Journal: bioRxiv

Article Title: Dynamic Regulation OF The Chromatin Environment By Ash1L Modulates Human Neuronal Structure And Function

doi: 10.1101/2024.12.02.625500

Figure Lengend Snippet: (A) Diagram of ASH1L protein domains showing the location of the pathogenic variant E2148* (blue) in ASH1L catalytic domain and itsr associated clinical phenotypes. (B) Illustration depicts the dual SMAD inhibition protocol used to generate cortical excitatory human neurons. ( C ) ASH1L expression was quantified by qPCR using human neurons at day 35 of neuronal induction. Fold change is normalized to control. Bar represents the mean and individual measures from four independent experiments are shown for control (grey with open circles), and E2148* (light blue with solid blue circles). Samples were analyzed as a ratio of the control. Statistical analysis was conducted using unpaired t-test. **** P < 0.0001. ( D ) Representative images are shown for human neurons from control, and E2148* cultures at day 35 of neuronal induction. Neurons stained with MAP2 are shown in black and white for ease of viewing. Calibration bars represent 20µm. ( E-H ) Morphogenesis measures are shown for four independent experiments for control neurons (grey bar with open circles), and E2148* mutant neurons (light blue bars with solid dark blue circles). Individual points represent the average of 4 independent experiments, an average of 30 neurons were measured per experiment. ( E ) Mean neurite length is shown for control (n=124 neurons; 56.9 ± 2.41), and E2148* (n=118 neurons; 47.47 ± 1.99). Grouped statistical analysis was conducted using unpaired t-test, **P < 0.004. ( F ) Total neurite length is shown for control (n=124 neurons; 182.7 ± 6.39), and E2148* (n=118 neurons; 139.3 ± 4.66). Grouped statistical analysis was conducted using unpaired t-test, **** P < 0.0001. ( G ) Neuronal morphology analyzed by measuring the complexity index (see methods). Calculations were conducted after identifying outliers using the ROUT 1% method for control (n=115; 289.5 ± 18.21), and E2148* (n=112; 228.8 ± 13.42). Grouped statistical analysis was conducted unpaired t-test ** P < 0.0099. ( H ) Cell soma size was analyzed for three independent experiments by measuring the area for control (n=96; 77.67 ± 3.47), and E2148* (n=91; 69.15 ± 2.51). Statistical analysis was conducted using unpaired t-test P=0.056. ( I ) Sholl analysis was used to measure neuronal arborization. The number intersections away from the cell soma were measured every 10µm and are shown for control (open gray circles), and E2148* (solid dark blue circles) neurons from 10µm to 120µm. Statistical analysis was conducted using a mixed model effects *** P < 0.0006, and **** P < 0.0001. ( J-L ) Analysis of H3K36me2 and H3K4me3 levels on chromatin fraction for four independent experiments is shown for neurons at day 41 of neuronal induction. ( J ) Representative western blot shows H3K36me2, H3K4me3 and histone H3 for control, and E2148* neurons. H3 Histone marks were normalized to histone H3 levels for analysis. ( K ) H3K36me2 protein levels are shown for control (1± 0), and E2148* (0.67 ± 0.26). ( L ) H3K4me3 protein levels are shown for control (1± 0), and E2148* (0.68± 0.11). (K -L ) Statistical analysis was conducted using unpaired t-test *P< 0.025. Not significant P value is not shown.

Article Snippet: Primary antibody for MAP2 (Aves lab, #MAP) was added and cells were incubated overnight at 4°C, followed by secondary antibody incubation for 1 hour at room temperature using Alexa Fluor 647 (Thermo Scientific, #A-21449) at a dilution of 1:1000.

Techniques: Variant Assay, Inhibition, Expressing, Control, Staining, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Dynamic Regulation OF The Chromatin Environment By Ash1L Modulates Human Neuronal Structure And Function

doi: 10.1101/2024.12.02.625500

Figure Lengend Snippet: ( A ) PCA plots shows biological replicates (n=4) for control (green), and E2148* (salmon) neurons RNA seq experiments. ( B ) Heatmap shows top 100 DEGs for control (green), and E2148* (salmon) neurons at day 35 (n=4 biological replicates). The top 15 DEGs are listed. ( C ) Volcano plots showing DEGs in the heterozygous E2148* mutant iPSC-derived neurons. Log 2 fold changes (LFC) gene expression (x-axis) and -log 10 adjusted P values (y-axis) generated from DESeq2 differential gene expression analysis are shown. Vertical dotted lines represent 0.58 LFC (1.5 FC) and horizontal dotted line shows adjusted P=0.05. Significant DEGs are shown in red with the top 20 labelled in the plot. ( D-F ) Functional enrichment analysis by EnrichR for biological process ( D ), cellular compartment ( E ), and molecular function ( F ) show enrichment for all DEGs, upregulated and downregulated DEGs in E2148* mutant neurons vs. control neurons. Circle size represents the number of DEGs in that category and the color represents the adjusted P value. ( G ) Correlation of gene length to fold change analyzed for all significant DEGs in E2148* (blue line) mutant neurons. Grey shade shows the variability across samples. ( H ) Analysis of gene length in upregulated (blue) and downregulated (red) DEGs for E2148* neurons. ( I ) Analysis of de novo transcription by EU click chemistry at day 41 of neuronal differentiation. Representative images of human neurons that incorporated EU (gray), stained with neuronal marker MAP2 (cyan) and nuclear marker DAPI (blue) are shown for control (top row), and E2148* (bottom row). Enlarged nuclei stained with EU is shown. Calibration bars are 20µm. ( J-K ) Measurements of EU incorporation are shown for control neurons (grey bars with open circles), and E2148* (light blue bars with solid deep blue circles) mutant neurons. Mean and standard error are shown with individual dots representing the average of individual measures for five independent experiments. ( I ) Pearsons’ correlation coefficient analysis is shown for five independent experiments for control (n=187; 0.785 ± 0.003), and E2148* (n=115; 0.746 ± 0.005) neurons. ( J ) EU nuclear intensity normalized to control is shown for five independent experiments for control (n=187; 1.017 ± 0.029), and E2148* (n=115; 0.817 ± 0.027) neurons. ( I-J ) Grouped data analyzed using unpaired t test with Welch’s correction, ****P < 0.0001. Not significant P values are not shown.

Article Snippet: Primary antibody for MAP2 (Aves lab, #MAP) was added and cells were incubated overnight at 4°C, followed by secondary antibody incubation for 1 hour at room temperature using Alexa Fluor 647 (Thermo Scientific, #A-21449) at a dilution of 1:1000.

Techniques: Control, RNA Sequencing, Mutagenesis, Derivative Assay, Gene Expression, Generated, Functional Assay, Staining, Marker

Journal: bioRxiv

Article Title: Dynamic Regulation OF The Chromatin Environment By Ash1L Modulates Human Neuronal Structure And Function

