Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Cell & Bioscience

Article Title: KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

doi: 10.1186/s13578-019-0344-5

Figure Lengend Snippet: Cytokinetic phenotype in Kif5b knockdown ATDC5 cells. a Western blot of protein extracts from single cell clones with stable expression of sh-ctl or sh-kif5b constructs. b Immunofluorescence of KIF5B (green) in sh-Kif5b clone #4. Scale bar: 10 μm. c sh-ctl and sh-Kif5b cells stained with α-tubulin (red) and DAPI (blue). Yellow asterisks denote typical binucleated cells. Scale bar: 20 μm. d Quantification of the bi- and multi-nucleation rate in control cell clones ( n = 3, sh-ctl clone #1–3) and Kif5b-knockdown cell clones ( n = 5, sh-Kif5b clone #4–8). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. e Re-introduced GFP tagged full length KIF5B reduced the bi- and multi-nucleation rate in Kif5b knockdown ATDC5 cells clone #4 ( n = 10 independent experiments) and #5 ( n = 5 independent experiments). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. f Time-lapse images of sh-ctl and sh-Kif5b cells in mitosis. Scale bar: 10 μm. g Quantification of duration of cytokinesis in sh-ctl cells ( n = 84 cells from sh-ctl clone #1–3) and sh-Kif5b cells ( n = 68 cells from sh-Kif5b clone #4, #5 and #8). *** P < 0.0001; two-tailed Mann–Whitney U -test. The whisker plot shows median (lines), interquartile range (boxes) and 5% to 95% percentile (whiskers). Duration of cytokinesis was calculated from the furrow ingression to the final separation of the two daughter cells. Cells fusing back were not included in this analysis

Article Snippet: Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLightTM Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes).

Techniques: Knockdown, Western Blot, Clone Assay, Expressing, Construct, Immunofluorescence, Staining, Control, Two Tailed Test, MANN-WHITNEY, Whisker Assay

Journal: Cell & Bioscience

Article Title: KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

doi: 10.1186/s13578-019-0344-5

Figure Lengend Snippet: Midbody microtubule organization is impaired in Kif5b deficient cells. a Immunofluorescence of α-tubulin in primary chondrocytes in late cytokinesis. Scale bar: 10 μm. b Quantification of tubulin intensity in midbody in primary chondrocytes ( Kif5b fl/ + cells: n = 24; Col2cre ; Kif5b fl/ − cells: n = 27). *** P < 0.0001; two-tailed Mann–Whitney U -test. The whisker plot shows median (lines), interquartile range (boxes) and 10% to 90% percentile (whiskers). c Electron micrographs of midbody regions from sh-ctl and sh-Kif5b ATDC5 cells. Red lines denote the region of Flemming body. Yellow arrows and boxes denote the broken regions in Flemming body. Scale bar: 0.5 μm. d Quantification of the length of Flemming body in sh-ctl and sh-Kif5b cells (sh-ctl cells: n = 12; sh-Kif5b cells: n = 9). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. e Live imaging of sh-ctl and sh-Kif5b cells expressing GFP-tubulin in cytokinesis. Scale bar: 10 μm. f Quantification of the bi- and multi-nucleation rate in sh-Kif5b cells transiently expressing GFP, GFP-Kif5b and GFP-Kif5bΔMT ( n = 5 independent experiments). *** P < 0.0001; NS, P = 0.4039; unpaired two-tailed t-test. Data are mean ± S.D. g A model showing the function of KIF5B in cytokinesis. In late cytokinesis, KIF5B alone or together with other unknown molecules cross-links microtubules in the midbody, and therefore the structure of midbody can be stably maintained

Article Snippet: Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLightTM Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes).

Techniques: Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Whisker Assay, Imaging, Expressing, Stable Transfection

Journal: PLOS Pathogens

Article Title: Genomes of the autonomous parvovirus minute virus of mice induce replication stress through RPA exhaustion

doi: 10.1371/journal.ppat.1011203

Figure Lengend Snippet: (A) Schematic of the timeline of Single-Molecule DNA Fiber Assays during MVM infection. Mouse A9 fibroblasts are synchronized in Isoleucine deficient media for 42 hours before being released into complete DMEM and concurrently infected with MVMp at an MOI of 25 or treated with hydroxyurea (HU) at a concentration of 2μM. At the indicated timepoints post-infection (12, 18, and 24 hours post infection), cells were sequentially pulsed with IdU and CldU and processed for DFA. (B, C) Each datapoint represents the length of a single IdU and/or CldU labelled DNA fiber. The median length of many measurements of IdU and CldU labelled fibers are represented by red and green horizontal bars respectively. The experiment was performed as described by the schematic depicted in (A). At least 150 individual fibers were measured for each condition. Similar results were obtained for three independent biological replicates of MVM infection. Statistical significance was determined by Mann Whitney Wilcoxon test, **** represents P ≤ 0.0001, ns represents not statistically significant. (D) Representative DFA images of single fibers in Mock infected A9 cells (top panel) compared with MVM infected A9 cells at 24 hpi (bottom panel) with the respective measurements of the IdU and CldU lengths indicated in white text. (E) Western blot showing the levels of NS1 and γH2AX for viral replication and DNA damage, respectively, over the time-course of MVM infection. The respective band intensities are shown below the sample. The top row of γH2AX is taken with a 10 minute exposure (short) and the bottom with a 20 minute exposure (long). Cells pulsed with Hydroxyurea at 2μM concentration is a positive control for DNA damage, as has been previously described . Tubulin serves as the loading control for the Western blots. (F) Categorization of DNA fiber types as percentages out of a total of 100% as determined by presence of IdU or CldU, divided into percentages that are progressing replication forks (black), stalled replication forks (red) and new origins (green with black stripes). (G,H) Non-synchronous U2OS cells were infected with wild-type MVMp or the NS2-deficient (MVM ΔNS2 ) mutant of MVM at an MOI of 25 for 24 hours before being pulsed with IdU/CldU as described in the schematic in (A), and processed for imaging. At least 150 individual fibers were measured for each condition. Similar results were obtained for three independent biological replicates of MVM infection. Statistical significance was determined by Mann Whitney Wilcoxon test, **** represents P ≤ 0.0001.

