tspan14  (Proteintech)

Journal: bioRxiv

Article Title: Integration of Alzheimer’s GWAS, 3D genomics, and single-cell CRISPRi non-coding screen implicates causal variants in a microglial enhancer regulating TSPAN14

doi: 10.1101/2025.04.01.646442

Figure Lengend Snippet: a: Schematic of the screen. Three sgRNAs were designed to target each of 74 candidate regulatory regions (CREs) from our V2G mapping effort. We created a CRISPRi helper line expressing dCas9-KRAB in HMC3 cells and transduced it with a lentiviral pool of the sgRNAs at low MOI so that most cells would only receive a single perturbation. After selection and expansion, we performed scRNA-seq with 10x Genomics. Upon successful targeting of a CRE using CRISPRi, we anticipate RNA expression knockdown of the target gene. b, c, d: QQ plots from SCEPTRE differential expression analysis showing b ) good calibration, c) significant signal, and d ) volcano plot of the hits. These plots refer to the V2G “union” analysis. e , f , g : a candidate region containing three AD-associated SNPs (rs7080009, rs1870138, and rs1870137) regulates TSPAN14 expression in microglial but not in neuronal cells. CRISPRi ( e ) and CRISPRa ( f ) were performed in HMC3 cells stably expressing dCas9-KRAB and dCas9-VPR, respectively, via lentiviral delivery of three gRNAs targeting the region (T1, T2, and T3) and three non-targeting control guides (N1, N2, and N3). TSPAN14 expression was assessed by qPCR (N=3; bar plots show mean TSPAN14 expression with SEM error bars normalized to their respective control line with no guide. Significance by one-way ANOVA followed by pairwise t-test. ** P <5×10 −5 ; * P <0.05. A similar CRISPRi experiment was performed in the neuronal ReNcell VM line, showing no effect on TSPAN14 expression ( c ). P: a guide targeting TSPAN14 promoter was used as a positive control.

Article Snippet: The HiFi cloning approach (NEBuilder HiFi DNA Assembly Master Mix, cat# E2621S) was implemented to clone a PCR amplified TSPAN14 enhancer region encompassing rs7080009, rs1870138 and rs1870137 (hg19 coordinates: chr10:82,268,673-82,270,263, 1,591bp) in two different plasmids which would be used for dual luciferase reporter assays: pNL[Nlucp/minP/hygro] (Promega; custom-made) containing a minimal promoter (minP) and the Nanoluc reporter cassette (the pNL-minP plasmid), and pGL4.10[luc2] (Promega) containing the endogenous promoter of the TSPAN14 gene, which was digested out of a commercially available plasmid (Genecopoeia, cat# HPRM34820-PF02) using restriction endonucleases EcoRI (NEB, cat# R3101S) and SacI (NEB, cat# R3156S) (the resulting plasmid: pGL4.10-TSPAN14promoter), and the firefly luciferase reporter cassette (the resulting plasmid: pGL4.10-TSPAN14promoter/enhancer).

Techniques: Expressing, Selection, RNA Expression, Knockdown, Stable Transfection, Control, Positive Control

Journal: bioRxiv

Article Title: Integration of Alzheimer’s GWAS, 3D genomics, and single-cell CRISPRi non-coding screen implicates causal variants in a microglial enhancer regulating TSPAN14

doi: 10.1101/2025.04.01.646442

Figure Lengend Snippet: a: genomic location of the three AD SNPs (red) showing the chromatin loop with the TSPAN14 promoter. b, c : in luciferase assays, a 1.6 kb intronic region containing the three AD SNPs increases reporter luminescence in microglial cells ( b ; HMC3 cells; two-sample t-test P =8×10 −4 ), but not in neuronal cells ( c ; SH-SY5Y cells). minP: minimal promoter only (no enhancer region). NNN: enhancer region with 3 SNPs with non-risk alleles. d : constructs containing AD risk alleles significantly increase enhancer activity compared to TSPAN14 promoter alone (* P <0.05, ** P <0.005), while a construct with all non-risk allele does not. Cell line: HMC3; DNN: rs7080009 risk allele only; NDN: rs1870138 risk allele only; NND: rs1870137 risk allele only; DDD: 3 SNPs with risk alleles; Prom: TSPAN14 promoter only (no enhancer region). Bar plots show mean reporter activity with SEM error bars normalized to promoter only control. Statistical analysis by one-way ANOVA ( P =1.7×10 −3 ) followed by pairwise t-test with BH correction, N=3. e, f, g: rs7080009, rs1870138, and rs1870137 AD-risk alleles affect binding of specific transcription factors in microglial cells. TF binding analysis was performed with motifbreakR using the “default” scoring method. In each panel, sequence logo diagrams for TF binding site motifs matching each SNP locus are plotted with respect to the reference genome (top, non-risk allele; bottom, risk allele). Motifs on top have stronger binding to the non-risk allele and motifs on bottom to the risk allele; both are ordered by the magnitude of the difference in binding affinities. The name of the TF is adjacent to each motif. Motifs depicted have expression > 10 TPM in at least two of the three cell lines analyzed (HMC3, iMg, monocytes).

