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

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 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: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Structure prediction of TMEM95 protein (left) using IZUMO1 (right) as template, created by SWISS-MODEL software. ( B ) Tmem95 KO allele generated following CRISPR-mediated edition. CRISPR target sequence and PAM are depicted in blue and purple letters, respectively. ( C ) The deletion of 10 bp altered Tmem95 ORF. Large letters indicate the aminoacid sequence corresponding to the codons (DNA sequence) shown in smaller letters below. ( D ) Western Blot images for TMEM95, IZUMO1 and β-tubulin proteins from protein extracts from WT or KO sperm. Graph on right indicates the abundance of IZUMO1 in WT and KO extracts. ( E ) Immunocytochemistry images of KO and WT sperm stained with an antibody against TMEM95 and the acrosomal stain PNA. TMEM95 localized to the acrosomal cap in acrosome intact sperm and in the equatorial segment after acrosome reaction. ( F ) Immunocytochemistry images of acrosome intact (upper images) or reacted (lower images) WT sperm stained against IZUMO1 and TMEM95. Both proteins relocalize to the equatorial segment following acrosome reaction.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Software, Generated, CRISPR, Sequencing, Western Blot, Immunocytochemistry, Staining

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Immunoblotting of protein extracts obtained from WT and KO epididymal sperm samples following two lysis protocols. In lysis protocol #1, sperm were re-suspended in 4X reducing SDS Sample Buffer and boiled for 10 min. In lysis protocol #2, sperm were re-suspended in 1% Octyl β-D-glucopyranoside solution in PBS and incubated on ice for 30 min . Supernatants were probed with anti-TMEM95 (MyBiosource MBS7004333), anti-IZUMO1 (Abcam ab211623) or anti-β-TUBULIN (Sigma T8328) antibodies. ( B ) Gel electrophoresis of PCR products amplified from cDNA obtained from testis, seminal vesicle (S.V.) prostate (prost.), epididymis (epid.) or a negative control testis RNA not retrotranscribed (RT-) to detect Gapdh and Tmem95 transcripts. ( C ) Immunoblotting of protein extracts obtained from WT epididymal sperm, testis or accessory glands (seminal vesicle and prostate). Same antibodies than ( A ). ( D ) Uncropped images of the WB used to generate . ( E ) WB images used for the quantification of IZUMO1 and β-TUBULIN shown in . Four samples were used for quantification (marked with asterisk), as β-TUBULIN band on samples #4 was dispersed, leading to inaccurate quantification.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Western Blot, Lysis, Incubation, Nucleic Acid Electrophoresis, Amplification, Negative Control

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Immunocytochemistry images of WT sperm stained with PNA and IZUMO1 antibody (upper row) or PNA and TMEM95 antibody (lower row) in the absence of permeabilizing agents. Despite the absence of permeabilizing agents (30 min fixation in 4% PFA without Triton X-100), TMEM95 and inner acrosomal (PNA) and acrosomal membrane (IZUMO1) markers were detected. ( B ) IZUMO1 relocates to the equatorial segment following acrosome reaction in TMEM95 KO sperm. Upper row shows one acrosome intact spermatozoon. Lower rows show WT and KO acrosome reacted sperm where IZUMO1 has relocated to the equatorial segment.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Immunocytochemistry, Staining

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Binding analysis using the AVEXIS assays shows that the soluble recombinant TMEM95 ectodomain does not interact with JUNO nor with IZUMO1. The entire ectodomains of the named proteins were expressed in HEK293-6E cells either as biotinylated baits or as pentameric beta-lactamase-tagged preys. Bait proteins were immobilised on streptavidin-coated plates and captured prey proteins quantified by measuring the absorbance of a colorimetric reaction product of the beta-lactamase substrate, nitrocefin. The CD200R (bait)-CD200 (prey) binding pair was used as positive control. The same prey, CD200R, was tested against TMEM95 and is shown as negative control. Bars represent means + s.d.; n = 3. ( B ) HEK293 cells stably expressing the N-terminal half of GFP (GFP1-7) and mouse JUNO stained with a highly avid IZUMO1 probe. ( C ) HEK293 cells stably expressing the C-terminal half of GFP (GFP8-11) and mouse IZUMO1 stained with a highly avid JUNO. ( D ) TMEM95 does not induce fusion when expressed in HEK293T cells in the presence of JUNO and IZUMO1 using a GFP-complementation cell fusion assay. HEK293T cells expressing either half of GFP and either JUNO or IZUMO1 were mixed and their fusogenic ability visualized by GFP fluorescence. The IZUMOI-expressing cells were either mock transfected prior to mixing (Control), transfected with Syncitin a , as a positive fusion control, or Tmem95 . By contrast to the cells transfected with Syncytin a , Tmem95 did not induce cell fusion. Cell nuclei are stained with DAPI and scale bar represents 20 µm.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Binding Assay, Recombinant, Positive Control, Negative Control, Stable Transfection, Expressing, Staining, Cell Fusion Assay, Fluorescence, Transfection

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Amino acid sequence of FKBP1A underlined in blue fused via a short linker to the N-terminal region of GFP (GFP 1-7 ) (green underline). ( B ) Amino acid sequence of the C-terminal region of GFP (GFP 8-11 ) highlighted in green, fused to RB, underlined in blue. (C, C´, D and D´) The cells expressing the GFP1-7 stably express mouse Juno and the cells expressing the GFP8-11 stably express mouse Izumo1. To establish functional cell surface expression of both Juno and Izumo1 on the cells, noth cell lines were stained with the corresponding pentameric FLAG-tagged Izumo1 and Juno preys. The cells expressing mouse Juno were stained with the Izumo1 prey but not Juno, and the cells expressing mouse Izumo1 stained with the Juno prey but not Izumo1. Images are representative of at least three independent replicates. Nuclei are stained with DAPI (blue) and scale bars represents 10 µm.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Sequencing, Expressing, Stable Transfection, Functional Assay, Staining

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Generated, CRISPR, Recombinant, Plasmid Preparation, Sequencing

Journal: Cell reports

Article Title: Synthetic Lethal Interaction between the ESCRT Paralog Enzymes VPS4A and VPS4B in Cancers Harboring Loss of Chromosome 18q or 16q

doi: 10.1016/j.celrep.2020.108493

Figure Lengend Snippet:

Article Snippet: RAB7 , Rabbit , Clone D95F2 , , Cell Signaling Technology , 1 , #9367S.

Techniques:

Journal: Cell reports

Article Title: Synthetic Lethal Interaction between the ESCRT Paralog Enzymes VPS4A and VPS4B in Cancers Harboring Loss of Chromosome 18q or 16q

doi: 10.1016/j.celrep.2020.108493

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: RAB7 , Rabbit , Clone D95F2 , , Cell Signaling Technology , 1 , #9367S.

Techniques: Virus, Recombinant, CRISPR, Mouse Assay, Luciferase, Software, Simple Western, Quantitative Proteomics