Article Snippet: Male murine A9 fibroblasts and female human U2OS osteosarcoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose; Gibco) supplemented with 5% Serum Plus (Sigma Aldrich) and 50 μg/ml gentamicin (Gibco).

Techniques: Infection, Concentration Assay, MANN-WHITNEY, Western Blot, Positive Control, Mutagenesis, Imaging

Journal: PLOS Pathogens

Article Title: Genomes of the autonomous parvovirus minute virus of mice induce replication stress through RPA exhaustion

doi: 10.1371/journal.ppat.1011203

Figure Lengend Snippet: (A) Schematic of the Single-Molecule DNA Fiber Assays using the CDC7 inhibitor PHA 767491 (Millipore Sigma). Mouse A9 fibroblasts were synchronized in Isoleucine deficient media for 42 hours before being released into complete DMEM media and concurrently infected with MVMp at an MOI of 25. At the indicated 20 hpi timepoint, 5μM of iCDC7 was added to the cells for 4 hours. At 24 hpi, MVM infected iCDC7 pulsed cells were sequentially pulsed with IdU and CldU for 20 minutes each before being processed for DFA analysis. (B, C) Each datapoint represents the length of a single IdU and/or CldU labelled DNA fiber. The median length of many measurements of IdU and CldU labelled fibers are represented by red and green horizontal bars respectively. The experiment was performed as described by the schematic depicted in (A). At least 150 individual fibers were measured for each condition. Similar results were obtained for three independent biological replicates of MVM infection. Statistical significance was determined by Mann Whitney Wilcoxon test, **** represents P ≤ 0.0001. (D) Categorization of DNA fiber types as percentages out of a total of 100% as determined by presence of IdU or CldU, divided into percentages that are progressing replication forks (black), stalled replication forks (red) and new origins (green with black stripes). (E) Western blot showing the levels of NS1 and γH2AX for viral replication and cellular DNA damage, respectively, upon pulsing with iCDC7 as shown in the schematic in (A) prior to MVM infection. Activity of the CDC7 inhibitor activity was verified by phospho-MCM2 levels and Tubulin was served as loading control for the western blots. The respective band intensities are shown below the sample. (F) Samples from lanes 3 and 4 in panel (E) were processed for formation of viral replication centers by NS1 staining (red) and phospho MCM2 levels (green) measured for CDC7 inhibitor activity. Cells infected with MVM at 24 hpi under mock-treated conditional (top panel) were compared to those pulsed with iCDC7 (bottom panel) by NS1 staining (red). White broken line demarcates the nuclear border, which was identified by DAPI staining (blue). The white scalebar represents 10 microns.

Article Snippet: Male murine A9 fibroblasts and female human U2OS osteosarcoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose; Gibco) supplemented with 5% Serum Plus (Sigma Aldrich) and 50 μg/ml gentamicin (Gibco).

Techniques: Infection, MANN-WHITNEY, Western Blot, Activity Assay, Staining

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Integrated multiomic analysis identifies TRIP13 as a mediator of alveolar epithelial type II cell dysfunction in idiopathic pulmonary fibrosis

doi: 10.1016/j.bbadis.2024.167572

Figure Lengend Snippet: Key resources.

Article Snippet: Mouse IgG1 anti-human CD326 (EpCAM)-conjugated microbeads , Miltenyi Biotec , Cat# 130–061–101 RRID: AB_2832928.

Techniques: Plasmid Preparation, Magnetic Beads, Recombinant, Blocking Assay, Lysis, Protease Inhibitor, Red Blood Cell Lysis, Software, Microscopy, Fluorescence, Imaging

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 2. Neuronal activity bidirectionally modulates the phosphorylation state of Shank3. (A) The experiment protocol for extraction of Shank3 from rat cultured neocortical neurons for further quantitative mass spectrometry (MS) or Western blot analyses. (B) Volcano plot of quantitative MS data showing Shank3 residues that were differentially phosphorylated in tetrodotoxin (TTX)-treated samples compared to untreated controls. The log2 values of fold changes, if below zero, indicated hypophosphorylation (paired t-test: S1586, adjusted p=0.034142, S1614/5, 0.014444). (C) Top: diagram showing the location of S1586 and S1615 within the rat Shank3 protein. Functional domains: ANK = ankyrin repeat; SH3 = SRC homology 3; PDZ = PSD-95/Disc Large/ZO-1; Pro-rich = proline rich; SAM = sterile alpha motif. Bottom: homology comparison of sequences flanking rat S1586 and S1615 (matching mouse S1539) across species (human Shank3: NP_001358973.1; rat Shank3: NP_067708.2; mouse Shank3: UniprotKB: Q4ACU6.3). Phosphosites of interest are labeled in red; the only residue not conserved is shown in blue. (D, E) Representative Western blot using an antibody specific for phosphorylated S1615, showing changes in Shank3 phosphorylation after 10 min (D) or 24 hr (E) treatment with TTX or picrotoxin (PTX). (F) Quantification of the fold change of Shank3 S1615 phosphorylation in (D). Dashed line indicates the baseline untreated control (one-sample t-test: TTX, ***p=0.0005, PTX, **p=0.0035, n = 5 and 10 biological replicates, respectively). (G) Quantification of the fold change of Shank3 S1615 phosphorylation in (E) (one-sample t-test: TTX, ****p<0.0001, PTX, p = 0.6336, n = 7 and 7 biological replicates, respectively). Solid colored horizontal