Article Snippet: The HiFi cloning approach (NEBuilder HiFi DNA Assembly Master Mix, cat# E2621S) was implemented to clone a PCR amplified TSPAN14 enhancer region encompassing rs7080009, rs1870138 and rs1870137 (hg19 coordinates: chr10:82,268,673-82,270,263, 1,591bp) in two different plasmids which would be used for dual luciferase reporter assays: pNL[Nlucp/minP/hygro] (Promega; custom-made) containing a minimal promoter (minP) and the Nanoluc reporter cassette (the pNL-minP plasmid), and pGL4.10[luc2] (Promega) containing the endogenous promoter of the TSPAN14 gene, which was digested out of a commercially available plasmid (Genecopoeia, cat# HPRM34820-PF02) using restriction endonucleases EcoRI (NEB, cat# R3101S) and SacI (NEB, cat# R3156S) (the resulting plasmid: pGL4.10-TSPAN14promoter), and the firefly luciferase reporter cassette (the resulting plasmid: pGL4.10-TSPAN14promoter/enhancer).

Techniques: Luciferase, Construct, Activity Assay, Control, Binding Assay, Sequencing, Expressing

Journal: bioRxiv

Article Title: Integration of Alzheimer’s GWAS, 3D genomics, and single-cell CRISPRi non-coding screen implicates causal variants in a microglial enhancer regulating TSPAN14

doi: 10.1101/2025.04.01.646442

Figure Lengend Snippet: a: via CRISPR-Cas9 editing, we generated two clonal HMC3 lines harboring ∼780 bp homozygous genomic deletions spanning the three AD SNPs. b: DNA agarose gel shows sizes of the PCR amplicons for clones 1 and 2 and a control clone with no deletion. c: TSPAN14 expression levels by qPCR in clones 1 and 2 (blue) and control lines (grey). TSPAN14 expression was significantly decreased (60-80%) in both deletion clones (two-sample t-test P =2×10 −3 ). NTC1, NTC2, NTC3: control lines, each with a different non-targeting guide. N=3. Bar plots show mean TSPAN14 expression levels with SEM error bars normalized to the average of the control lines. d: Volcano plot showing differentially expressed genes in the same two KO lines vs three control lines expressing non-targeting guides. The dashed lines show thresholds for Fold Change (1.5) and FDR (0.05). e: PANTHER enrichment analysis using the GO-Slim Biological Process annotation set. Top (red): significantly upregulated pathways. Bottom (blue): significantly downregulated pathways. Correction: FDR<0.05. For each significantly enriched term, only the top term of the GO hierarchy (i.e., the most specific) is shown.

Article Snippet: The HiFi cloning approach (NEBuilder HiFi DNA Assembly Master Mix, cat# E2621S) was implemented to clone a PCR amplified TSPAN14 enhancer region encompassing rs7080009, rs1870138 and rs1870137 (hg19 coordinates: chr10:82,268,673-82,270,263, 1,591bp) in two different plasmids which would be used for dual luciferase reporter assays: pNL[Nlucp/minP/hygro] (Promega; custom-made) containing a minimal promoter (minP) and the Nanoluc reporter cassette (the pNL-minP plasmid), and pGL4.10[luc2] (Promega) containing the endogenous promoter of the TSPAN14 gene, which was digested out of a commercially available plasmid (Genecopoeia, cat# HPRM34820-PF02) using restriction endonucleases EcoRI (NEB, cat# R3101S) and SacI (NEB, cat# R3156S) (the resulting plasmid: pGL4.10-TSPAN14promoter), and the firefly luciferase reporter cassette (the resulting plasmid: pGL4.10-TSPAN14promoter/enhancer).