doi: 10.1101/2024.12.02.625500

Figure Lengend Snippet: (A) Representative images are shown for day 35 human neurons from control, and E2148* cultures treated for 3 days with DMSO, Tazemetostat (0.5µM) and Vorinostat (0.1µM). Neurons stained with MAP2 are shown in black and white for ease of viewing. Calibration bars represent 30µm. ( B-F ) Morphogenesis analysis is shown for at least 4 independent experiments (unless otherwise annotated) in which we measured at least 30 neurons per experiment for control (grey bar with open circles) and E2148* (light blue bars with solid dark blue circles) neurons treated with either DMSO, Tazemetostat (TAZ) or Vorinostat (VOR). Individual points represent the average of multiple independent experiments. ( B ) Total neurite length is shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=93 neurons; 231.9 ± 7.27); control + TAZ (n=119; 184.4± 5.46); control + VOR (n=118 neurons; 227.3± 6.8); E2148* + DMSO (n=113 neurons; 163.3 ± 4.83); E2148* + TAZ (n=112; 200.5± 5.94); E2148* + VOR (n=129 neurons; 225.2± 8.38). ( C ) Mean neurite length is shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=90 neurons; 69.86 ± 2.409); control + TAZ (n=117; 64.16± 2.04); control + VOR (n=118 neurons; 81.28± 2.75); E2148* + DMSO (n=112 neurons; 55.75 ± 1.88); E2148* + TAZ (n=111; 66.16 ± 2.47); E2148* + VOR (n=126 neurons; 76.02 ± 2.58). ( D ) Complexity index measurements were first analyzed using the “identify outliers” ROUT function in graph pad and are shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=90 neurons; 390± 26.88); control + TAZ (n=111; 329.4 ± 22.40); control + VOR (n=116 neurons; 461.8 ± 25.45); E2148* + DMSO (n=116 neurons; 261.9 ± 18.80); E2148* + TAZ (n=105; 357.6 ± 20.83); E2148* + VOR (n=125 neurons; 458.3 ± 29.66). ( B-D ) Statistical analysis of grouped measurements was conducted using TWO-way ANOVA with Tukey’s test for multiple comparisons: * P < 0.04 ** P < 0.009, *** P < 0.0006, **** P < 0.0001. ( E ) Sholl analysis was used to measure neuronal arborization across three different treatments in the E2148* mutant neurons. The number intersections away from the cell soma were measured every 10µm and are shown for E2148* + DMSO (inverted dark blue triangles), E2148* + TAZ (open triangles), and E2148* + VOR (solid light blue triangles) neurons. Statistical analysis by TWO-way ANOVA with mixed model effects * P < 0.05, ** P < 0.009, and *** P = 0.0008. Green asterisk (E2148* +DMSO vs. E2148* + VOR), red asterisk (E2148* + DMSO vs. E2148* + TAZ). ( F ) Sholl analysis is shown to compare the most effective treatment (vorinostat) to the untreated control and E2148* mutant neurons. The number of intersections away from the cell soma were measured every 10µm and are shown for control+ DMSO (open gray circles), E2148* + DMSO (solid dark blue circles) and E2148* + VOR (half pink/light blue circles) neurons. Statistical analysis by TWO-way ANOVA with mixed model effects * P < 0.05, ** P < 0.005, *** P = 0.0005, and **** P < 0.0001. Green asterisk (E2148*+DMSO vs. Control + DMSO), red asterisk (E2148* + DMSO vs. E2148* + VOR). ( G-K ) Analysis of nuclear levels of H3K27me3 and H4K16ac in four independent experiments (unless otherwise indicated) across all treatments is shown for neurons at day 35 of neuronal induction. ( G ) Representative images of nuclear H3K27me3 (red) are shown for either DMSO (left column) or Tazemetostat (right column) treated control, or E2148* mutant neurons stained with MAP2 (cyan) and nuclei is stain with DAPI (blue). ( H ) Quantification of H3K27me3 nuclear levels measured by mean gray value is shown for all treatments. Measurements from at least 3 independent experiments with at least 30 neurons analyzed per experiment were analyzed as a group and are shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=134 neurons; 547.4 ± 13.19); control + TAZ (n=154; 258.1 ± 10.83); control + VOR (n=101 neurons; 545.5 ± 19.07); E2148* + DMSO (n=141 neurons; 471.0 ± 10.87); E2148* + TAZ (n=140; 301.1 ± 8.03); E2148* + VOR (n=103 neurons; 539.4 ± 18.79). ( I ) Representative images of nuclear H4K16ac (red) are shown for either DMSO (left column) or Tazemetostat (right column) treated control, and E2148* mutant neurons stained with MAP2 (cyan) and nuclei is stain with DAPI (blue). ( J ) Quantification of H4K16ac nuclear levels measured by mean gray value is shown for all treatments. Measurements from at least 3 independent experiments with at least 30 neurons analyzed per experiment were analyzed as a group and are shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (N= 4 experiments; n=114 neurons; 322.8 ± 12.85); control + TAZ (n=95; 360.5 ± 13.04); control + VOR (n=145 neurons; 581.6 ± 16.45); E2148* + DMSO (n=166 neurons; 270.3 ± 10.33); E2148* + TAZ (n=106; 350.4 ± 11.81); E2148* + VOR (n=158 neurons; 654.7 ± 9.49). ( H and J ) Statistical analysis of grouped measurements was conducted using TWO-way ANOVA with Tukey’s test for multiple comparisons: * P < 0.05, *** P < 0.005, *** P < 0.0005, **** P < 0.0001.

Article Snippet: Primary antibody for MAP2 (Aves lab, #MAP) was added and cells were incubated overnight at 4°C, followed by secondary antibody incubation for 1 hour at room temperature using Alexa Fluor 647 (Thermo Scientific, #A-21449) at a dilution of 1:1000.

Techniques: Control, Staining, Mutagenesis

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A) Serial 10-fold dilutions of haploid strains with the indicated genotypes were plated onto rich media (YPD) containing different MMS concentrations. rad52-L264P * denotes the original isolated mutant strain and rad52-L264P ** denotes a strain in which the RAD52 gene was replaced by the mutant newly generated by directed mutagenesis. (B) Conservation of a motif comprising L264. The primary structure of Rad52 is schematized showing the conserved N-terminus moiety containing the major DNA binding and self-association domains (black, amino acids 1 to 179) as well as the C-terminus part (white, amino acids 180 to 471) containing the RPA (amino acids 275 to 278) and the Rad51 (amino acids 376 to 379) binding domains. The alignment of the Rad52 protein in Hemiascomycetes species shows the conservation of a domain containing the non-essential L264 residue and the QDDD residues essential for RPA binding and consequently for Rad52 mediator activity. The color code used in the alignment follows the default ClustalX color scheme as implemented in JalView (see Material and Methods). Cyan is for fully hydrophobic (I, L, V, M, F), turquoise for aromatic residues containing polar moieties (Y, H), green for small polar (T, S), purple for acidic (D, E), orange for glycine (G) and dark yellow for proline (P) residues.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Isolation, Mutagenesis, Generated, Binding Assay, Residue, Activity Assay

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A to E) Spot assay of haploid cells with the indicated genotype on rich medium (YPD) containing increasing MMS concentrations. Note that the deletion of the SIZ2 gene (A) and the rad52-3KR allele (B) cannot suppress the MMS sensitivity of srs2 Δ cells. Therefore, the MMS resistance of rad52-L264P siz2 Δ srs2 Δ and rad52-3KR-L264P srs2 Δ cells is only related to rad52-L264P . (D) Note that the haploid strain spotted at the bottom contains both rad52-3KR and RAD52 alleles.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Spot Test

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A) Tetrad analysis of crosses between haploid rad52-L264P srs2 Δ strains and haploid mutants synthetically lethal with srs2 Δ. Double mutant spores, which do not contain rad52-L264P , are indicated by white squares. The white circles mark triple mutants. (B) siz2 Δ and rad52-3KR do not suppress the synthetic lethality of srs2 Δ sgs1 Δ and srs2 Δ rrm3 Δ mutants. In crosses involving siz2 Δ, white squares display spores of srs2 Δ sgs1 Δ or srs2 Δ rrm3 Δ genotypes and white circles indicate triple mutants. To analyze the genetic interaction between rad52-3KR inserted at the URA3 locus and the synthetically lethal rrm3 Δ srs2 Δ double mutant, diploids homozygous for rad52 Δ were sporulated, in order to avoid the co-expression of RAD52 and rad52-3KR . The white square indicates srs2 Δ rrm3 Δ rad52 Δ triple mutants and white circles indicate unviable srs2 Δ rrm3 Δ rad52 Δ ura3 :: rad52-3KR monosporic colonies.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Mutagenesis, Expressing

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: The HO endonuclease was induced in WT or srs2 Δ cells that express Rad52-FLAG, Rad52-L264P-FLAG or Rad52-SUMO-FLAG to create a single DSB that can be repaired by SSA. Samples were taken before induction and at 2, 4, 6 and 8 hours after galactose addition. Antibodies against the RPA complex, the FLAG epitope or Rad51 were used to precipitate protein-bound chromatin. Quantitative PCR was performed with primers located at 0.6 kb or 7.6 kb from the DSB site, using the immunoprecipitated chromatin (IP) and input DNA as template. As a control, primers specific for the ARG5,6 locus were used. The relative enrichment represents the ratio of the PCR enrichment in the IP fraction to the input fraction. The median value of at least 3 experiments is shown and error bars represent the upper and lower values observed.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: FLAG-tag, Real-time Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation, Control

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A) To test the interaction with RPA, Rad52 or Rad52-L264P was immunoprecipitated with a rabbit anti-Rad52 polyclonal antibody from 1 mg of whole cell extracts (without DNAse treatment) prepared from RAD52 , rad52-L264P or rad52 Δ strains. To test the robustness of the interaction, increasing NaCl concentrations were added to the cell extracts. Proteins from the whole extracts (50 µg) and from the immunoprecipitated fractions were separated by SDS-PAGE and immunoblotted with rabbit anti-Rad52 polyclonal antibody or rabbit anti-RPA polyclonal antibody (allowing detection of the Rfa1 subunit of RPA). The signals corresponding to immunoprecipitated Rad52 or Rad52-L264P were quantified in three independent experiments and plotted as a fraction of the signal intensity measured in the 150 mM NaCl experiment. (B) To assess the interaction between Rad52-L264P and Rad51, Rad51 was immunoprecipitated from 1 mg of whole cell extracts (without DNAse treatment) from cells expressing Rad52-FLAG or Rad52-L264P-FLAG. The strength of the interaction was also evaluated against increasing NaCl concentrations. The proteins from whole cell extracts (50 µg) and from immunoprecipitated fractions were separated by SDS-PAGE and immunoblotted with rabbit anti-Rad51 polyclonal antibody and mouse anti-FLAG monoclonal antibody. The presence of Rad51 in the immunoprecipitated fraction cannot be detected to validate the efficiency of the immunoprecipitation because it migrates at the same level as the IgG anti-Rad51 used for the immunoprecipitation. However, the absence of Rad52 in the rad51 Δ immunoprecipitate confirmed that the Rad52-FLAG signals observed are related to the Rad52-Rad51 interaction. The signals corresponding to immunoprecipitated Rad52 or Rad52-L264P were quantified in three independent experiments and plotted as in (A).