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay, Phospho-proteomics, Extraction, Cell Culture, Mass Spectrometry, Western Blot, Functional Assay, Sterility, Comparison, Labeling, Residue, Control

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 3. Phosphorylation state modulates activity-dependent changes in the synaptic enrichment of Shank3. (A) Representative images of synaptic puncta colocalized with surface GluA2 (sGluA2) and Shank3 in neuron dendrites ± tetrodotoxin (TTX) (scale bar = 5 µm). (B) Quantification of synaptic sGluA2 intensity changes induced by scaling up and down protocols (number of neurons: untreated, n = 77, TTX, n = 40, picrotoxin [PTX], n = 29; Kruskal–Wallis test with post-hoc Dunn’s multiple comparison tests: Un vs. TTX, **p=0.0034, Un vs. PTX, *p=0.0408, TTX vs. PTX, ****p<0.0001). (C) Quantification of synaptic Shank3 intensity during scaling up and down protocols (Kruskal–Wallis test with post-hoc Dunn’s tests: Un vs. TTX, *p=0.0155, Un vs. PTX, *p=0.0205, TTX vs. PTX, ****p<0.0001). (D) Representative images of synaptic localization of wild-type Shank3 and Shank3 phospho-mutants (scale bar = 5 µm). (E) Quantification of synaptic intensity of Shank3 phospho-mutants (number of neurons: WT, n = 33, AA, n = 30, DD, n = 24; Kruskal–Wallis test with post-hoc Dunn’s tests: WT vs. AA, p>0.9999, WT vs. DD, *p=0.0395, AA vs. DD, **p=0.0039). (F) Quantification of the density of synaptic puncta containing Shank3 phospho-mutants (number of neurons: WT, n = 32, AA, n = 30, DD, n = 24; Kruskal–Wallis test: p=0.2814). For imaging experiments here and below, each data point represents a single pyramidal neuron, and data were collected from at least four independent experiments. Also see Figure 3—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Phospho-proteomics, Activity Assay, Comparison, Imaging

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 4. Increased PP2A activity maintains tetrodotoxin (TTX)-induced Shank3 hypophosphorylation. (A) Diagram showing the potential roles of kinases and phosphatases in regulating activity-dependent Shank3 phosphorylation. (B) Representative Western blot showing the impacts of inhibiting CAMKII (KN62, KN93) or PKA (H89) on Shank3 phosphorylation at baseline and upon TTX treatment. (C) Quantification of S1615 phosphorylation in (B) (two-way ANOVA with post-hoc Tukey’s test: DMSO vs. KN62, p>0.9999, DMSO vs. KN93, p=0.8148, DMSO vs. H89, p=0.9112, DMSO vs. picrotoxin (PTX), *p=0.0406, PTX vs. PTX/KN62, **p=0.0040, PTX vs. PTX/KN93, ****p<0.0001, PTX vs. PTX/H89, ****p<0.0001, n = 5 biological replicates). Dashed line indicates the DMSO control. (D) Quantification of PP2A activity after 1 hr TTX treatment (Un, n = 5, TTX, n = 5; paired t-test: **p=0.0018). (E) Quantification of PP2A activity after 24 hr TTX treatment (Un, n = 7, TTX, n = 7; paired t-test: *p=0.0129). (F, G) Western blot analyses showing changes in S1615 phosphorylation after 1 hr (F) or 24 hr (G) TTX treatment, with inhibition of PP2A by okadaic acid (OKA, 50 nM) during the

