Journal: Cell reports

Article Title: A multiplexed in vivo approach to identify driver genes in small cell lung cancer

doi: 10.1016/j.celrep.2023.111990

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: NCI-H146 , ATCC , HTB-173; RRID:CVCL_1473.

Techniques: Plasmid Preparation, Virus, Recombinant, Modification, Labeling, Amplification, Polymer, Staining, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Simple Western, Membrane, DNA Extraction, Purification, Sequencing, Generated, Software

Journal: iScience

Article Title: Tumor microenvironment mimicking 3D models unveil the multifaceted effects of SMAC mimetics

doi: 10.1016/j.isci.2023.106381

Figure Lengend Snippet: SM induces human dendritic cell maturation which is further enhanced by cancer cell necroptosis (A) Schematic representation of the in vitro co-culture assay. moDCs alone (B) or in co-culture with spheroids (tumor cells and hd-fibroblasts) (D and E) were treated with the compound conditions depicted. 72 h post-treatment, cells were harvested and analyzed via flow cytometry. (B) Relative proportion of activated vs non-activated moDC treated alone. (C) TSNE plot depicting islands of phenotypically similar cells based on CD45, CD11c, HLA-DR, CD86, CD83, and PDL1 as markers. Included are the moDCs treated in monoculture, as well as the different co-cultures. Analyzed cells separate into three subpopulations: activated moDC – blue, non-activated moDC - light gray, tumor cells - black. (D) Relative proportion of tumor and moDCs in the different co-cultures. (E) Relative proportion of activated vs non-activated moDCs in co-culture. Error bars, mean ± SD of triplicate independent wells for each condition of at least 2 independent experiments using different donors. ∗∗p value <0.01, by two-tailed t-test. TNFα: 0.1 ng/mL, SM: 1 μM, zVad: 5 μM. (F) Heatmap depicting cytokines measured in the supernatant of spheroids composed of tumor cells with or without CAFs, 72 h post-treatment. Data are averages from 3 independent wells and black squares indicate levels measured below the detection limit. SM: 0.25-1 μM; TNFα: 0.1 ng/mL, zVad: 20 μM.

Article Snippet: The human dermal fibroblasts (hd-fibroblasts - ATCC® PCS-201-010TM) were cultured in Fibroblast Basal Medium (ATCC PCS201030) supplemented 2% heat-inactivated FBS and Fibroblast Growth Kit-Serum Low (ATCC, #PCS-201-041) without HLL Supplement and rh EGF/TGTb1.

Techniques: In Vitro, Co-culture Assay, Co-Culture Assay, Flow Cytometry, Two Tailed Test

Journal: iScience

Article Title: Tumor microenvironment mimicking 3D models unveil the multifaceted effects of SMAC mimetics

doi: 10.1016/j.isci.2023.106381

Figure Lengend Snippet: SM modulates fibroblasts by downregulating myofibroblast-like CAF markers and upregulating pro-inflammatory soluble mediators CAF 2D monoculture was treated with increasing concentrations of SM or TGFβ. (A) Fold change in FAP and αSMA expression, analyzed via flow cytometry, 72 h post-treatment. (B) CAF confluence monitored overtime using IncuCyte. Error bars, mean ± SD of 3 independent experiments. (C) Heatmap depicting fold change in cytokine and chemokine concentration in the supernatant of treated CAFs (normalized to control). Results from 2 independent experiments. Black squares indicate levels measured below the detection limit.

Article Snippet: The human dermal fibroblasts (hd-fibroblasts - ATCC® PCS-201-010TM) were cultured in Fibroblast Basal Medium (ATCC PCS201030) supplemented 2% heat-inactivated FBS and Fibroblast Growth Kit-Serum Low (ATCC, #PCS-201-041) without HLL Supplement and rh EGF/TGTb1.

Techniques: Expressing, Flow Cytometry, Concentration Assay, Control

Journal: iScience

Article Title: Tumor microenvironment mimicking 3D models unveil the multifaceted effects of SMAC mimetics

doi: 10.1016/j.isci.2023.106381

Figure Lengend Snippet: Key resources table

Article Snippet: The human dermal fibroblasts (hd-fibroblasts - ATCC® PCS-201-010TM) were cultured in Fibroblast Basal Medium (ATCC PCS201030) supplemented 2% heat-inactivated FBS and Fibroblast Growth Kit-Serum Low (ATCC, #PCS-201-041) without HLL Supplement and rh EGF/TGTb1.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software, Simple Western, High Content Screening

Journal: Cell

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

doi: 10.1016/j.cell.2023.06.005

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

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

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

Journal: Cell

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

doi: 10.1016/j.cell.2023.06.005

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

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

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

Journal: Cell

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

doi: 10.1016/j.cell.2023.06.005

Figure Lengend Snippet:

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

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

Journal: STAR Protocols

Article Title: Examining PI3K-signaling-dependent regulation of lens organelle free zone formation via immunolocalization and immunoblotting in chick embryos

doi: 10.1016/j.xpro.2023.102569

Figure Lengend Snippet:

Article Snippet: Goat anti-mouse IgG HRP conjugate , Bio-Rad , 1706516.