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, SDS Page, Expressing

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: ( A ) The purity of recombinant Rad52 and Rad52-L264P (2 µg/each) was assessed by separation on 8% SDS-PAGE and staining with Coomassie blue. (B) Binding of Rad52-L264P to ssDNA. Protein titration reactions were performed by incubating 0.27 µM of a 62-nucleotides long Cy5-labeled ssDNA fragment with various amounts of Rad52-L264P at 37°C for 10 min (protein/bases: 1/10, 1/5, 1/2.5, 1/1.25, 1/0.6, 1/0.3, 1/0.15). Quantification of free ssDNA is shown. The data were fitted into a sigmoidal curve by using the PRISM software (GraphPad). (C) Rad52-L264P-mediated DNA annealing. Representative gels of Rad52 or Rad52-L264P-promoted DNA annealing are shown in the upper panel (Rad52/bases: 1/100, 1/42, 1/14, 1/6; same DNA as in (B) with reverse-complement, 340 nM each). The dsDNA/total DNA ratio at 10 min is shown in the lower panel. (D) RPA bound to ssDNA inhibits equally Rad52- and Rad52-L264P-catalyzed annealing reactions. Reactions were carried out with primers 25 and 26 (200 nM each, see Material and Methods) that were first incubated with 30 nM RPA (1/13 bases) at 30°C for 5 minutes, followed by addition of 40 nM Rad52 (1/10 bases). Self-annealing of the primers incubated without proteins and reactions performed without RPA or Rad52 are also shown. (E) Over-stimulation of DNA strand exchange by Rad52-L264P. Upper panel , diagram of the reaction substrates and products. Middle panel , ethidium bromide-stained DNA gel. As shown by the standard reaction (st), Rad51 efficiently catalyzes the formation of nicked circular products. Pre-bound RPA inhibits this reaction (line 2). Increasing amounts of Rad52 (lines 3–6, Rad52/bases: 1/55, 1/27, 1/18, 1/14) or Rad52-L264P (lines 7–10, Rad52/bases: 1/880, 1/220, 1/110, 1/55) overcome the inhibitory effect of pre-bound RPA. In line 1, only RPA was added to the reaction. Lower panel , the ratio of the nicked circular product over the sum of the linear dsDNA substrate and the nicked circular product is shown. (F) Salt titration of Rad51-Rad52/Rad52-L264P-ssDNA complex formation. The nucleoprotein complexes were assembled by incubating 0.8 µM Rad51 with 0.09 µM Rad52 or Rad52-L264P and 0.08 µM RPA pre-bound to 2.5 µM Cy5-labeled ssDNA (400 nucleotides) in the presence of the indicated NaCl concentrations at 37°C for 15 min. The Cy5 signals after nucleoprotein gel eletrophoresis are shown. Quantifications are shown below. Data were fitted into a third order polynomial curve. Western blot analysis of the gel using antibodies against Rad51 or Rad52 is also shown. Stars indicate signals corresponding to proteins not bound to ssDNA. (G) Transmission electron microscopy images of protein-DNA complexes showing the association of Rad52 with complete Rad51 filaments. Positive (left) and negative (right) staining images are shown for each type of filaments. The proportion of each type of complete Rad51 filaments formed by Rad52 or Rad52-L264P at different NaCl concentrations is shown and compared to a control reaction without Rad52. 100 molecules were examined in each experiment.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Recombinant, SDS Page, Staining, Binding Assay, Titration, Labeling, Software, Incubation, Western Blot, Transmission Assay, Electron Microscopy, Control

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A) Spot assay of haploid cells over-expressing SIZ2 . Serial 10-fold dilutions were plated on minimal media lacking uracil with or without MMS. Strains of the indicated genotype were transformed with an empty vector or with the same plasmid containing the SIZ2 gene. (B) Over-expression of SIZ2 stimulates Rad52 sumoylation. Proteins conjugated with a His 7 -SUMO radical were pull-down on Ni-NTA from 5 mg of extracts of RAD52 - FLAG cells over-expressing His7- SMT3 . Pull-downs were carried out from strains transformed with a SIZ2 -containing multi-copy vector or with an empty vector. Cells treated with 0.3% of MMS were also tested as a positive control of sumoylation. rad52-L264P-FLAG strains were also subjected to pull-down analysis. Proteins from the whole extracts (3 µg) and from the pull-down fractions were separated by SDS-PAGE and immunoblotted with an anti-FLAG mouse monoclonal antibody.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Spot Test, Expressing, Transformation Assay, Plasmid Preparation, Over Expression, Positive Control, SDS Page

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A) Schematic representation of the two kinds of toxic recombination intermediates eliminated by Srs2 in WT cells. We found that srs2 Δ haploid cells sensitivity to DNA damage is related to recombination-deficient Rad51 nucleoprotein filaments. The toxicity of such filaments disappears in rad52-L264P srs2Δ cells, or alternatively, the intermediates themselves are not formed. However, srs2 Δ cells sensitivity to DNA damage related with toxic intertwined HR intermediates cannot be suppressed by this allele. Unproductive Rad51 filaments can be formed after resection of a DSB located in a unique sequence in the genome. In a situation where homologous dsDNA cannot be found by the recombinase, Srs2 is essential to remove Rad51 filaments to allow alternative repair pathways such as SSA. Srs2 could also edit Rad51 filaments improperly nucleated by Rad52. In srs2 Δ cells, nonrecombinogenic Rad51 filaments could also accumulate on ssDNAs generated from the uncoupling between the helicase complex opening replicative dsDNA and the DNA synthesis machinery in replicative mutants such as mrc1 Δ. Finally, when a stable paranemic joint cannot be processed from a plectonemic joint because of mutations in genes involved in late recombination steps, Srs2 is necessary to address lesions to other DNA repair pathways. Intertwined recombination intermediates that occur between homologous chromosomes in diploid cells or between ectopic chromosomes in haploid cells cannot be suppressed by rad52-L264P . (B) Unproductive Rad51 filaments mediated by Rad52-SUMO are not toxic even in srs2 Δ cells. When mediated by Rad52, Rad51 filaments that cannot complete strand invasion have to be removed by Srs2 in order to allow SSA or post-replication repair processes (PRR). Conversely, Rad52-SUMO (or Rad52-L264P) might lower (or shorten) Rad51 filaments. These modified mediators might also change Rad51 filament properties as indicated by Rad52 occupancy on Rad51 filaments. These changes might suppress Rad51 filaments toxicity, thereby bypassing the need for Srs2. Rad51 filaments might be removed by a Srs2-independent process.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Sequencing, Generated, DNA Synthesis, Modification

Journal: Cell Cycle

Article Title: Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion

doi: 10.1080/15384101.2016.1261765

Figure Lengend Snippet: Genome-wide RNAi screen comparing normal and tumor cells. (A) Schematic representation of genome-scale RNAi screening in human immortalized (hTERT-RPE1) and cancer (U2OS) cell lines. (B) Hierarchical clustering of cell cycle regulators (z-score for at least one parameter is >5 or <−5) based on their phenotypic scores (for full data, see Supplementary Table S1). Note that known genes required for cell division (e.g., CDK1, KIF11, PLK1) cluster together for both cell lines. (C) S-phase z-scores in hTERT-RPE1 and U2OS cell lines. Most S-phase arrested hits for hTERT-RPE1 are genes involved in DNA replication and S-phase progression (marked black). Transcriptional regulators of histone genes are marked in red.

Article Snippet: U2OS human osteosarcoma (from M. Laiho lab, University Helsinki), HeLa human cervix adenocarcinoma (from M. Laiho lab, University Helsinki), HT1080 human fibrosarcoma (from ATCC, CRL-121), PC3 prostate adenocarcinoma (from Gonghong Wei, University Oulu), SK-N-MC human brain neuroepithelioma (from Gonghong Wei, University Oulu), SW480 colorectal adenocarcinoma (from Gonghong Wei, University Oulu) cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin).