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Control, Inhibition

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 5. PP2A activity is required for tetrodotoxin (TTX)-induced synaptic enrichment of Shank3. (A) Representative images of synaptic enrichment of endogenous Shank3 upon treatment with TTX and PP2A inhibitor fostriecin (FST) (scale bar = 10 µm). (B) Quantification of synaptic Shank3 intensity in (A) (number of neurons: DMSO, n = 26, FST, n = 28, TTX, n = 28, TTX/FST, n = 29; Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. FST, p>0.9999, DMSO vs. TTX, ***p=0.0002, FST vs. TTX/FST, p=0.1259, TTX vs. TTX/FST, p=0.1292). (C) Quantification of density of synapses containing Shank3 in (A) (Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. FST, p=0.9458, DMSO vs. TTX, **p=0.0051, FST vs. TTX/FST, p=0.2446, TTX vs. TTX/FST, *p=0.0273). (D) Representative images of synaptic enrichment of endogenous Shank3 upon treatment with TTX and PP1 inhibitor tautomycetin (TAUT) (scale bar = 10 µm). (E) Quantification of synaptic Shank3 intensity in (D) (number of neurons: DMSO, n = 26, TAUT, n = 21, TTX, n = 28, TTX/ TAUT, n = 32; Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. TAUT, *p=0.0315, DMSO vs. TTX, ***p=0.0006, TAUT vs. TTX/TAUT, ***p=0.0002, TTX vs. TTX/TAUT, *p=0.0392). (F) Quantification of density of synapses containing Shank3 in (D) (Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. TAUT, p=0.2450, DMSO vs. TTX, *p=0.0116, TAUT vs. TTX/TAUT, p=0.6552, TTX vs. TTX/TAUT, ***p=0.0007). Also see Figure 5—figure supplement 1 and Figure 5—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 6. Changes in the phosphorylation state of Shank3 are crucial for bidirectional synaptic scaling. (A, B) Representative miniature excitatory postsynaptic current (mEPSC) recordings from neurons overexpressing Shank3 WT (A) or DD mutant (B) during scaling up. (C) Quantification of average mEPSC amplitude in (A) (WT, n = 8, WT + tetrodotoxin [TTX], n = 9; unpaired two-tailed t-test: **p=0.0074). (D) Quantification of average mEPSC amplitude in (B) (number of neurons: DD, n = 12, DD + TTX, n = 14; unpaired two-tailed t-test: p=0.5708). (E, F) Representative traces of mEPSCs recorded from neurons overexpressing Shank3 WT (E) or AA mutant (F) during scaling down. (G) Quantification of average mEPSC amplitude in (E) (number of neurons: WT, n = 8, WT + bicuculline [BIC], n = 8; Mann–Whitney test: *p=0.0148). (H) Quantification of average mEPSC amplitude in (F) (AA, n = 9, AA + BIC, n = 14; unpaired two-tailed t-test: p=0.8612). Also see Figure 6—figure supplement 1, Figure 6—figure supplement 2, and Figure 6—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Phospho-proteomics, Mutagenesis, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 7. Brief PP2A inactivation reverses scaling up. (A) Representative images showing the effects of 1 hr fostriecin (FST) treatment on synaptic sGluA2 intensity in neurons expressing Shank3 WT or AA, after 24 hr of tetrodotoxin (TTX) to scale up synaptic strengths (scale bar = 10 µm). (B) Quantification of synaptic sGluA2 intensity in (A) (number of cells: WT/TTX, n = 22, WT/TTX/FST, n = 23, AA/TTX, n = 26, AA/TTX/FST, n = 25; Mann–Whitney test: WT/TTX vs. WT/TTX/FST, ***p=0.0007, AA/TTX vs. AA/TTX/FST, p=0.3739). (C) Quantification of synaptic Shank3 intensity in (A) (Mann–Whitney test: WT/TTX vs. WT/TTX/FST, **p=0.0090, AA/TTX vs. AA/TTX/FST, p=0.7296). (D) Quantification of the density of puncta containing sGluA2 and Shank3 (Mann–Whitney test: WT/TTX vs. WT/TTX/FST, **p=0.0016, AA/TTX vs. AA/TTX/FST, p=0.7017). Each data point indicates a cell, and the total number (n) was pooled from five independent experiments. Also see Figure 7—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Expressing, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Schematic representation of luciferase screening approach. Upper panel shows the four target predictions software used for in silico analysis. Bottom panel indicates the main steps performed in the high‐throughput screening. B Upper panel, sequence interaction of miR‐182‐3p with the target site of the wild type 3′UTR of TRF2 in human. Bottom panel, generation of mutant 3′UTR of TRF2 luciferase construct containing the deletion of target site for miR‐182‐3p. C–E Luciferase reporter assay in HeLa cells using the synthetic miR‐Control or miR‐182‐3p in combination with the wild type (C) or the mutant 3′UTR of TRF2 construct (D) or the wild type 3′UTR of TRF1 (E). F, G Western blotting for TRF2 expression in telomerase‐positive (HeLa, HCT116, MDA‐MB‐231, MDA‐MB‐436) and ALT‐positive (U2‐OS, Saos‐2) cells transiently transfected with miR‐Control or miR‐182‐3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control. H U2‐OS cells transiently transfected with the miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor were assayed by quantitative immunofluorescence for TRF2 3 days post‐transfection. Left panel, representative images. Scale bar: 10 μm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N = number of analyzed nuclei. Red bar indicates mean value. I U2‐OS cells transfected as described in (H) were assayed by immunofluorescence combined with telomeric FISH. Left panel, representative images of co‐localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10 μm. Right panel, co‐localizations were analyzed using ImageJ software. N = number of analyzed nuclei. Data information: For (C–G and I), data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by Student's t ‐test; for (H), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Luciferase, Software, In Silico, High Throughput Screening Assay, Sequencing, Mutagenesis, Construct, Reporter Assay, Western Blot, Expressing, Transfection, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Results of high‐throughput luciferase screening performed in Hela cells using the wild type 3′UTR‐TRF2 vector in combination with each of the 54 miRNAs selected by in silico analysis. Three days post‐transfection, luciferase ratio (Renilla:Firefly) of each miRNA was calculated, the control miRNA was set “1.” Renilla:Firefly ratios < 1 indicate target specificity of candidate miRNAs for the 3′UTR of TRF2. miRNAs near to the ratio of 0.5 were considered for further analysis. Two biological replicates were performed. B HeLa cells transiently transfected with the indicated miRNAs (miR‐Control, miR‐182‐3p, miR‐519e‐5p, miR‐296‐3p) were assayed by western blotting. Upper panel, quantification of TRF2 expression. Bottom panel, representative images of TRF2, TRF1 and RAP1 are shown, actin was used as loading control. C Analysis of TRF2 mRNA expression performed by qPCR in four different cancer cell lines (HeLa, MDA‐MB‐231, MDA‐MB‐436, U2‐OS) 3 days post‐transfection with miR‐Control or miR‐182‐3p. The control miRNA was set “1.” Three independent experiments were performed. D, E Telomeric ChIP assay in MDA‐MB‐231 (D) and U2‐OS cells (E). Quantification of TRF2 enrichment at telomeric repeats, in the different conditions, is shown in the table under the respective figure. Alu probe and Rabbit IgG were used as negative control for the assay. Data information: For (A), data are presented as mean values. For (B, C), data are presented as mean values ± SD and Student t‐ test was used to calculate statistical significance. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: High Throughput Screening Assay, Luciferase, Plasmid Preparation, In Silico, Transfection, Western Blot, Expressing, Negative Control

