WT ADAMTS17 (red) or enriched in conditioned medium from WT ADAMTS17 cocultures compared with the cocultures with proteolytically inactive ADAMTS17-EA suggest ADAMTS17 substrates. (D) Venn diagram showing overlap of ADAMTS17-cleaved proteins (TAILS) from coculture systems (left) and binding partners for the ADAMTS17 ancillary domain (17-AD) identified by yeast-2-hybrid screening with a human placenta–derived cDNA library (right). Note that fibronectin (FN1) and COL6 were independently identified in both screens. (E) Domain organization of fibronectin (FN1, NP_997647 ) showing the localization of the domains that interacted with 17-AD (grey box, bolded amino acid sequence) and the localization of the peptide identified by TAILS (red bar, red amino acid sequence). (F) MS2 spectrum of the N-terminally labeled FN1 peptide (GNSVNEGLNQPTDDSCFDPYTVSHYAVGDEWER) showing b- and y ions. (G) Western blot detection of endogenous fibronectin in conditioned medium (Med) and cell lysates (Lys) collected after coculture of 17- or 17-EA–expressing HEKs with HDFs. A monoclonal (green) and four different polyclonal (red) anti-fibronectin antibodies were used. (H) Western blot detection of recombinant fibronectin (rFN) in conditioned medium (Med) and cell lysate (Lys) collected after co-expression of 17 or 17-EA with rFN in HEK293 cells. A polyclonal anti-fibronectin antibody (red) and a monoclonal anti V5-tag antibody (green) were used to detect rFN.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Residue, Construct, Stable Transfection, Expressing, Cotransfection, Labeling, Binding Assay, Derivative Assay, cDNA Library Assay, Sequencing, Western Blot, Recombinant

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of COL6A2 (NP_0018403) showing the localization of the domains that interacted with 17-AD (grey box, bolded amino acid sequence). (B) Domain organization of COL6A3 ( NP_004360 ) showing the localization of the peptide identified by MS (red bar, red amino acid sequence). (C) MS2 spectrum of the N-terminally labeled ADAMTS17-digested COL6A3 peptide (SDDEVDDPAVELkQFGVAPF) showing b- and y ions. (D) Western blot of endogenous (end.) COL6 (red) in conditioned medium (Med) and cell lysate (Lys) collected from cocultures of 17- or 17-EA–expressing HEK293 cells with HDFs. (E) Micrographs of endogenous COL6A1 deposition (red) in the ECM of HDFs cocultured with 17- or 17-EA–expressing HEK293 cells. Nuclei were stained with DAPI (blue). (F) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates). (G) Micrographs of endogenous COL6A1 deposition (red) in the ECM of HDF after culture in the presence of conditioned medium from 17- or 17-EA–expressing HEK293 cells. Nuclei were stained with DAPI (blue). (H) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates). (I) Western blot of recombinant COL66A2 (rCOL6) in conditioned medium (Med) and cell lysate (Lys) collected after co-expression of 17 or 17-EA and rCOL6A2 in HEK293 cells using a monoclonal anti FLAG-tag antibody (green). (J) Micrographs of HDFs cultured in the presence of 50 μg/ml of purified recombinant 17-PCD and 17-AD protein (see for domain organization) costained for endogenous COL6A1 (red) and the Myc-tag of the recombinant ADAMTS17 protein fragments (green). Nuclei were stained with DAPI (blue). (K) Micrographs of adult HDFs and Weill–Marchesani syndrome (WMS) patient–derived dermal fibroblasts (WMS-DF) for endogenous COL6A1 (red). Nuclei were counterstained with DAPI (blue). (L) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates, 2–3 fields of view). (M) Western blot of endogenous COL6A1 (red) and GAPDH (green) in conditioned medium (Med) and cell lysate (Lys) collected from HDF and WMS-DF cultures. (N) Quantification of COL6A1 band mean fluorescence intensities normalized to GAPDH. In (E, G, M), bars represent the mean value and whiskers the SD. In (K), the floating bars indicate the 25th–75th percentile range, the lines the mean value, and whiskers the SD. Statistical differences in (E, G, K, M) were determined using a two-sided t test.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Sequencing, Labeling, Western Blot, Expressing, Staining, Fluorescence, Recombinant, FLAG-tag, Cell Culture, Purification, Derivative Assay