Techniques: CRISPR, Generated, Agarose Gel Electrophoresis, Clone Assay, Control, Expressing

Journal: bioRxiv

Article Title: Integration of Alzheimer’s GWAS, 3D genomics, and single-cell CRISPRi non-coding screen implicates causal variants in a microglial enhancer regulating TSPAN14

doi: 10.1101/2025.04.01.646442

Figure Lengend Snippet: a, b: Secreted IL-6 and IL-8 protein levels are strongly reduced in TSPAN14 enhancer KO microglial cells with deletions encompassing AD-risk variants rs7080009, rs1870138, and rs1870137. ELISA was performed on cell culture media of two HMC3 clonal lines (clone 1 and 2, blue). IL-6 or IL-8 protein levels are normalized to the amounts of total protein in each well. Controls are lines harboring a non-targeting guide and/or an empty vector (grey). Bar plots show means with SEM error bars; N=3. *** P <5×10 −3 ; two-sample t-test. c : The same KO lines with TSPAN14 enhancer deletions (blue) showed a significant reduction (∼25-30%) in intensity of phalloidin (F-actin) staining as compared to three control lines (grey). Single cells were segmented, quantified, and the fluorescence intensities of all cells in each well were averaged. Three wells were measured per condition, with 200-500 cells quantified per well. Bar plots show average fluorescence intensity with SEM error bars. *** P <5×10 −3 ; two-sample t-test. d: Representative images of NTC1 and clone 1 are shown, with notably less F-actin staining observed for clone 1, which harbors the TSPAN14 enhancer deletion.

Article Snippet: The HiFi cloning approach (NEBuilder HiFi DNA Assembly Master Mix, cat# E2621S) was implemented to clone a PCR amplified TSPAN14 enhancer region encompassing rs7080009, rs1870138 and rs1870137 (hg19 coordinates: chr10:82,268,673-82,270,263, 1,591bp) in two different plasmids which would be used for dual luciferase reporter assays: pNL[Nlucp/minP/hygro] (Promega; custom-made) containing a minimal promoter (minP) and the Nanoluc reporter cassette (the pNL-minP plasmid), and pGL4.10[luc2] (Promega) containing the endogenous promoter of the TSPAN14 gene, which was digested out of a commercially available plasmid (Genecopoeia, cat# HPRM34820-PF02) using restriction endonucleases EcoRI (NEB, cat# R3101S) and SacI (NEB, cat# R3156S) (the resulting plasmid: pGL4.10-TSPAN14promoter), and the firefly luciferase reporter cassette (the resulting plasmid: pGL4.10-TSPAN14promoter/enhancer).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Plasmid Preparation, Staining, Control, Fluorescence

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: Clinicopathological parameters of the 40 NSCLC patients.

Article Snippet: After washing in PBS, the samples were blocked with 0.5% bovine serum albumin solution in PBS (BSA) for 1 h and incubated overnight at 4 °C with polyclonal anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:50 in 0.5% BSA or kept overnight only with 0.5% BSA (for determination of relative Tspan14 expression).

Techniques: Expressing

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: Decreased expression of TSPAN14 in NSCLC patients is correlated with a lower survival rate. ( a ) TSPAN14 gene expression in tumor tissue and the surrounding normal lung tissue in NSCLC patients. TSPAN14 expression was normalized to HPRT as endogenous control. Normalized ΔCt values are presented in a logarithmic scale. p < 0.05 (*) indicate a statistically significant difference in TSPAN14 expression in tumor samples compared to normal samples. ( b ) Kaplan-Meier survival curves show the NSCLC patients with TSPAN14 decreased expression (“ TSPAN14 low expression”) and the NSCLC patients with higher TSPAN14 expression (“ TSPAN14 others”). Low TSPAN14 gene expression is indicated with a 3-fold decrease in the tumor compared to the matching normal tissue sample, and the differences were statistically significant ( p < 0.05). ( c ) Kaplan-Meier plotter graph survival curves showing OS of NSCLC patients with TSPAN14 low expression and the NSCLC patients with high TSPAN14 expression (n = 1144); ( d ) Kaplan-Meier plotter graph showing PFS of NSCLC patients with TSPAN14 low expression and the high TSPAN14 expression (n = 591).

Article Snippet: After washing in PBS, the samples were blocked with 0.5% bovine serum albumin solution in PBS (BSA) for 1 h and incubated overnight at 4 °C with polyclonal anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:50 in 0.5% BSA or kept overnight only with 0.5% BSA (for determination of relative Tspan14 expression).