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A MDA‐MB‐231 cells were transiently transfected with the indicated miRNAs or siRNA. The indicated DNA damage markers were assayed by western blotting. Actin was used as loading control. B Telomeric DNA FISH performed in MDA‐MB‐231 transiently transfected with the indicated miRNAs. Telomere length was measured by TLF software and indicated as arbitrary fluorescence unit (a.f.u). N = number of analyzed nuclei. Black bar indicates mean value. C DNA damage markers were assayed by western blotting in HeLa cells. Actin was used as loading control. D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. Co‐localizations of γH2AX with telomeres are indicated as mean number of TIFs per nucleus. E Representative images and enlargements of co‐localizations of experiment described in D. F Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. G Representative images of co‐localizations relative to the experiment described in (F). H, I MDA‐MB‐231 and HeLa cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with miR‐Control or miR‐182‐3p. TRF2, pATM and γH2AX expression were assayed by western blotting. Actin was used as loading control. Data information: For (D) and (F), data are presented as mean values ± SD. Three independent replicates were performed. Scale bar: 10 μm. At least 60 nuclei were analyzed in (D) and (F). A Student t‐ test was used to calculate statistical significance. For (B), P values are determined by Mann–Whitney t ‐test. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Western Blot, Software, Fluorescence, Immunofluorescence, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analyzed. Representative images and enlargements of co‐localizations (white arrowheads) relative to the experiment described in (A). Scale bar: 10 μm. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images of co‐localizations (white arrowheads) relative to the experiment described in (C). Scale bar: 10 μm. Quantification of TIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified. Representative images and enlargements relative to the experiment described in (E). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Quantification of PIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images relative to the experiment described in (G). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Data information: For (A, C, E, G) data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. At least 60 nuclei were analyzed for each experimental condition. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐436 and MDA‐MB‐231 cells underwent two rounds of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24 h up to a maximum of 3 days. The percentage of cell confluence was analyzed. C, D Cell number of MDA‐MB‐436 (C) and MDA‐MB‐231 (D) cells and TRF2 expression were analyzed by automatic cell count and by western blotting at the end of the experiment described in (A) and (B). Actin was used as loading control. E Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐436 at the end of the second cycle of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Red boxes indicate early and late apoptotic cells. F Quantification of Annexin V‐positive cells (%) of experiment described in (E). G Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐231 as described in (E). H Quantification of Annexin V‐positive cells (%) of experiment described in (G). I, J MDA‐MB‐436 cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72 h post‐transfection. Data information: For (A, B) data are shown as mean ± SEM. For (C, D, F, H, I, J), data are shown as mean ± SD. For (A–D) and (I), three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. For (F), (H) and (J), two different biological replicates were performed. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Cell Counting, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Western blotting for TRF2 expression in BJ cells transiently transfected with miR‐Control or miR‐182‐3p. The graph represents the quantification of three independent experiments. Representative images are shown, Actin was used as loading control. Unspecific bands are indicated with (*). B, C Mean of γH2AX foci per nucleus was analyzed in BJ cells 72 h post‐transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in (C). D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72 h post‐transfection with the indicated miRNAs. Left panel: The mean number of TIFs per nucleus was analyzed. Right panel: Representative images and enlargements of co‐localizations. E Cell number of BJ cells was analyzed by automatic cell count at the end of the second round of transfection with miR‐Control or miR‐182‐3p. F FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in (E). G β‐Galactosidase assay in BJ cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. H–J IL‐6 (H), CXCL1 (I), IL‐8 (J) factors were analyzed by ELISA to evaluate the senescence‐associated secretory phenotype (SASP) in BJ cells treated as indicated in (G). Data information: For (A, B, D, E and G–J), a student t‐ test was used to calculate statistical significance. Scale bars (10 μm). P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Cell Counting, Staining, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A TRF2 and γH2AX expression after two rounds of transfection with the indicated miRNAs, was analyzed by western blotting in MCF10A cells. Actin was used as loading control. B–E The mean number of γH2AX foci (B) and TIFs (D) per nucleus were analyzed 72 h post‐transfection with the indicated mimic miRNAs in MCF10A cells. Representative images (C) and (E) are referred to the experiment showed in (B) and (D) respectively. F, G Cell confluence (F) of MCF10A was monitored by Incucyte, every 24 h starting from the day of the second transfection, and cell number (G) was counted at the end of experiment (day 4). H–I Cell cycle progression analysis by PI staining (H) and cell death analysis by Annexin V assay (I) were performed in MCF10A upon two rounds of transfection with the indicated miRNAs. J β‐Galactosidase assay in MCF10A cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. Data information: Panels (B, D, F, G, J) data are presented as mean values ± SD. A Student t‐ test was used to calculate statistical significance. P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Transfection, Western Blot, Staining, Annexin V Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐231 (A) and MDA‐MB‐436 (B) tumor xenografts were treated with LNPs‐empty, LNPs‐miR‐Control or by LNPs‐miR‐182‐3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20 μg of LNPs‐miR‐Control, LNPs‐miR‐182‐3p or equivalent volume of LNPs‐empty as indicated in the scheduling. The mean of tumor volumes ( n = 5 per group) is shown. C, D Tumors from mice treated in (A) and (B) were processed to measure miR‐182‐3p expression by TaqMan qPCR. E Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA‐MB‐231 human breast cancer xenografts. Scale bar: 50 μm. F The histograms show the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, TUNEL or CD31 staining. The analyses were performed on three mice per group, and the points represent the number of field analyzed for each condition. G, H Luminescent MDA‐MB‐436 cells were injected into the brain and monitored by IVIS imaging system. After 1 week from implant, treatment with LNPs‐miR‐Control and LNPs‐miR‐182‐3p was performed as indicated in (A) and (B). Representative images from in vivo (upper panel) or ex‐vivo (bottom panel) brain tumors are shown in (G). Boxplots (H) show the measurement of photons for each brain tumor ( n = 5 per group) acquired at the indicated times. Data information: For (A, B, F), data are shown as mean ± SD. For (C, D, H), the line in the middle of the box plot denotes a median value, the limits of box represent the interquartile range (25 th to 75 th percentiles), while, the whiskers denote the minimum to maximum values. For (A–D) and (H), P values are determined by unpaired two‐tailed t‐ test; for (F), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, TUNEL Assay, Staining, Injection, Imaging, In Vivo, Ex Vivo, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: The organs (brain, liver, kidney) taken from mice, previously engrafted with MDA‐MB‐231 cells and treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p, were assayed for miR‐182‐3p expression by TaqMan qPCR. Representative images show IHC analysis on tumor samples, from mice bearing MDA‐MB‐436 human breast cancer xenografts, with the indicated markers. Scale bar: 50 μm. The histograms show the expression of TRF2 indicated as immunoreactivity score (IRS) and the percentage of positive cells to γH2AX, TIUNEL or CD31 