Techniques: Genome Wide

Journal: Cell Cycle

Article Title: Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion

doi: 10.1080/15384101.2016.1261765

Figure Lengend Snippet: Regulation of DNA synthesis and expression of histone genes by CASP8AP2, NPAT and HINFP. (A) Flow cytometric analysis of DNA content (x-axis) and DNA replication (EdU incorporation; y-axis) shows partial or complete DNA synthesis progression 3 d after knockdown of CASP8AP2, NPAT and HINFP in tumor (U2OS) and normal (hTERT-RPE1) cells. Note that in both cell lines, CASP8AP2 RNAi results in formation of a population of S-phase cells with low EdU incorporation (red arrowheads). (B) Analysis of expression of histone genes following knockdown of the indicated genes in U2OS and hTERT-RPE1 cells. Replication-independent histone genes are marked with an asterisk. (C) Location-analysis of transcriptional regulators at histone gene cluster on chromosome 6p22. Cell lines and antibodies used in ChIP-Seq are indicated on the left, and signal intensity as number of reads is shown in parentheses above each track. Note that CASP8AP2 and NPAT co-bind to transcription start sites of replication-dependent histone genes (indicated in bottom) in this cluster.

Article Snippet: U2OS human osteosarcoma (from M. Laiho lab, University Helsinki), HeLa human cervix adenocarcinoma (from M. Laiho lab, University Helsinki), HT1080 human fibrosarcoma (from ATCC, CRL-121), PC3 prostate adenocarcinoma (from Gonghong Wei, University Oulu), SK-N-MC human brain neuroepithelioma (from Gonghong Wei, University Oulu), SW480 colorectal adenocarcinoma (from Gonghong Wei, University Oulu) cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin).

Techniques: DNA Synthesis, Expressing, Knockdown, ChIP-sequencing

Journal: Cell Cycle

Article Title: Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion

doi: 10.1080/15384101.2016.1261765

Figure Lengend Snippet: CASP8AP2 siRNA triggers activation of a p53 dependent S-phase checkpoint in normal but not in tumor cells. (A) Analysis of p53 target gene expression 3 d after knockdown of the indicated transcriptional regulators of histone genes. (B, C) Flow cytometry analysis of CASP8AP2 siRNA treated hTERT-RPE1 (B) and U2OS (C) cells indicates that non-replicated cells arrested in S-phase express a marker for DNA damage signaling (γ-H2AX; low right hand corner). (D, E) hTERT-RPE1 (D) but not U2OS (E) cells with activated DNA damage signaling have activated p53, based on expression of the p53 target-gene p21 (upper right hand corner).

Article Snippet: U2OS human osteosarcoma (from M. Laiho lab, University Helsinki), HeLa human cervix adenocarcinoma (from M. Laiho lab, University Helsinki), HT1080 human fibrosarcoma (from ATCC, CRL-121), PC3 prostate adenocarcinoma (from Gonghong Wei, University Oulu), SK-N-MC human brain neuroepithelioma (from Gonghong Wei, University Oulu), SW480 colorectal adenocarcinoma (from Gonghong Wei, University Oulu) cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin).

Techniques: Activation Assay, Targeted Gene Expression, Knockdown, Flow Cytometry, Marker, Expressing

Journal: Nature communications

Article Title: Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids.

doi: 10.1038/ncomms7867

Figure Lengend Snippet: Figure 1 | Genome editing to produce caveolin-1-GFP and cavin-1-mCherry does not perturb caveolar morphology or abundance. (a) NIH3T3 cells untransfected, overexpressing either caveolin-1-GFP or cavin-1-mCherry via CMV-promoter plasmid and transient transfection, or expressing either caveolin-1-GFP or both caveolin-1-GFP and cavin-1-mCherry from endogenous loci via genome editing to insert GFP or mCherry, were analysed by western blotting using the antibodies as shown. All the samples were obtained and analysed in duplicate. *Note that caveolin-1-GFP expressed from endogenous loci appears in two isoforms, similarly to endogenous untagged protein (visible at shorter exposures). (b) Electron micrographs of wild-type NIH3T3 cells or genome-edited NIH3T3 cells expressing both caveolin-1-GFP and cavin-1-mCherry. Bars are 100 nm. (c) Quantification of the abundance of caveolae in genome-edited cell lines used in this study. Each point represents one cell perimeter outlined in 10–30 individual electron micrographs. Bars are mean and s.d. (d) Confocal image of live NIH3T3 cells expressing both caveolin-1-GFP and cavin-1-mCherry from endogenous loci. Bar is 10 mm, the white box delineates the region shown at higher magnification in the right-hand panels.

Article Snippet: HeLa (ATCC) and NIH3T3 cells (a gift from the lab of Julian Sale, MRC-LMB) were grown in DMEM (Gibco) supplemented with penicillin and streptomycin, and 10% fetal calf serum.

Techniques: Plasmid Preparation, Transfection, Expressing, Western Blot

Journal: Nature communications

Article Title: Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids.

doi: 10.1038/ncomms7867

Figure Lengend Snippet: Figure 2 | Genome-edited caveolin-1-GFP and cavin-1-mCherry are present in dynamic intracellular vesicles. (a) Time-lapse TIR imaging of live NIH3T3 cells expressing both genome-edited caveolin-1-GFP and cavin-1-mCherry shows that these proteins are present on motile structures. Bars are 2 mm. Note that in the upper series of images the motile structure is brighter than minimum quantal caveolin-1-GFP þcavin-1-mCherry puncta visible in the same images, while in the lower series the motile structure is more likely to represent one quantal assembly of caveolin-1-GFP þcavin-1-mCherry. See also Supplementary Movies 2 and 3. (b) Time-lapse confocal imaging of a typical FRAP experiment in genome-edited cells expressing caveolin-1-GFP shows fluorescence recovery due to movement of vesicular structures, rather than association of new molecules to existing structures in the bleached area. See also Supplementary Movie 4. (c) FRAP experiments comparing the mobility of genome-edited caveolin-1-GFP in untransfected cells to cells expressing EHD2-I157Q-mCherry. N ¼ 58 for control, 17 for mutant, bars are 95% confidence intervals. (d) FRAP experiments comparing the mobility of genome- edited caveolin-1-GFP in untransfected cells to cells expressing AP180-C-myc. N ¼ 58 for control, 19 for mutant, bars are 95% confidence intervals. The control data are the same as in c.

Article Snippet: HeLa (ATCC) and NIH3T3 cells (a gift from the lab of Julian Sale, MRC-LMB) were grown in DMEM (Gibco) supplemented with penicillin and streptomycin, and 10% fetal calf serum.

Techniques: Imaging, Expressing, Control, Mutagenesis

Journal: Nature communications

Article Title: Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids.

doi: 10.1038/ncomms7867

Figure Lengend Snippet: Figure 5 | Caveolin-positive endosomes include both classical early endosomes and separate organelles. (a) Confocal image of an NIH3T3 cell expressing genome-edited caveolin-1-GFP and FYVE-mCherry, labelled with streptavidin to detect internalized sulfo-NHS-SS-biotin after 15 min uptake. The boxes denote regions shown in b. Bar is 15 mm. (b) Examples of caveolin-1-GFP-positive endosomes that do not recruit FYVE-mCherry, taken from the cell shown in A, yellow circles. One example of an endosome that does recruit FYVE-mCherry is also shown, white circle. (c) Confocal image of an NIH3T3 cell expressing genome-edited caveolin-1-GFP, labelled with streptavidin to detect internalized sulfo-NHS-SS-biotin after 15 min uptake, and with transferrin internalized continuously for the same length of time. The boxes denote regions shown in d. Bar is 15 mm. (d) Examples of caveolin-1-GFP-positive endosomes that do not contain transferrin, yellow circles, and one endosome that does contain transferrin, white circle, taken from the cell shown in c. (e) Confocal image of an NIH3T3 cell expressing genome-edited caveolin-1-GFP and Rab5-mCherry, labelled with streptavidin to detect internalized sulfo-NHS-SS-biotin after 15 min uptake. The boxes denote regions shown in b. Bar is 15 mm. (f) Examples of caveolin-1-GFP-positive endosomes that do not recruit Rab5-mCherry, taken from the cell shown in A, yellow circles. One example of an endosome that does recruit Rab5-mCherry is also shown, white circle.

Article Snippet: HeLa (ATCC) and NIH3T3 cells (a gift from the lab of Julian Sale, MRC-LMB) were grown in DMEM (Gibco) supplemented with penicillin and streptomycin, and 10% fetal calf serum.