Techniques: Expressing, Gene Expression, Control

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: The expression of Tspan14 in normal and NSCLC cell lines and the cell lines’ potential to invade and migrate. ( a ) Quantitative real-time PCR analysis of TSPAN14 gene expression, normalized to HPRT endogenous control, and ( b ) flow cytometry analysis of Tspan14 protein expression in permeabilized NCI-H460, A549, and NCI-H661 cancer cells, relative to normal human keratinocytes (HaCaT); ( c ) Representative images of gelatin degradation by HaCaT, NCI-H460, A549, and NCI-H661, attributed to histograms show a degraded gelatin area normalized to the total number of cells (relative degraded area). The scale bar in the figures marks a length of 50 μm. ( d ) Representative images of HaCaT, NCI-H460, A549, and NCI-H661 that migrated through the Matrigel ® matrix to the other side of the porous membrane; the histograms represent an average number of cells per field, in 10 independent fields per membrane, from three independent experiments. The scale bar in the pictures marks a length of 100 μm. All results are from at least three independent experiments. p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) indicate statistically significant differences between cancer cell lines and a normal cell line.

Article Snippet: After washing in PBS, the samples were blocked with 0.5% bovine serum albumin solution in PBS (BSA) for 1 h and incubated overnight at 4 °C with polyclonal anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:50 in 0.5% BSA or kept overnight only with 0.5% BSA (for determination of relative Tspan14 expression).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Control, Flow Cytometry, Membrane

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: Transfection with siRNA TSPAN14 for 24 h reduces TSPAN14 expression in NCI-460. ( a ) Quantitative real-time PCR analysis of the TSPAN14 expression normalized to HPRT as endogenous control, in non-transfected NCI-H460 cells (control), the cells transfected with non-coding siRNA (siRNA NC), and siRNA TSPAN14 transfected NCI-H460 cells; ( b ) Representative micrographics of specific fluorescent labeling of Tspan14 protein (green) with the ascribed histogram quantification in control, siRNA NC and siRNA TSPAN14 NCI-H460 cells. Cell nuclei are labeled with Hoechst 33342 (blue). The scale bar in the pictures marks a length of 100 μm. The histogram shows the Tspan14 expression normalized to the control. All results are from at least three independent experiments. p < 0.05 (*), p < 0.001 (***) and p < 0.0001 (****) indicate statistically significant difference between siRNA NC and siRNA TSPAN14 transfected cells relative to control, while p < 0.001 (###) and p < 0.0001 (####) indicates statistically significant difference between siRNA NC and siRNA TSPAN14 .

Article Snippet: After washing in PBS, the samples were blocked with 0.5% bovine serum albumin solution in PBS (BSA) for 1 h and incubated overnight at 4 °C with polyclonal anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:50 in 0.5% BSA or kept overnight only with 0.5% BSA (for determination of relative Tspan14 expression).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Control, Labeling

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: NCI-H460 cells with silenced TSPAN14 expression demonstrate higher migratory and invasive potential. ( a ) Representative images of gelatin degradation by control, siRNA NC, and siRNA TSPAN14 transfected cells, attributed to histograms, show a degraded surface of gelatin under the cell per total surface of the cell. The scale bar in the figures marks a length of 50 μm. ( b ) Representative images of control, siRNA NC, and siRNA TSPAN14 cells that migrated through Matrigel ® matrix to the other side of the porous membrane; the histograms represent an average number of cells per field, in 10 independent fields per membrane, from three independent experiments. The scale bar in the pictures marks a length of 100 μm. All results are from at least three independent experiments. p < 0.05 (*) indicates a statistically significant difference between siRNA TSPAN14 transfected cells and control cells.

Article Snippet: After washing in PBS, the samples were blocked with 0.5% bovine serum albumin solution in PBS (BSA) for 1 h and incubated overnight at 4 °C with polyclonal anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:50 in 0.5% BSA or kept overnight only with 0.5% BSA (for determination of relative Tspan14 expression).

Techniques: Expressing, Control, Transfection, Membrane

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: The qPCR analysis of matrix metalloproteinases MMP2 and MMP9 gene expression in control, the siRNA NC, and siRNA TSPAN14 transfected NCI-H460 cells. The gene expression of MMP2 and MMP9 was normalized to ACTB as endogenous control. All results are from four independent experiments. p < 0.01 (**) and p < 0.001 (***) indicate statistically significant differences between siRNA TSPAN14 transfected cells and control cells, while p < 0.001 (###) indicates a statistically significant difference between siRNA NC and siRNA TSPAN14 transfected cells.