staining in MDA‐MB‐436 xenografts. Three mice per group were analyzed, the points represent the number of field analyzed for each condition. Data information: For (A, C), data are presented as mean values ± SD. Statistical significance using unpaired (A) or Mann–Whitney t‐ test (C) was calculated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B PDTCs #1 and #2 underwent two rounds of transfection with miR‐Control or miR‐182‐3p. Three days after the second transfection, miR‐182‐3p and TRF2 expression were analyzed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. C, D Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50 μm. At least 85 3D cells were analyzed for each experimental condition. E NSG mice implanted with breast PDTX (#2) were treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p as indicated in the scheduling. Caliper measurement of tumors was taken at the indicated days. The mean of tumor volumes ( n = 5 per group) is shown. F miR‐182‐3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. G Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in (E). Scale bar: 50 μm. H The histograms show the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and TUNEL. The analysis was performed on three mice per group, the points represent the number of field analyzed for each condition. Data information: For (A–F) and (H), data are shown as mean ± SD. For (A–F), P values are determined by unpaired two‐tailed t‐ test; for (H), P values are determined by Mann–Whitney t ‐test. For the experiments showed in (A, B) and (C, D) two or three biological replicates were performed, respectively. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Western Blot, TUNEL Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Representative images of intestine sections from mice previously treated with LNPs‐Empty or LNPs‐miR‐182‐3p. H&E staining (scale bar: 200 μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50 μm). B, C Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX‐positive cells (%) (C) on intestine samples. D Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of skin samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). E, F Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX‐positive cells (%) (F) on skin samples. G Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). H, I Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX‐positive cells (%) (I) on bone marrow samples. Data information: For (B, C, E, F, H, I), data are shown as mean ± SD. A Mann–Whitney test t‐ test was used to calculate statistical significance. Four mice per group were analyzed, the points represent the number of field analyzed for each condition. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Immunofluorescence imaging of immune synapses between H1299 lung cancer cells and γδ T cells by phosphotyrosine (pTyr) staining. H1299 lung cancer cells are pretreated with phosphate- buffered saline (PBS) or DAC prior to coculture with γδ T cells. Quantifications of immune synapses per cancer cell on eight randomly taken high power fields for each treatment are shown in the dot plots (mean ± SD). Scale bar: 100 μm. p value is calculated by the Mann-Whitney test. ( B ) A scatter plot of DAC-induced surface proteomes in H1299 (y-axis) and A549 (x-axis) human lung cancer cells following daily treatment of 100 nM DAC for 72 hours and culture in drug-free medium for 3 days (D3R3). ICAM-1 is among the top upregulated surface proteins by DAC in both cells. ( C ) Western blot analyses of ICAM-1 protein expression in mock-treated vs. DAC-treated human lung cancer cells. D3: daily treatment of 100 nM decitabine for 72 hours. D3R3: daily treatment for 72 hours, followed by a 3-day rest period in drug-free medium. β-actin: loading control. ( D ) Immunofluorescence staining of ICAM-1 and immune synapse molecules (e.g., LFA-1, LAT) at immune synapses formed between γδ T cells and DAC-treated H1299 lung cancer cells. Scale bar: 10 μm. ( E ) Representative flow cytometric dot plot showing H1299 lung cancer cells with CRISPR-knockout of ICAM1 (KO-ICAM1) subject to γδ T cell killing for 2 hours. The effector to target (E: T) ratio is 3:1. Lung cancer cells are pre-treated with mock, DAC alone, γδ T cells alone or a combination of DAC and γδ T cells. The X-axis denotes surface ICAM1 levels. Y-axis represents signal intensities of propidium iodide. ( F ) Bar graphs showing percent cell death of human lung cancer cell lines (i.e., H1299, CL1-0, and A549) with CRISPR-knockout of ICAM-1 subject to γδ T cell killing for 2 hours. Cell death is measured by Annexin V and propidium iodide apoptosis assays (mean ± SEM, n = 3). Statistical significance is determined by one-way ANOVA test. ( G ) Representative flow cytometric dot plot showing H1299 lung cancer cells with a Tet-on expression system of ICAM1 (OV-ICAM1) subject to γδ T cell killing for 2 hours. Doxycycline (1 μg/mL) is added 24 hours prior to coculture to induce ICAM-1 protein expression. Cell death is measured by Annexin V (x-axis) and propidium iodide (y-axis) apoptosis assays. ( H ) Bar graphs showing cell death of human lung cancer cell lines (i.e., H1299, CL1-0, and A549) with ICAM-1 over-expression subject to γδ T cell killing for 2 hours. E:T ratio is 3:1. Cell death is measured by Annexin V and propidium iodide apoptosis assays. Statistical significance is determined by one-way ANOVA test (* p < 0.05, ** p < 0.01, ***, p < 0.001). ( I ) Immunofluorescence imaging of immune synapses between H1299 KO-ICAM1 lung cancer cells and γδ T cells by phosphotyrosine (pTyr) staining. Scale bar: 100 μm. Quantifications of immune synapses per cancer cell on six randomly taken high power fields for each treatment are shown in the dot plots (mean ± SD). p value is calculated by the Mann-Whitney test.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Immunofluorescence, Imaging, Staining, Saline, MANN-WHITNEY, Western Blot, Expressing, Control, CRISPR, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: Sequencing results of the KO-ICAM1 lung cancer cells are aligned against the reference sequence of the ICAM1 genome locus. Alignment gaps are denoted as hyphens (-) to mark the lost (knockout) regions of the edited ICAM1 genome locus.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Sequencing, Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Immunofluorescence staining of F-actin (red), ICAM-1 (green), pTyr (phosphotyrosine, white) at immune synapses between γδ T cells and DAC-pretreated H1299 lung cancer cells at D3R3. Accumulation of F-actin beneath the cell membrane is noted in DAC-pretreated lung cancer cells. DAPI: 4′,6-diamidino-2-phenylindole, as a nuclear counterstain. Scale bar: 10 μm. ( B ) Representative immunofluorescence images of the interfaces between γδ T cells and H1299 lung cancer cells (parental vs. ICAM-1 knockout (KO-ICAM1)). Signals of F-actin (red) in the periphery of H1299 cancer cells are shown in two- and-a-half-dimensional (2.5D) images in the lower panels. Scale bar: 10 μm. ( C ) Dot plots of signal intensities of F-actin (left panel) and ICAM-1 (right panel) from five pTry-positive immune synapses between γδ T cells and H1299 lung cancer cells (parental or KO-ICAM1). p value is calculated by two-way ANOVA test. ( D ) Immunofluorescence images of immune synapses between γδ T cells (marked with T) and H1299 lung cancer cells (marked with C) stained for ICAM-1 (green), F-actin (Red) and phosphotyrosine (pTyr, white). Lung cancer cells (parental or KO-ICAM1) are pretreated with PBS (Mock) or 100 nM DAC and cocultured with γδ T cells at D3R3. ( E ) Dot plots of F-actin signal intensities at immune synapses between γδ T cells and H1299 cells. H1299 cells are pretreated with PBS (Mock), DAC alone or combination of DAC pretreatment (D3R3) and 1 μg/mL Cyto B (cytochalasin B, an inhibitor of actin filament polymerization) for 1.5 hours prior to coculture with γδ T cells (mean ± SD). p value is calculated by one-way ANOVA with Tukey’s multiple comparisons test (***, p < 0.001; ****, p < 0.0001). ( F ) Representative immunofluorescence images of immune synapses (pTyr staining) between γδ T and H1299 cells pretreated with PBS (Mock), DAC alone, and combination of DAC and Cyto B. Blow-up images of the square areas for each treatment are shown in the lower panels. Arrows denote immune synapses between γδ T and H1299 cells. Scale bar: 100 μm (upper) and 20 μm (lower panels). ( G ) Dot plots showing numbers of immune synapses per cancer cell on eight randomly taken high power fields for H1299 cells pretreated with PBS (Mock), DAC, and combination of DAC and Cyto B (mean ± SD). p value is calculated by one-way ANOVA with Tukey’s multiple comparisons test (*, statistical significance).