Techniques: Expressing

Journal: Nature communications

Article Title: Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids.

doi: 10.1038/ncomms7867

Figure Lengend Snippet: Figure 6 | Budding of caveolae from the plasma membrane delivers endocytosed cargo to caveolar endosomes. (a) Genome-edited caveolin-1-GFP cells were transfected with myc-AP180-C. Cells were labelled with streptavidin to detect internalized sulfo-NHS-SS-biotin after 15 min uptake and MESNA treatment. Transfected cells are outlined with a dashed line. The region shown by a white box is shown in the magnified panels to the right-hand side. (b) Quantification of total endocytosis of sulfo-NHS-SS-biotin, detected by streptavidin labelling after MESNA treatment, in control and AP180-C expressing cells. Bars are mean and s.d. (c) Quantification of caveolar endosomes abundance. Caveolar endosomes were scored by eye as structures clearly containing both caveolin-1-GFP and internalized sulfo-NHS-SS-biotin as shown in A. Bars are mean and s.d. (d) Confocal image of an NIH3T3 cell expressing genome-edited caveolin-1-GFP, labelled with streptavidin to detect internalized sulfo-NHS-SS-biotin after 90 s uptake. The boxes denote regions shown in E. Bar is 15 mm. (e) Individual caveolar endocytic vesicles after 90 s uptake, taken from d. (f) Quantification of co-localization between caveolin-1- GFP and internalized protein labelled with sulfo-NHS-SS-biotin and MESNA treatment. Internalization was for 90 s. To establish empirically the degree of overlap between internalized protein and relevant marker expected by chance, quantification was carried out both with the images in the correct register and also with one channel manually offset 500 nm from the other. Quantification of offset images is shown as grey dots, correct registration as black dots, statistically significant increase in co-localization with images in the correct register indicates biologically significant co-localization. Bars are mean and s.d., each point is one cell region. (g) Confocal image of an NIH3T3 cells expressing genome-edited caveolin-1-GFP, labelled with streptavidin to detect internalized sulfo-NHS-SS-biotin after 15 min uptake. Cells were treated with 500 nM okadaic acid as shown. Arrows indicate co-localization in caveolar endosomes. (h) Quantification of co-localization between caveolin-1-GFP and internalized protein labelled with sulfo-NHS-SS-biotin and MESNa treatment. Internalization was for 15 min, cells were treated with 500 nM okadaic acid.

Article Snippet: HeLa (ATCC) and NIH3T3 cells (a gift from the lab of Julian Sale, MRC-LMB) were grown in DMEM (Gibco) supplemented with penicillin and streptomycin, and 10% fetal calf serum.

Techniques: Clinical Proteomics, Membrane, Transfection, Control, Expressing, Marker

Journal: Nature communications

Article Title: Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids.

doi: 10.1038/ncomms7867

Figure Lengend Snippet: Figure 7 | Caveolae exclude bulk membrane proteins. (a) Cartoon to illustrate how increased membrane convolution within caveolae will cause local increase in fluorescence intensity in images of proteins with a uniform distribution per unit area of membrane, such as GPI-linked proteins. In contrast, proteins that are excluded from caveolae will appear at uniform fluorescence intensity across the region containing caveolae. (b) Confocal image of NIH3T3 cells expressing caveolin-1-GFP, transiently transfected for 24 h with syntaxin-2-mCherry and SNAP–GPI. Cells were incubated at 4 C for 15 min with membrane impermeant SNAP Cell Surface Reagent 647 to label cell surface-associated GPI proteins before fixation and confocal imaging. Bar is 2 m. (c,d,e,f) Quantification of intensity profiles across caveolin-1-GFP-positive regions as shown in b. The intensity of caveolin-1-GFP, SNAP-GPI labelled as in A, and the fluorescent membrane protein shown in red, was quantified for 20 different puncta, and plotted as mean þ / s.e.m. Distance (x axis) and fluorescence intensities (y axis) are in arbitrary units. (g) Electron micrographs of cells expressing CD9-APEX. Reactive oxygen generated by the APEX tag triggers polymerization of DAB, leading to the electron-dense deposit all along the plasma membrane. The white arrow indicates plasma membrane of a non-transfected cell. Yellow arrows indicate caveolar membrane bulb, and black arrows highlight the extra electron density specifically generated by the APEX tag.

Article Snippet: HeLa (ATCC) and NIH3T3 cells (a gift from the lab of Julian Sale, MRC-LMB) were grown in DMEM (Gibco) supplemented with penicillin and streptomycin, and 10% fetal calf serum.

Techniques: Membrane, Expressing, Transfection, Incubation, Imaging, Generated, Clinical Proteomics

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: PD-L1 is modified by UBL3. ( a ) Co-immunoprecipitation (IP) analysis of using MDA-MB-231 cell lysates expressing Flag-tagged PD-L1 (PD-L1-Flag) and biotinylated-tagged UBL3 (Biotin-UBL3). ( b ) Co-IP was performed using cell lysates expressing PD-L1-Flag or PD-L1 mutants-Flag (C250A, C272A, C250/272A) along with Biotin-UBL3 in MDA-MB-231 cells. IB analysis was conducted using the indicated antibodies. ( c ) the relative intensity of UBL3 modification (Flag/SA-HRP) calculated as the ratio of UBL3-modified PD-L1 signals (> 70 kDa) to input SA-HRP signals, normalized to the mean of cells transfected with Biotin-UBL3 and wild-type PD-L1. Data are presented as mean ± s.e.m., with dots representing individual experiments. One-way ANOVA with Tukey’s multiple comparisons test. ns, not significant, * P < 0.05, ** P < 0.001, *** P < 0.0005, PD-L1 vs. C250A, P = 0.1708; PD-L1 vs. C272A, P = 0.0009; PD-L1 vs. C250/272A, P = 0.0002; C250A vs. C272A, P = 0.0135; C272A vs. C250/272A, P = 0.5847; TMD, transmembrane domain; Flag, Flag-tag; βME-, without 2-mercaptoethanol; βME+, with 2-mercaptoethanol; SA-HRP, streptavidin-horseradish peroxidase.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Modification, Immunoprecipitation, Expressing, Co-Immunoprecipitation Assay, Transfection, FLAG-tag

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: UBL3 regulates PD-L1 levels in sEVs. ( a ) Immunoblot (IB) analysis. The cell lysates and sEVs from the conditioned medium of H1299 cells transfected with 3xFlag-UBL3 vectors were blotted with various antibodies. ( b ) IB analysis. The cell lysates and sEVs from the stable UBL3 knockdown H1299 cell lines were blotted with various antibodies. CD9 and CD63 were used as sEV markers, as they are tetraspanin proteins abundantly expressed on the sEV surface . Relative intensity of PD-L1 in sEVs was calculated as PD-L1/CD63 and presented as fold change, normalized to the mean value of the respective control (mock for a; shNega for b). Data are presented as mean ± s.e.m., with dots representing individual experiments. Two-tailed unpaired t-test. *** P < 0.005.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

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

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: Inhibition of UBL3 modification and PD-L1 sorting by statins. ( a and b ), Effect of pitavastatin treatment on UBL3 modification in WM9 ( a ) and H1299 ( b ) cells. Five hours after transfection, 10 µM pitavastatin was added. ( c and d ), IB analysis of the cell lysates and sEVs from the conditioned medium of H1299 cells transfected with 3xFlag-UBL3 vectors ( c ) or untransfected ( d ) were blotted with various antibodies. Five hours after gene transfection, 0.2 µM pitavastatin was added. Pit, Pitavastatin. Right panels, the relative intensity of PD-L1 in sEVs (c: PD-L1/CD63 [upper] normalized to the mean of mock − untreated control, and UBL3/CD63 [lower] normalized to the mean of Flag-UBL3 − untreated control; d: PD-L1/CD63 [upper] and UBL3/CD63 [lower], both normalized to the mean of untreated control). Data are presented as mean ± s.e.m., with dots representing individual experiments. As inhibition of UBL3 modification was observed even at low concentrations of pitavastatin (Supplementary Fig. S4b), a low concentration of pitavastatin was used in sEV purification experiments. IB analysis using the indicated antibodies. Flag, Flag-tag; GAPDH, internal control; CD63 and CD9, sEVs markers; PD-L1, endogenous PD-L1; UBL3, endogenous UBL3. βME−, without 2-mercaptoethanol. βME+, with 2-mercaptoethanol. ( c ), one-way ANOVA with Tukey’s multiple comparisons test. ( d ), Two-tailed unpaired t-test. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Inhibition, Modification, Transfection, Control, Concentration Assay, Purification, FLAG-tag, Two Tailed Test