Article Snippet: After washing in PBS, the samples were blocked with 0.5% bovine serum albumin solution in PBS (BSA) for 1 h and incubated overnight at 4 °C with polyclonal anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:50 in 0.5% BSA or kept overnight only with 0.5% BSA (for determination of relative Tspan14 expression).

Techniques: Gene Expression, Control, Transfection

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: Clinicopathological parameters of the 40 NSCLC patients.

Article Snippet: Primers and probes specific to TSPAN14 and HPRT were obtained from Applied Biosystems as following Assay-on-Demand Gene Expression Products: TSPAN14 (Hs00229502_m1) and HPRT1 (Hs01003267_m1).

Techniques: Expressing

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: Decreased expression of TSPAN14 in NSCLC patients is correlated with a lower survival rate. ( a ) TSPAN14 gene expression in tumor tissue and the surrounding normal lung tissue in NSCLC patients. TSPAN14 expression was normalized to HPRT as endogenous control. Normalized ΔCt values are presented in a logarithmic scale. p < 0.05 (*) indicate a statistically significant difference in TSPAN14 expression in tumor samples compared to normal samples. ( b ) Kaplan-Meier survival curves show the NSCLC patients with TSPAN14 decreased expression (“ TSPAN14 low expression”) and the NSCLC patients with higher TSPAN14 expression (“ TSPAN14 others”). Low TSPAN14 gene expression is indicated with a 3-fold decrease in the tumor compared to the matching normal tissue sample, and the differences were statistically significant ( p < 0.05). ( c ) Kaplan-Meier plotter graph survival curves showing OS of NSCLC patients with TSPAN14 low expression and the NSCLC patients with high TSPAN14 expression (n = 1144); ( d ) Kaplan-Meier plotter graph showing PFS of NSCLC patients with TSPAN14 low expression and the high TSPAN14 expression (n = 591).

Article Snippet: Primers and probes specific to TSPAN14 and HPRT were obtained from Applied Biosystems as following Assay-on-Demand Gene Expression Products: TSPAN14 (Hs00229502_m1) and HPRT1 (Hs01003267_m1).

Techniques: Expressing, Gene Expression, Control

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: The expression of Tspan14 in normal and NSCLC cell lines and the cell lines’ potential to invade and migrate. ( a ) Quantitative real-time PCR analysis of TSPAN14 gene expression, normalized to HPRT endogenous control, and ( b ) flow cytometry analysis of Tspan14 protein expression in permeabilized NCI-H460, A549, and NCI-H661 cancer cells, relative to normal human keratinocytes (HaCaT); ( c ) Representative images of gelatin degradation by HaCaT, NCI-H460, A549, and NCI-H661, attributed to histograms show a degraded gelatin area normalized to the total number of cells (relative degraded area). The scale bar in the figures marks a length of 50 μm. ( d ) Representative images of HaCaT, NCI-H460, A549, and NCI-H661 that migrated through the Matrigel ® matrix to the other side of the porous membrane; the histograms represent an average number of cells per field, in 10 independent fields per membrane, from three independent experiments. The scale bar in the pictures marks a length of 100 μm. All results are from at least three independent experiments. p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) indicate statistically significant differences between cancer cell lines and a normal cell line.

Article Snippet: Primers and probes specific to TSPAN14 and HPRT were obtained from Applied Biosystems as following Assay-on-Demand Gene Expression Products: TSPAN14 (Hs00229502_m1) and HPRT1 (Hs01003267_m1).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Control, Flow Cytometry, Membrane

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: Transfection with siRNA TSPAN14 for 24 h reduces TSPAN14 expression in NCI-460. ( a ) Quantitative real-time PCR analysis of the TSPAN14 expression normalized to HPRT as endogenous control, in non-transfected NCI-H460 cells (control), the cells transfected with non-coding siRNA (siRNA NC), and siRNA TSPAN14 transfected NCI-H460 cells; ( b ) Representative micrographics of specific fluorescent labeling of Tspan14 protein (green) with the ascribed histogram quantification in control, siRNA NC and siRNA TSPAN14 NCI-H460 cells. Cell nuclei are labeled with Hoechst 33342 (blue). The scale bar in the pictures marks a length of 100 μm. The histogram shows the Tspan14 expression normalized to the control. All results are from at least three independent experiments. p < 0.05 (*), p < 0.001 (***) and p < 0.0001 (****) indicate statistically significant difference between siRNA NC and siRNA TSPAN14 transfected cells relative to control, while p < 0.001 (###) and p < 0.0001 (####) indicates statistically significant difference between siRNA NC and siRNA TSPAN14 .