Journal: Cell host & microbe

Article Title: The Listeriolysin O PEST-like Sequence Co-opts AP-2-Mediated Endocytosis to Prevent Plasma Membrane Damage during Listeria Infection

doi: 10.1016/j.chom.2018.05.006

Figure Lengend Snippet: (A) Schematic diagrams of different baits (LLO truncations) constructs tested in the yeast 2-hybrid assay. (B) Quantification of secreted alpha-galactosidase activity following a GAL4-based two-hybrid interaction in Y2HGold yeast colonies. The results represented the percentage of alpha-galactosidase activity of positive control (p53+ SV40 large T antigen). Bars and error represent mean ± SD of replicate measurements. N=4 biological repeats *** p<0.001 (Student’s t-test). Results shown represent at least 3 independent experiments. (C) HEK 293T and HeLa cells were co-transfected with LLO-Myc tag and Ap2a2-HA tag plasmids. At 24 hours post transfection, cells were permeabilized and stained with anti-Myc (red), anti-HA antibodies (Green), and nuclear dye DAPI (blue). Scale bars are 10 μm. (B) Co-immunoprecipitation of Ap2a2 from transfected HEK293T cells using an anti-Myc-LLO antibody. Results shown represent 3 independent experiments. See also Figure S3.

Article Snippet: Human: HEK293T cell line , Lab stock , https://www.atcc.org/Products/All/CRL-11268.aspx.

Techniques: Construct, Y2H Assay, Activity Assay, Positive Control, Transfection, Staining, Immunoprecipitation

Journal: Cell host & microbe

Article Title: The Listeriolysin O PEST-like Sequence Co-opts AP-2-Mediated Endocytosis to Prevent Plasma Membrane Damage during Listeria Infection

doi: 10.1016/j.chom.2018.05.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: HEK293T cell line , Lab stock , https://www.atcc.org/Products/All/CRL-11268.aspx.

Techniques: Virus, Recombinant, Staining, Flow Cytometry, Modification, Lysis, Transfection, Immunoprecipitation, Plasmid Preparation, CRISPR, Software, Microscopy

Journal: Experimental Hematology & Oncology

Article Title: Ubiquitin ligase subunit FBXO9 inhibits V-ATPase assembly and impedes lung cancer metastasis

doi: 10.1186/s40164-024-00497-4

Figure Lengend Snippet: FBXO9-mediated ATP6V1A ubiquitination impairs metastasis of lung cancer cells. A , B Ubiquitination assays were performed in HEK293T cells by co-transfection with full-length HA-ATP6V1A, HA-ATP6V1A-KR (lysine residues removed), HA-ATP6V1A-K393 (K393 reintroduced into the ATP6V1A-KR mutant), or HA-ATP6V1A-K393R (single lysine substitution mutant), in the presence or absence of FLAG-FBXO9. After 48 h, cells were lysed, followed by Ni-NTA bead pull-down and immunoblotting for HA-ATP6V1A. C Alignment of ATP6V1A K393 conservation across species. D Ubiquitination analysis of K393 in HA-Atp6v1a by co-expressing Flag-Fbxo9 and His-Ub with indicated HA-Atp6v1a constructs (WT, K393, or K393R) in HEK293T cells. Cells were lysed after 48 h, followed by Ni-NTA bead pull-down and immunoblotting for HA-Atp6v1a. E V-ATPase complex reconstitution was achieved in H1299 cells by introducing sgRNA-resistant ATP6V1A (wild-type or K393R mutant) using a Tet-Off system. CRISPR was then used to deplete endogenous ATP6V1A, allowing the V-ATPase complex to be reconstituted. Immunoblot analysis confirmed comparable expression levels of the added ATP6V1A and endogenous subunits ATP6V1B and ATP6V1D1, in the experimental group compared to the control cells, after 48 h of doxycycline treatment. F–I Recombinant cell lines H1299-KO-V1A-wt, H1299-KO-V1A-K393R, and H1299-EGFP-sgCtrl were used in a transwell migration assay (F, G) and tumor sphere formation assay (H, I) to evaluate the effects of ATP6V1A ubiquitination on cell migration and tumor sphere growth( P < 0.001). J–N To assess the effects of ATP6V1A ubiquitination on cell migration and tumor sphere growth, V1A-23aa was introduced into A549 cells to inhibit ATP6V1A ubiquitination (J) which was followed by transwell migration assay (K, L) and tumor sphere formation assay (M, N) to evaluate the impact on cell migration and tumor sphere growth, respectively ( P < 0.001). O, P Impact of ATP6V1A ubiquitination inhibition by FBXO9 on in vivo lung metastasis was assessed. A549 cells expressing V1A-23 aa were injected into mice via the tail vein. After 6 weeks, the lungs of the mice were harvested for examination and H&E staining. Metastasis nodules were quantified and statistically analyzed using fluorescence microscopy. Error bars represent the mean ± SD. **** P < 0.0001 indicates the significance level