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: UBL3 and PD-L1 expression levels influence survival in lung cancer patients. ( a ) Kaplan–Meier survival curves for the lung squamous cell carcinoma cohort, stratified by UBL3 and PD-L1 expression. ( b ) Survival curves for the same cohort, stratified by UBL3 and PD-1 expression. Hazard ratios and p-values from log-rank (Mantel–Cox) tests are summarized in Supplementary Figure S7a.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

doi: 10.1093/nar/gky071

Figure Lengend Snippet: eIF4A interacts with the base of the DLP RNA in the 48S complex. ( A ) Crosslinking assays using SV-DLP U1 and SFV-DLP U1 mRNAs in the presence of hippuristanol or polyI:C. Samples were treated with 1 μM of hippuristanol or 0.5 ng/μl poly I:C to block eIF4A or to induce eIF2α phosphorylation, respectively, as indicated. Then, P100 fractions were obtained and equivalent cpms were analyzed by SDS-PAGE and autoradiography. The lower panel shows typical eIF2α phosphorylation induced by poly I:C treatment of RRL. ( B ) Denaturing IP with anti-eIF4A and anti-RPS6 antibodies. 48S complexes were assembled with SV-DLP U1 mRNA, crosslinked and the WRF was denatured and subjected to IP with the indicated antibodies (as described in ‘Materials and Methods’ section).

Article Snippet: Western blots were carried out as described previously ( ) using the following primary antibodies: α-myc (Clontech), α-SV E1, α-SV C, α-eIF2 (Santa Cruz Biotech.), α-RPS7 (Santa Cruz Biotech.), α-RPS2 (Santa Cruz Biotech.), α-RPS6 (Santa Cruz Biotech.), mouse α-eIF4A (a gift of M. Altmann, University of Bern, Switzerland) and rabbit α-eIF4A (St John’s lab, STJ27247).

Techniques: Blocking Assay, Phospho-proteomics, SDS Page, Autoradiography

Journal: Nucleic Acids Research

Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

doi: 10.1093/nar/gky071

Figure Lengend Snippet: eIF4A crosslinking with SV mRNA is DLP position dependent. ( A ) Variants of SV-DLP U1 mRNA where the distance from the AUG was shortened ( d = 19) or extended ( d = 37) were used. Quantification of eIF4A crosslinking from three independent experiments (mean ± SEM) is shown in the bottom panel. ( B ) Model of 43S complex bound to mRNA showing the approximate positions of DLP structures at d = 19, 28 and 37, assuming that mRNA penetrates through the ES6S A and ES6S B helices of the 40S subunit. The positions of eIF3 and the ternary complex (TC) are also shown. ( C ) Translation activity of DLP structures located 19, 28 and 37 nt from the AUG. Recombinant virus expressing an EGFP reporter mRNA (SV-EGFP) bearing the DLP structure located at the indicated position (d) or lacking it (−) were obtained and described previously . 3T3 cells were infected with the indicated viruses at an MOI of 10, extracts were made at 6 hpi and analyzed by western blot with the indicated antibodies.

Article Snippet: Western blots were carried out as described previously ( ) using the following primary antibodies: α-myc (Clontech), α-SV E1, α-SV C, α-eIF2 (Santa Cruz Biotech.), α-RPS7 (Santa Cruz Biotech.), α-RPS2 (Santa Cruz Biotech.), α-RPS6 (Santa Cruz Biotech.), mouse α-eIF4A (a gift of M. Altmann, University of Bern, Switzerland) and rabbit α-eIF4A (St John’s lab, STJ27247).

Techniques: Activity Assay, Recombinant, Virus, Expressing, Infection, Western Blot

Journal: Nucleic Acids Research

Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

doi: 10.1093/nar/gky071

Figure Lengend Snippet: Effect of VIC oligo-4 on the interaction of eIF4A in the 48S complex with the base of the DLP structure. ( A ) 43S model showing the binding site of VIC oligo-4 to the ES6S C-D helix. VIC oligo-4 binding to purified rabbit 40S subunit was analyzed by RNAse H cleavage as described previously . The resulting 18S rRNA fragments were analyzed by electrophoresis in a 0.8% agarose gel. ( B ) Effect of VIC oligo-4 addition (10 μM) on translation programmed with SV DLP capsid and SV ΔDLP capsid mRNAs in RRL. Note that elimination of the DLP structure impaired AUG recognition, giving rise to aberrant products (gray arrowheads) that resulted from spurious initiation at downstream AUGs, as reported previously . ( C ) Crosslinking assays using SV DLPU1 mRNA in the presence of 10 μM of VIC oligo-4. Ribosomal fractions (P100) and post-ribosomal fractions (S100) were analyzed. The change in intensity of the eIF4A band was measured from three independent experiments; data are represented as the mean ± SEM. ( D ) Effect of VIC oligo-4 on 48S formation and eIF4A crosslinking. The experiment was performed as in Figure ; as VIC oligo-4 reduced 48S formation, the volume of fraction 11 was adjusted to analyze an equivalent cpm. Arrows shows the direction of sedimentation.

Article Snippet: Western blots were carried out as described previously ( ) using the following primary antibodies: α-myc (Clontech), α-SV E1, α-SV C, α-eIF2 (Santa Cruz Biotech.), α-RPS7 (Santa Cruz Biotech.), α-RPS2 (Santa Cruz Biotech.), α-RPS6 (Santa Cruz Biotech.), mouse α-eIF4A (a gift of M. Altmann, University of Bern, Switzerland) and rabbit α-eIF4A (St John’s lab, STJ27247).

Techniques: Binding Assay, Purification, Electrophoresis, Agarose Gel Electrophoresis, Sedimentation

Journal: Nucleic Acids Research

Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

doi: 10.1093/nar/gky071

Figure Lengend Snippet: eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for RNase H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ) Effect of eIF4A inhibition on 48S toeprinting generated by SV-DLP U1 and SV-ΔDLP U1 mRNAs. Assays were carried out in the absence of presence of 1 μM of hippuristanol; positions of resulting primer extensions are annotated with respect to the A (+1) position of AUG, and they have been assigned with a precision of ±1 nt. The bottom shows a 2D structural model of the first 100 nt of 26S mRNA, showing the secondary structure derived from SHAPE analysis . Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.

Article Snippet: Western blots were carried out as described previously ( ) using the following primary antibodies: α-myc (Clontech), α-SV E1, α-SV C, α-eIF2 (Santa Cruz Biotech.), α-RPS7 (Santa Cruz Biotech.), α-RPS2 (Santa Cruz Biotech.), α-RPS6 (Santa Cruz Biotech.), mouse α-eIF4A (a gift of M. Altmann, University of Bern, Switzerland) and rabbit α-eIF4A (St John’s lab, STJ27247).

Techniques: Activity Assay, Inhibition, Generated, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

doi: 10.1093/nar/gky071

Figure Lengend Snippet: EM localization of eIF4A and mRNA in the 48S complex. ( A ) Analysis of the eIF composition of 48S complexes assembled with SV-DLP U1 mRNA. A small amount of fluorescein-labeled SV-DLP U1 mRNA was included in the reaction to identify the fraction corresponding to 48S peaks. Fractions 6–12 were precipitated in acetone–chloroform and analyzed by western blot using the indicated antibodies. Fractions 8–10 were pooled and subjected to immuno-EM using an α-eIF4A antibody followed by incubation with 5 nm GPA. ( B ) Gallery of most abundant views grouped into three classes. Photographs were taken at 60K (except otherwise indicated) and the corresponding projections on the 43S model (EM-5658) are shown Scale bar corresponds to 10 nm. ( C ) Gallery of most abundant views of the 48S complex assembled with SV ΔDLP mRNA bound to a 5′-biotinylated antisense RNA oligo (see for details). In this case, the samples were incubated with streptavidin-5 nm gold as described in the ‘Materials and Methods’ section. ( D ) Superposition of all labels for eIF4A (red) and for mRNA (green), showing the most likely placement areas.

Article Snippet: Western blots were carried out as described previously ( ) using the following primary antibodies: α-myc (Clontech), α-SV E1, α-SV C, α-eIF2 (Santa Cruz Biotech.), α-RPS7 (Santa Cruz Biotech.), α-RPS2 (Santa Cruz Biotech.), α-RPS6 (Santa Cruz Biotech.), mouse α-eIF4A (a gift of M. Altmann, University of Bern, Switzerland) and rabbit α-eIF4A (St John’s lab, STJ27247).