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Immunofluorescence, Staining, Membrane, Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: Parental or ICAM-1 knockout (KO-ICAM1) H1299 cells are pretreated daily with PBS (Mock) or 100 nM DAC for 72 hours followed by 3-day drug-free culture before coculture with γδ T cells. Signal intensities of each protein (F-actin, red; ICAM-1, green; phosphotyrosine, pTyr, white) along the immune synapse area are graphed on the right. DAPI: 4’,6-diamidino-2-phenylindole, as nuclear counterstain. Scale bar: 10 μm.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Visualization of multi-omics data (i.e., mRNA-seq, Omni-ATAC-seq, and MethylationEPIC arrays) for DAPK3 , EVPLL, and TUBE1 in H1299 lung cancer cells. ( B ) Promoter methylation status and mRNA expression levels of the ICAM1 gene measured by Infinium MethylationEPIC arrays (left panels) and mRNA-seq (right panels) in human lung cancer cells treated without and with DAC 100 nM DAC for 3 days followed by a 3-day drug-free culture. ( C ) Open chromatin regions in the promoter areas of the ICAM1 gene in human lung cancer cells upon 100 nM DAC treatment analyzed by Omni-ATAC-seq. The green bar represents a CpG island. ( D ) Validation of Omni-ATAC-seq by quantitative real-time PCR on transposase-accessible chromatin at the ICAM1 promoter of human lung cancer cells subject to daily treatment of 100 nM DAC treatment for 3 days, followed by a 3-day drug-free culture. Experiments are performed in triplicates, and data are presented as mean ± SD. p value was calculated by unpaired t test (*, p < 0.05). ( E ) IPA Network analysis of mRNA expression changes in human lung cancer cells treated by DAC reveals coordinated changes of the immune-related surface molecules and the cytoskeleton-associated genes. ( F ) IPA upstream regulator analysis of mRNA expression changes in human lung cancer cells treated by DAC. T cell effector cytokines such as TNF-α and IFN-γ may enhance DAC-induced expression changes of immune-related molecules and ICAM-1 in lung cancer cells. TP53 is a potential master regulator for cancer cytoskeleton reorganization essential for DAC-potentiated γδ T cell killing.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Biomarker Discovery, Methylation, Expressing, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Diagram of transcription factor binding sites at the ICAM1 promoter derived from the ENCODE ChIP-seq data ( https://www.encodeproject.org ). Visualizations of ATAC-seq peaks at the ICAM1 promoter in PC9 and CL1-5 lung cancer cell lines subject to DAC treatment are shown above. ( B ) Promoter methylation status and mRNA expression levels of putative transcription factors (i.e., RELB, NFKB2, STATS, and RUNX3) at the ICAM1 promoter in A549, H1299, PC9, and CL1-5 lung cancer cells. Dot and line plots represent methylation levels (β values) of promoter probes measured by Infinium MethylationEPIC arrays. The promoter probes with β values greater or equal to 0.5 at baseline (Mock) are shown. Bar graphs represent relative mRNA expression levels based on normalized FPKM measured by mRNA-seq.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Binding Assay, Derivative Assay, ChIP-sequencing, Methylation, Expressing