Article Snippet: Subsequently, the puromycin-resistant cells were treated with doxycycline for 72 h. Furthermore, for the knockdown of human FBXO9, cancer cells were transiently transfected with siRNA oligonucleotides using Lipofectamine RNAiMAX (Thermo Fisher), following the manufacturer’s guidelines.

Techniques: Ubiquitin Proteomics, Cotransfection, Mutagenesis, Western Blot, Expressing, Construct, CRISPR, Control, Recombinant, Transwell Migration Assay, Tube Formation Assay, Migration, Inhibition, In Vivo, Injection, Staining, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: SOX-5 activates a novel RORγt enhancer to facilitate experimental autoimmune encephalomyelitis by promoting Th17 cell differentiation

doi: 10.1038/s41467-020-20786-w

Figure Lengend Snippet: a The ranking of predicted transcriptional factors (TFs) capable of binding to RORCE was performed based on relative scores predicted with the JASPAR database ( http://jaspar.genereg.net ). b Red vertical line shows SOX-5 binding site (SOX-5-BS) in RORCE, which is very close to TS3. c , d A ChIP-qPCR assay was performed to determine the enrichment of SOX-5 in SOX-5-BS and RORγt promoter (RORγt p) in PI-stimulated EL4 cells ( c ) and WT Th17-polarized cells ( d ). e – h A ChIP-loop assay with an anti-SOX-5 antibody or control mouse IgG was performed to quantify relative interaction efficiency between the AS and TS3 mediated by SOX-5 in PI-stimulated EL4 ( e ), Th17-polarized ( f ), Th1-polarized ( g ), and Th2-polarized cells ( h ) from WT mice. i The sequence of SOX-5-BS (AACAA) in RORCE was deleted by using CRISPR/Cas9 technology (top). The knockout allele was confirmed by sequencing (bottom). j A ChIP-qPCR assay was performed to test the enrichment of SOX-5 in SOX-5-BS in Th17-polarized cells from SOX-5-BS-deficient (SOX-5-BS −/− ) or WT mice. k , l Quantitative analysis of 3C-qPCR ( k ) and ChIP-loop assays ( l ) evaluating the interaction between TS3 and AS in Th17-polarized cells from SOX-5-BS-deficient or WT mice was performed. Th17 D, Th1 D, and Th2 D represent Th17, Th1, or Th2 differentiation under the corresponding polarization conditions, respectively, and N represents naïve cells. Mean ± SEM are shown, n = 4 independent experiments, unpaired two-tailed Student’s t -test ( b – h , j – l ). Experimental mice were between 8 and 12 weeks of age, with no preference to gender and were maintained on a C57BL/6 background. Source data are provided as a Source Data file.