Techniques: Labeling, Western Blot, Incubation

Journal: Nucleic Acids Research

Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

doi: 10.1093/nar/gky071

Figure Lengend Snippet: Ectopic expression of eIF4A in viral factories reduced SV mRNA translation. ( A ) Exclusion of eIF4A from SFV viral factories. IF of eIF4A in mock and SFV-infected cells, with SFV C protein stained in red and eIF4A in green. Note the exclusion of eIF4A from viral factories as revealed by C protein staining (arrowheads). About 75% of analyzed cells showed this pattern, and a representative field is shown. ( B ) Schematic representation of recombinant SV expressing human eIF4A-myc. Cells were infected with two independent SV-eIF4A clones and analyzed by western blot against myc epitope (right upper panel), by IF against SV C and myc epitopes (left lower), and by metabolic labeling with [ 35 S]-Met. The position of viral protein bands is indicated; in all cases, samples were analyzed at 6 hpi, with two SV clones expressing EGFP used as controls.

Article Snippet: Western blots were carried out as described previously ( ) using the following primary antibodies: α-myc (Clontech), α-SV E1, α-SV C, α-eIF2 (Santa Cruz Biotech.), α-RPS7 (Santa Cruz Biotech.), α-RPS2 (Santa Cruz Biotech.), α-RPS6 (Santa Cruz Biotech.), mouse α-eIF4A (a gift of M. Altmann, University of Bern, Switzerland) and rabbit α-eIF4A (St John’s lab, STJ27247).

Techniques: Expressing, Infection, Staining, Recombinant, Clone Assay, Western Blot, Labeling

Journal: Nucleic Acids Research

Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

doi: 10.1093/nar/gky071

Figure Lengend Snippet: Model of PIC scanning. ( A ) Canonical model of PIC scanning with eIF4F complex placed on ES6S region of 40S subunit (EM-5658) , interacting with both 3′and 5′regions of mRNA (orange). The scaffolding eIF4G factor is in yellow, interacting with both ES6S and eIF3 as reported before ( , ). In this model two molecules of eIF4A (red rollers) are placed on eIF4G according to previous data ( , ). The 4E subunit of eIF4F (eIF4E) is dissociated from the cap structure upon 48S complex formation as suggested recently . ( B ) SV DLP-mediated stalled PIC showing one eIF4A molecule jammed by DLP structure on ES6S region. The rest of factors and details were omitted for simplicity. The position of VIC oligo-4 is also shown (red).

Article Snippet: Western blots were carried out as described previously ( ) using the following primary antibodies: α-myc (Clontech), α-SV E1, α-SV C, α-eIF2 (Santa Cruz Biotech.), α-RPS7 (Santa Cruz Biotech.), α-RPS2 (Santa Cruz Biotech.), α-RPS6 (Santa Cruz Biotech.), mouse α-eIF4A (a gift of M. Altmann, University of Bern, Switzerland) and rabbit α-eIF4A (St John’s lab, STJ27247).

Techniques: Scaffolding

Journal: The Journal of Cell Biology

Article Title: Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFα

doi: 10.1083/jcb.200612131

Figure Lengend Snippet: Expression of IL-6 and TNFα in macrophages. (A) RAW264.7 cells were stimulated with 100 ng/ml LPS and 500 pg/ml IFNγ, and samples of the medium were removed every 2 h over a 6-h time course. The graph shows the levels of IL-6 as determined by cytokine-specific ELISA secreted by macrophages over the time course. (B) Macrophages were activated with LPS and IFN for 6 h before fixation, permeabilization, and staining for intracellular IL-6 (red) and TNFα (green). Epifluorescence staining shows the coexpression of endogenous IL-6 and TNFα and their coaccumulation in the perinuclear Golgi complex. (C) Immuno-EM of newly synthesized cytokines in Golgi complexes (G), demonstrating IL-6–GFP (arrows) alone or together with TNFα (arrowheads), labeled with 5 or 10 nm gold particles as indicated. (D) Cells were activated with combinations of LPS and IFN for 6 h in the presence or absence of 5 μg/ml brefeldin A, and IL-6 in cell extracts was determined by Western blotting. Consistent with the ELISA data, increased levels of intracellular IL-6 are stimulated in the presence of IFN and LPS together, and disruption of the Golgi complex leads to the intracellular accumulation of IL-6. Bars (B), 5 μm; (C) 100 nm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody to mouse IL-6 (Serotec), rabbit polyclonal and rat monoclonal anti–mouse TNFα antibodies (Calbiochem and Auspep, respectively), monoclonal antibodies to Stx6 and Vti1b (Translabs; BD Biosciences), monoclonal antibody to TfnR (Zymed Laboratories and Invitrogen), and a polyclonal rabbit antibody to VAMP3 (Abcam).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Synthesized, Labeling, Western Blot, Disruption

Journal: The Journal of Cell Biology

Article Title: Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFα

doi: 10.1083/jcb.200612131

Figure Lengend Snippet: IL-6 exits the TGN in carriers both with and without TNFα. (A and B) Live epifluorescence dual-color imaging of macrophages transfected with TNFα-mCherry (red) and IL-6–GFP (green) demonstrating IL-6 leaving the TGN in a carrier devoid of TNFα (A) and in a tubule also containing TNFα (B). Cells were stimulated with LPS and IFNγ before imaging. In the enlarged images, arrows are used to highlight single trafficking events, and the time at which each still frame was collected is labeled. (C) Cell lysates of activated macrophages were fractionated to separate Golgi membranes and cytosol. Golgi membranes were then incubated with the cytosol in an in vitro Golgi budding reaction as described in Materials and methods. Samples from the budded vesicle fractions (labeled 1–9) were resolved and analyzed by Western blotting to detect γ-adaptin as a vesicle-associated protein and IL-6 and TNF as cargoes. Bar, 10 μm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody to mouse IL-6 (Serotec), rabbit polyclonal and rat monoclonal anti–mouse TNFα antibodies (Calbiochem and Auspep, respectively), monoclonal antibodies to Stx6 and Vti1b (Translabs; BD Biosciences), monoclonal antibody to TfnR (Zymed Laboratories and Invitrogen), and a polyclonal rabbit antibody to VAMP3 (Abcam).

Techniques: Imaging, Transfection, Labeling, Incubation, In Vitro, Western Blot

Journal: The Journal of Cell Biology

Article Title: Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFα

doi: 10.1083/jcb.200612131

Figure Lengend Snippet: A large proportion of vesicular IL-6 colocalizes with TNFα. (A) Live epifluorescence microscopy of an IL-6–GFP-transfected macrophage showing fusion of a post-TGN carrier (red circles) with a large preexisting IL-6–containing structure (green circles), possibly a recycling endosome. A yellow circle highlights the resulting fused structure. An intensity-based color display pallet was used to emphasize vesicular structures, and the time each was captured is indicated. (B) High resolution confocal microscopy on macrophages cotransfected with IL-6–GFP (green) and TNFα-mCherry (red) revealed overlap in the localization of IL-6 and TNFα in structures toward the cell periphery (yellow arrows). Green arrows highlight structures that contain IL-6 alone. Quantification of colocalization revealed that in macrophages, ∼60–70% of the IL-6–positive structures also contain either endogenous TNFα or overexpressed TNFα. More than 20 IL-6–GFP-expressing cells were measured to generate each set of data, and error bars represent means ± SEM. (C) Immuno-EM of IL-6–GFP (arrows) and TNFα (arrowheads) labeled with 10 nm or 5 nm gold particles, respectively, in peripheral endosomes (E), some of which are in the vicinity of the plasma membrane (pm). Individual endosomes have IL-6–GFP alone or together with TNFα. Bars (A), 10 μm; (B) 5 μm; (C) 100 nm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody to mouse IL-6 (Serotec), rabbit polyclonal and rat monoclonal anti–mouse TNFα antibodies (Calbiochem and Auspep, respectively), monoclonal antibodies to Stx6 and Vti1b (Translabs; BD Biosciences), monoclonal antibody to TfnR (Zymed Laboratories and Invitrogen), and a polyclonal rabbit antibody to VAMP3 (Abcam).