Techniques: Recombinant, Electron Microscopy, Western Blot, BIA-KA, Software, Simple Western, Microscopy, Electrophoresis, Plasmid Preparation, Chromatography, Cell Culture, Blocking Assay

Journal: Cell reports

Article Title: PAI-1 uncouples integrin-β1 from restrain by membrane-bound β-catenin to promote collagen fibril remodeling in obesity-related neoplasms

doi: 10.1016/j.celrep.2024.114527

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-SPARC , Cell Signaling Technology , Cat# 5420S; RRID: AB_10692794.

Techniques: Recombinant, Membrane, Cell Culture, Reverse Transcription, SYBR Green Assay, Clinical Proteomics, Protein Extraction, Extraction, Bicinchoninic Acid Protein Assay, In Situ, Blocking Assay, Migration, shRNA, Control, Software, Pyromark Assay, Western Blot, Simple Western

Journal: Cell reports

Article Title: PAI-1 uncouples integrin-β1 from restrain by membrane-bound β-catenin to promote collagen fibril remodeling in obesity-related neoplasms

doi: 10.1016/j.celrep.2024.114527

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-Pan-TEAD , Cell Signaling Technology , Cat# 13295S; RRID: AB_2687902.

Techniques: Recombinant, Membrane, Cell Culture, Reverse Transcription, SYBR Green Assay, Clinical Proteomics, Protein Extraction, Extraction, Bicinchoninic Acid Protein Assay, In Situ, Blocking Assay, Migration, shRNA, Control, Software, Pyromark Assay, Western Blot, Simple Western

Journal: Cell Reports Medicine

Article Title: Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis

doi: 10.1016/j.xcrm.2020.100056

Figure Lengend Snippet: Transcriptome Analysis Identifies a Common Metabolic Signature in Human Primary Cells Treated with TGF-β (A) Schematic overview of RNA-seq analyses of four human primary cell types with TGF-β treatment. 3 independent replicates were used for each time point collection. (B) RT-PCR analysis of αSMA gene expression in human primary cells upon TGF-β treatment (5 ng/mL for 24 h), which was almost completely reversed by the presence of ALK5 inhibitor, SB-525334 (10 μM). n = 3, Mean ± SD. (C) Common effects of TGF-β across four cell types. Shown are the 1,603 genes that met the ±1.2-fold change and FDR_BH p < 0.1 threshold at 24 h in all four cell types. The color gradient represents fold change compared to vehicle-treated cells (−3.0 to 3.0-fold). (D) GO term enrichment analysis of the 1,603 genes using the PANTHER enrichment test ( http://pantherdb.org ). (E) Gene expression ratios of fatty acid oxidation enzymes in human primary cells and fibrotic tissues. From left: cardiac fibroblasts, hepatic stellate cells (HSCs), NHLF, RPTEC, UUO kidney, CCl4 liver, and BDL liver. Heatmap was generated by using Morpheus software ( https://software.broadinstitute.org/morpheus/ ). The color gradient represents fold change compared treatment (TGF-β 24 h, surgery, or CCl4 treatment) to control (−8.5 to 8.5-fold). Gray, below detection limit. ∗Peroxisomal enzymes.

Article Snippet: The next day, 5ng/ml of recombinant human TGFβ1 (BioLegend, #580702) was added to the culture medium and cells were incubated at 37°C for the indicated time.

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Generated, Software, Control

Journal: Cell Reports Medicine

Article Title: Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis

doi: 10.1016/j.xcrm.2020.100056

Figure Lengend Snippet:

Article Snippet: The next day, 5ng/ml of recombinant human TGFβ1 (BioLegend, #580702) was added to the culture medium and cells were incubated at 37°C for the indicated time.

Techniques: Transduction, Virus, Ligation, Recombinant, Saline, Injection, Protease Inhibitor, Mass Spectrometry, Staining, cDNA Synthesis, Extraction, RNA Library Preparation, Enzyme-linked Immunosorbent Assay, Software, Simple Western