Article Snippet: Furthermore, Th17 cells that were differentiated from naïve CD4 + T cells under Th17-polarization condition were also subjected to IP with anti-STAT3 antibody (1:100, Cell Signaling Technology, 124H6) or control mouse IgG (1:100, Abcam, MOPC-173) and IB with anti-STAT3 or anti-SOX-5 antibody.

Techniques: Binding Assay, Sequencing, CRISPR, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: SOX-5 activates a novel RORγt enhancer to facilitate experimental autoimmune encephalomyelitis by promoting Th17 cell differentiation

doi: 10.1038/s41467-020-20786-w

Figure Lengend Snippet: a Red vertical line shows STAT3 binding site (STAT3-BS) in RORCE2, which is located approximately 50 bp downstream of the SOX-5-BS. b , c A ChIP-qPCR assay was performed to determine the enrichment of STAT3 in its binding site in RORCE2 in EL4 cells ( b ), WT Th17-polarized cells before and after STAT3 overexpression or knockdown (KD) and Th17-polarized cells generated from SOX-5-BS-deficient mice ( c ). Th17 D represents Th17-polarized cells, and N represents naïve cells. d Relative expression of Stat3 , RORγt , and IL-17A genes in WT Th17-polarized cells and IL-17A production in culture supernatants before and after Stat3 overexpression or knockdown (Stat3 KD) were determined by RT-qPCR and ELISA, respectively. e Luciferase assays were performed with different reporter constructs in EL4 cells. RORCE2 with a STAT3-BS deletion (E2-∆Stat3BS) was cloned upstream of RORγt promoter in the pGL3 vector, and transcriptional activity was assessed with a dual-luciferase system. f Hela cells were transfected with pcDNA3.1-SOX-5 Flag vector together with pcDNA3.1-STAT3 HA vector and then cultured for 48 h. After stimulated with PI and Biochanin A, whole-cell lysates from Hela cells were subjected to IP with anti-HA antibody or control mouse IgG, and IB with anti-HA or anti-Flag HRP antibody. Input proteins (input) were also IB with anti-HA or anti-Flag HRP antibody. g Whole-cell lysates from Th17 cells were subjected to IP with anti-STAT3 antibody or control mouse IgG and IB with anti-SOX-5 or anti-STAT3 antibody. Input proteins (input) were also IB with anti-SOX-5 or anti-STAT3 antibody. Mean ± SEM are shown, n = 5 independent experiments, unpaired two-tailed Student’s t -test ( b – e ). Experimental mice were between 8 and 12 weeks of age, with no preference to gender and were maintained on a C57BL/6 background. Source data are provided as a Source Data file.

Article Snippet: Furthermore, Th17 cells that were differentiated from naïve CD4 + T cells under Th17-polarization condition were also subjected to IP with anti-STAT3 antibody (1:100, Cell Signaling Technology, 124H6) or control mouse IgG (1:100, Abcam, MOPC-173) and IB with anti-STAT3 or anti-SOX-5 antibody.

Techniques: Binding Assay, Over Expression, Generated, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Luciferase, Construct, Clone Assay, Plasmid Preparation, Activity Assay, Transfection, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Schematic of CRISPR/Cas9-mediated PLXNB2 knockout (KO) with small guide (sg) RNA targeting second coding exon. (B) Western blots show Plexin-B2 expression in different SD2 GSCs, with β-actin as loading control. Note Plexin-B2 precursor at 240 kDa and mature form at 170 kDa. (C) IF images show Plexin-B2 expression in different SD2 GSCs, with Hoechst nuclear counterstain. (D) Left, schematic of atomic force microscopy (AFM) indentation method to probe cell stiffness by cantilever deflection. Middle, AFM indentation curves of different SD2 GSCs; right, box plots of cell stiffness, showing 25– 75% quartiles, median (line), and mean (plus sign). n= 6 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (E) Left, depiction of membrane tension measurement with optical tweezers. Middle, force measurements during tether extrusion (shaded box). Right, quantifications of tether extrusion forces. n=5 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (F) Left, schematic of FLIM of cell membranes labeled with Flipper-TR membrane dye, with low and high membrane tension associated with shorter and longer lifetimes, respectively. Middle top, representative FLIM images, with lifetime heatmap shown on right. Middle bottom, images show similar fluorescence intensities of Flipper-TR dye in WT and PB2 KO cells. Right top, violin plots show fluorescence lifetime from 3 images per group. Two-sided unpaired t-test. Right bottom, phasor plots of FLIM image data, with arrow indicating a shift to shorter lifetime values for PB2 KO cells. (G) Model of Plexin-B2 regulation of cortical contractility and membrane tension. Phalloidin staining show differences of F-actin network in WT and PB2 KO SD2 GSCs. DAPI for nuclear staining. Arrows point to stress fibers and spread-out contours of the WT GSCs.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: CRISPR, Knock-Out, Western Blot, Expressing, Control, Microscopy, Membrane, Labeling, Fluorescence, Staining