Techniques: Epifluorescence Microscopy, Transfection, Confocal Microscopy, Expressing, Labeling, Clinical Proteomics, Membrane

Journal: The Journal of Cell Biology

Article Title: Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFα

doi: 10.1083/jcb.200612131

Figure Lengend Snippet: IL-6 and TNFα colocalize in the recycling endosome. (A and B) RAW264.7 cells were transfected with IL-6–mCherry and either GFP-VAMP3 (A) or GFP-TNFα (B) and activated with LPS and IFN for 4 h before fixation. In addition, to label transfected cells with Tfn (blue), macrophages were incubated in the presence of AlexaFluor647-conjugated Tfn (B). Cells were imaged via high resolution confocal microscopy. (A) A large proportion of IL-6–positive structures colocalize with the recycling endosome markers VAMP3 (A), endocytosed Tfn, and TNFα (B). In A, the insets highlight single ring-shaped VAMP3-positive structures containing IL-6. In B, colored arrows are used to highlight three recycling endosome structures containing all three cargo proteins. (C) Immuno-EM of IL-6–GFP, endogenous TfnR, and TNFα labeled with 5, 10, or 15 nm gold particles. Peri-Golgi (left) or peripheral (middle and right) recycling endosomes (RE) are labeled with TfnR, which is recycling from the plasma membrane (pm; right). IL-6–GFP or TNFα (arrows) colocalized with TfnR (arrowheads) in recycling endosomes. G, Golgi complex. Bars (A), 10 μm; (B) 5 μm; (C) 100 nm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody to mouse IL-6 (Serotec), rabbit polyclonal and rat monoclonal anti–mouse TNFα antibodies (Calbiochem and Auspep, respectively), monoclonal antibodies to Stx6 and Vti1b (Translabs; BD Biosciences), monoclonal antibody to TfnR (Zymed Laboratories and Invitrogen), and a polyclonal rabbit antibody to VAMP3 (Abcam).

Techniques: Transfection, Incubation, Confocal Microscopy, Labeling, Clinical Proteomics, Membrane

Journal: The Journal of Cell Biology

Article Title: Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFα

doi: 10.1083/jcb.200612131

Figure Lengend Snippet: Compartmentalization of endosomal structures containing IL-6, TNFα, and Tfn. (A–C) Macrophages were transfected with fluorescently tagged IL-6 (C) or both IL-6–mCherry and TNF-GFP (A and B) and were subsequently incubated in the presence of fluorescently conjugated Tfn. In A, a fluorescence intensity line scan profile was generated along the red line, with fluorescence peaks for the four recycling endosomes highlighted. In IL-6–positive structures, the peak fluorescence appears to overlap with the peak of TNFα (structures 2 and 3), but the Tfn fluorescence is more dispersed in these structures compared with those containing TNFα and Tfn alone (structures 1 and 4). (B) Two confocal sections are shown highlighting recycling endosome structures, which are labeled A and B. The enlarged area represents a surface-rendered 3D image of the region highlighted. Images are viewed in the direction of the large arrows and in the cross section through the cell. This demonstrates examples of recycling endosomes with both overlapping and discrete domains for each of the markers. In C, transfected macrophages were additionally subjected to labeling using the DilC 18 (5)-DS lipophilic dye (blue) before incubation with Tfn (red) as described in Materials and methods. The enlarged area shows Tfn and IL-6 labeling separate domains within a single dye-positive endosomal structure (highlighted by white arrows). Bars (A), 5 μm; (B and C) 10 μm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody to mouse IL-6 (Serotec), rabbit polyclonal and rat monoclonal anti–mouse TNFα antibodies (Calbiochem and Auspep, respectively), monoclonal antibodies to Stx6 and Vti1b (Translabs; BD Biosciences), monoclonal antibody to TfnR (Zymed Laboratories and Invitrogen), and a polyclonal rabbit antibody to VAMP3 (Abcam).

Techniques: Transfection, Incubation, Fluorescence, Generated, Labeling

Journal: The Journal of Cell Biology

Article Title: Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFα

doi: 10.1083/jcb.200612131

Figure Lengend Snippet: SNARE and recycling endosomes in the trafficking and secretion of IL-6. (A–F) Exocytic trafficking was altered in macrophages by the overexpression of specific SNAREs (A and C), their knockdown by specific siRNA (B and D), or by HRP inactivation of the endocytic compartment (E and F). After transfection or treatments, the macrophages were activated with LPS and IFN, the medium was collected 2 h and 6 h after stimulation, and the levels of IL-6 and/or TNFα, respectively, were determined by cytokine-specific ELISAs. (A and C) Overexpression of Stx6, Vti1b, and VAMP3 but not Stx2 led to increased levels of IL-6 secretion from macrophages. Bar graphs represent means ± SEM (error bars) of triplicate transfections. (B and D) Targeted knockdown of the Q-SNAREs Vti1b, Stx6, and VAMP3 by siRNA resulted in significant decreases in the level of IL-6 secreted from activated macrophages. Lysates of macrophages subjected to siRNA knockdown were blotted for Vti1b, Stx6, VAMP3, and the loading control protein glyceraldehyde-3-phoshphate dehydrogenase (GAPDH). Bar graphs represent mean ± SEM of triplicate siRNA transfections. (E) HRP inactivation of the endosomal compartment as described in Materials and methods blocked the secretion of both TNFα and IL-6. (F) In addition, after 6 h of stimulation, coverslips were fixed, stained for intracellular TNFα and IL-6, and the percentage of cells expressing each cytokine was determined. 10 low magnification images were captured per assay condition, each containing at least 50 cells, and the percentage of cells producing TNFα and/or IL-6 was calculated (D). Bar graphs represent mean percentages ± SEM of the individual images captured. **, P < 0.05.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody to mouse IL-6 (Serotec), rabbit polyclonal and rat monoclonal anti–mouse TNFα antibodies (Calbiochem and Auspep, respectively), monoclonal antibodies to Stx6 and Vti1b (Translabs; BD Biosciences), monoclonal antibody to TfnR (Zymed Laboratories and Invitrogen), and a polyclonal rabbit antibody to VAMP3 (Abcam).

Techniques: Over Expression, Knockdown, Transfection, Control, Staining, Expressing

Journal: The Journal of Cell Biology

Article Title: Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFα

doi: 10.1083/jcb.200612131

Figure Lengend Snippet: Delivery of TNFα and VAMP3 but not IL-6 or TfnR from recycling endosomes to the phagocytic cup. (A) Dual-color live imaging of the phagocytosis of IgG-opsonized 3-μm latex beads by macrophages transfected with VAMP3-GFP (green) and IL-6–mCherry (red). An overview of all particle tracks over a 10-min time course (200 frames) is shown. The tracks were filtered so only those that were continuous for five or more frames are displayed. Each detected track is shown at its full length as a continuous color, with red sections being interpolated by the software to handle occlusion, exit, and entry of a particle along the trajectory. VAMP3 can be seen to have extensive movement around and toward the forming phagocytic cup, which is in contrast to IL-6, which never advances to the cup. The location of the particle being phagocytosed is indicated with an asterisk. (B) IFN-primed macrophages were incubated for 10 min with C. albicans and were fixed and stained with DAPI (blue; nucleus) and for F-actin (red; highlighting sites of phagocytic cup formation), VAMP3, TNFα, and transferrin receptor (TfnR). Top panels show phase-contrast images to highlight the location of the yeast. Boxed areas are enlarged to show staining at the actin-rich phagocytic cups. Bars (A), 5 μm; (B) 10 μm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody to mouse IL-6 (Serotec), rabbit polyclonal and rat monoclonal anti–mouse TNFα antibodies (Calbiochem and Auspep, respectively), monoclonal antibodies to Stx6 and Vti1b (Translabs; BD Biosciences), monoclonal antibody to TfnR (Zymed Laboratories and Invitrogen), and a polyclonal rabbit antibody to VAMP3 (Abcam).

Techniques: Imaging, Transfection, Software, Incubation, Staining

Journal: The Journal of Cell Biology

Article Title: Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFα

doi: 10.1083/jcb.200612131

Figure Lengend Snippet: Trafficking of IL-6 from recycling endosomes can be regulated independently of other recycling endosome functions. (A) Dual-color live imaging of macrophages transfected with IL-6–GFP (green) and TNFα-mCherry (red). Enlarged images demonstrate the tubulation of a structure containing both TNFα and IL-6 (yellow arrows), with IL-6 alone budding off (white arrows) to form a new vesicle. (B) Dual-color live imaging of the phagocytosis of IgG-opsonized 3-μm latex beads by macrophages transfected with VAMP3-GFP (green) and IL-6–mCherry (red). Frames from the dual-color video were extracted to highlight the movement of vesicles containing VAMP3 (yellow arrow) but not IL-6, budding off a recycling endosome containing both cargoes (white arrows), and trafficking toward the phagocytic cup. The phagocytosing bead is highlighted with asterisks. Boxed areas are magnified at the right. Bars (A), 10 μm; (B) 5 μm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody to mouse IL-6 (Serotec), rabbit polyclonal and rat monoclonal anti–mouse TNFα antibodies (Calbiochem and Auspep, respectively), monoclonal antibodies to Stx6 and Vti1b (Translabs; BD Biosciences), monoclonal antibody to TfnR (Zymed Laboratories and Invitrogen), and a polyclonal rabbit antibody to VAMP3 (Abcam).

Techniques: Imaging, Transfection