Journal: Scientific Reports

Article Title: P-Selectin preserves immune tolerance in mice and is reduced in human cutaneous lupus

doi: 10.1038/srep41841

Figure Lengend Snippet: ( a) Relative frequency of peripheral blood leukocyte populations of 1.5–3 month-old WT and P-Sel −/− mice. ( b , d ) Percentage of IL-10 + conventional dendritic cells (cDC), plasmacytoid DC (pDC), monocytes, granulocytes and B cells; and frequency of IL-10 and IL-17 producing CD4 + and CD8 + T lymphocytes, in 1.5-month-old ( b ) and >18-month-old ( d ) WT and P-Sel −/− mice. ( c) Representative dot plots of IL-10 + cDCs and IL-10 and IL-17 producing CD4 + T cells in 1.5-months old WT and P-Sel −/− mice. ( e) Phenotyping of CD4 + and CD8 + splenic T lymphocytes according to the expression of the naïve/memory/effector markers CD62L and CD44 in 1.5–3 months-old (upper panels) and >18 month-old (lower panels) WT and P-Sel −/− mice. ( f) Representative dot plots showing the distribution of 1.5–3-month-old mice splenic populations according to the expression of L-Selectin and CD44. In all cases, n = 4 mice per group. In all cases, n = 4 mice per group. Bars represent the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.005, by Student’s two tailed t test.

Article Snippet: Consecutive tissue sections of sun-exposed skin biopsies underwent immunohistochemistry with the Dako REAL EnVision Detection System Peroxidase/DAB+kit (Dako), using as primary antibodies anti-human P-Selectin (10 μg/ml) (R&D Systems; Minneapolis, MN, USA) and anti-human CD31 (1/50) (Abcam).

Techniques: Expressing, Two Tailed Test

Journal: Scientific Reports

Article Title: P-Selectin preserves immune tolerance in mice and is reduced in human cutaneous lupus

doi: 10.1038/srep41841

Figure Lengend Snippet: ( a) Representative photomicrographs (20×) of anti-CD31 and anti-P-Selectin stained skin biopsies of healthy donors and SLE patients (upper panels). Black arrows point blood vessels. ( b) 200% magnification of representative blood vessels from the original images are represented (lower panels). ( c) Classification and quantification of CD31+ dermal blood vessels according to the expression level of P-Selectin (healthy controls, n = 4; SLE patients, n = 4); bars show the mean ± SD. **p < 0.01 by Student’s two tailed t test.

Article Snippet: Consecutive tissue sections of sun-exposed skin biopsies underwent immunohistochemistry with the Dako REAL EnVision Detection System Peroxidase/DAB+kit (Dako), using as primary antibodies anti-human P-Selectin (10 μg/ml) (R&D Systems; Minneapolis, MN, USA) and anti-human CD31 (1/50) (Abcam).

Techniques: Staining, Expressing, Two Tailed Test