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Top, timeline for dextran uptake assay. Bottom, live-cell imaging of WT and PB2 KO SD2 GSCs labeled with SPY-Actin and exposed to dextran-Alexa488. Enlarged images of boxed areas are shown below. Quantification of the areas of dextran + clusters per cell are shown in box plots, with 25–75% quartiles, median (line), and mean (plus sign). n=85 cells for WT, n=44 cells for PB2 KO. Mann–Whitney–Wilcoxon test. (B) Top, live cell confocal plane images of WT and PB2 KO GSCs with side views of z-stacks showing intracellular localization of diffuse dextran-Alexa 488 signals in PB2 KO cells in addition to dextran endosome signals. In contrast, WT cells contained only dextran + endosomes. Bottom, histograms show fluorescence profiles showing bimodal distribution of dextran-Alexa 488 fluorescence intensities in PB2 KO GSCs (blue and brown arrows). n=177 cells for WT, n=161 cells for PB2 KO. Mann–Whitney–Wilcoxon test. (C, D) Left, schematic of myr-palm-GFP or -CFP attached to inner membrane leaflet. Right, live cell fluorescence imaging at 72 hr after transfection shows internalization of myr-palm-GFP or -CPF on endomembranes (arrow) in WT GSCs, in contrast to membrane retention of the probes (arrowhead) in PB2 KO GSCs. (E) Left, schematic of TauSTED super-resolution microscopy of GSCs labeled with MemGlow. Middle, TauSTED live-cell images show reduced endosomes (arrowheads) in PB2 KO cells compared to WT. Right, box plots show areas of MemGlow clusters in each cell. n=26 cells for WT, n=13 cells for PB2 KO. Two-sided unpaired t-test. (F) Working model of regulation of cortical and membrane tension by Plexin-B2, affecting endocytosis and membrane permeability in GSCs.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Live Cell Imaging, Labeling, MANN-WHITNEY, Fluorescence, Membrane, Imaging, Transfection, Super-Resolution Microscopy, Permeability

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Left, schematic of PH(PLCδ1)-GFP PIP2 probe. Right, live-cell imaging at 72 hr post transfection reveals that the PH(PLCδ1)-GFP probes were largely internalized in WT GSCs (arrow), but retained on membrane of PB2 KO GSCs (arrowhead). (B) Left, still images of videography show accumulation of the PH(PLCδ1)-GFP probes (arrow) in front of the nucleus (NucSpot) of migrating WT SD2 GSCs in tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Dashed lines delineate cell boundary. Long arrow denotes direction of migration. Right, quantifications of the ratio of PH(PLCδ1)-GFP fluorescence intensity at front vs. rear of GSCs during passage. n=13-16 cells per condition. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (C) Left, schematic of R(+8)-pre-GFP probe for negative surface charge of inner plasma membrane. Right, live-cell imaging at 72 hr post-transfection shows internalization of the probes (arrow) in WT GSCs, in contrast to the predominant membrane localization in PB2 KO GSCs (arrowhead). (D) Left, still images of videography show accumulation of the R(+8)-pre-GFP probes (arrow) at front zone of WT GSCs when traversing the 3 µm tunnel, but not in PB2 KO cells. Right, bar graphs show the ratio of R-pre-GFP fluorescence intensity at rear vs. front of GSCs when passing through tunnels. n=22 cells for WT, n=27 cells for PB2 KO. Mann–Whitney–Wilcoxon test. Data represent mean ± SEM. (E) Diagram illustrating voltage sensitive FluoVolt membrane dye, with fluorescent intensity quenched by voltage-sensitive electron transfer from electron-rich donor mediated by “molecular wire” in plasma membrane. (F) Left, FluoVolt live-cell imaging shows reduced FluoVolt fluorescent intensity in cell membrane of Plexin-B2 KO cells, consistent with higher negative charges of inner membrane. Right, box plots of membrane FluoVolt intensity. n=25 cells for WT, n=27 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. (G) Left, still images from videography show higher FluoVolt fluorescent signals at rear zone (arrowhead) of WT GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Migration direction is denoted by long arrow. Right, bar graphs show the ratio of FluoVolt intensity at rear vs. front during confined migration. n=15 cells per group. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (H) Live-cell images and quantifications show the effects of constitutive active (CA) RAP1B-V12 or dominant-negative (DN) RAP1B-N17 on FluoVolt intensity in WT or PB2 KO GSCs. n=25 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (I) Left, still images capture calcium localization (Fluo4-AM fluorescence, arrowhead) at the rear of WT GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Migration direction is denoted by long arrow. Right, bar graphs showing Fluo4-AM intensity ratio at rear vs. front in GSC during passage through tunnels. n=15-16 cells. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (J) Left, still images from videography show that calcium chelator BAPTA-AM disrupted the pattern of high FluoVolt signals at the rear of WT GSCs (arrowhead) during confined migration. Right, bar graphs show FluoVolt intensity ratio at rear vs. front of GSCs when traversing tunnels. n=21 cells for WT, n=16 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. (K) Model of Plexin-B2 signaling affecting membrane surface charge and electric field during polarized confined migration, with PIP2 enrichment at cell front and Ca 2+ at rear zone, leading to asymmetry of FluoVolt and R(+8)-pre-GFP.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Live Cell Imaging, Transfection, Membrane, Migration, Fluorescence, Comparison, Clinical Proteomics, MANN-WHITNEY, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Structure model of the extracellular domain of human Plexin-B2 show the locations of lock1 and lock2 mutations predicted to form disulfide bridges that lock the ring structure. (B) Western blots show absence of mature Plexin-B2 (170 kDa) in PB2 KO GSC, and expression of lock mutants in PB2 KO SD2 and SD3 GSCs. β-actin serves as a loading control. (C) Still images from videography show passage of GSCs (nuclei visualized by NucSpot) through microchannels with PB2 wildtype rescue construct but not lock mutants, nor PB2 with deletion of extracellular domain (dECTO). Chevrons point to 3 µm constrictions. (D) Box plots show velocity through constrictions, stalling time at constrictions, and sum of forward and backward movements, with 25–75% quartiles, minimal and maximal values (whiskers), median (line), and mean (cross). For velocity and sum of movements: n=17-20 cells per condition. For stalling time at constriction: n=14-28 cells per condition. One-way ANOVA followed by Dunnett’s multiple comparisons test. (E) Still images from videography show F-actin assembly (SPY-actin, arrowhead) at cell rear and MemGlow + endosomes (arrow) at cell front of SD3 GSCs with Plexin-B2 WT rescue but not mutant rescues when traversing 3 µm constrictions (chevrons). (F) Bar graphs showing fluorescence intensity ratio of SPY-actin and MemGlow at rear vs. front of GSCs during confined migration. n=10-18 cells per condition. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Data represent mean ± SEM. (G) Model of Plexin-B2 signaling and mechano-electrical regulation of membrane tension and membrane surface charge during polarized confined migration. Regionalized enrichment of endocytosis/PIP2 at cell front and F-actin/Ca 2+ at rear zone lead to asymmetry of FluoVolt and R(+8)-pre-GFP membrane probes.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Western Blot, Expressing, Control, Construct, Mutagenesis, Fluorescence, Migration, Membrane