Article Snippet: Primers and probes specific to TSPAN14 and HPRT were obtained from Applied Biosystems as following Assay-on-Demand Gene Expression Products: TSPAN14 (Hs00229502_m1) and HPRT1 (Hs01003267_m1).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Control, Labeling

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: NCI-H460 cells with silenced TSPAN14 expression demonstrate higher migratory and invasive potential. ( a ) Representative images of gelatin degradation by control, siRNA NC, and siRNA TSPAN14 transfected cells, attributed to histograms, show a degraded surface of gelatin under the cell per total surface of the cell. The scale bar in the figures marks a length of 50 μm. ( b ) Representative images of control, siRNA NC, and siRNA TSPAN14 cells that migrated through Matrigel ® matrix to the other side of the porous membrane; the histograms represent an average number of cells per field, in 10 independent fields per membrane, from three independent experiments. The scale bar in the pictures marks a length of 100 μm. All results are from at least three independent experiments. p < 0.05 (*) indicates a statistically significant difference between siRNA TSPAN14 transfected cells and control cells.

Article Snippet: Primers and probes specific to TSPAN14 and HPRT were obtained from Applied Biosystems as following Assay-on-Demand Gene Expression Products: TSPAN14 (Hs00229502_m1) and HPRT1 (Hs01003267_m1).

Techniques: Expressing, Control, Transfection, Membrane

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: The qPCR analysis of matrix metalloproteinases MMP2 and MMP9 gene expression in control, the siRNA NC, and siRNA TSPAN14 transfected NCI-H460 cells. The gene expression of MMP2 and MMP9 was normalized to ACTB as endogenous control. All results are from four independent experiments. p < 0.01 (**) and p < 0.001 (***) indicate statistically significant differences between siRNA TSPAN14 transfected cells and control cells, while p < 0.001 (###) indicates a statistically significant difference between siRNA NC and siRNA TSPAN14 transfected cells.

Article Snippet: Primers and probes specific to TSPAN14 and HPRT were obtained from Applied Biosystems as following Assay-on-Demand Gene Expression Products: TSPAN14 (Hs00229502_m1) and HPRT1 (Hs01003267_m1).

Techniques: Gene Expression, Control, Transfection

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: Clinicopathological parameters of the 40 NSCLC patients.

Article Snippet: The cells were washed in PBS, fixed in 4% PFA for 15 min at 4 °C, and blocked in 0.5% BSA for 1 h. Anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, USA) was applied at 1:50 dilution in 0.5% BSA, and the cells were incubated overnight at 4 °C.

Techniques: Expressing

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: Decreased expression of TSPAN14 in NSCLC patients is correlated with a lower survival rate. ( a ) TSPAN14 gene expression in tumor tissue and the surrounding normal lung tissue in NSCLC patients. TSPAN14 expression was normalized to HPRT as endogenous control. Normalized ΔCt values are presented in a logarithmic scale. p < 0.05 (*) indicate a statistically significant difference in TSPAN14 expression in tumor samples compared to normal samples. ( b ) Kaplan-Meier survival curves show the NSCLC patients with TSPAN14 decreased expression (“ TSPAN14 low expression”) and the NSCLC patients with higher TSPAN14 expression (“ TSPAN14 others”). Low TSPAN14 gene expression is indicated with a 3-fold decrease in the tumor compared to the matching normal tissue sample, and the differences were statistically significant ( p < 0.05). ( c ) Kaplan-Meier plotter graph survival curves showing OS of NSCLC patients with TSPAN14 low expression and the NSCLC patients with high TSPAN14 expression (n = 1144); ( d ) Kaplan-Meier plotter graph showing PFS of NSCLC patients with TSPAN14 low expression and the high TSPAN14 expression (n = 591).

Article Snippet: The cells were washed in PBS, fixed in 4% PFA for 15 min at 4 °C, and blocked in 0.5% BSA for 1 h. Anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, USA) was applied at 1:50 dilution in 0.5% BSA, and the cells were incubated overnight at 4 °C.

Techniques: Expressing, Gene Expression, Control

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: The expression of Tspan14 in normal and NSCLC cell lines and the cell lines’ potential to invade and migrate. ( a ) Quantitative real-time PCR analysis of TSPAN14 gene expression, normalized to HPRT endogenous control, and ( b ) flow cytometry analysis of Tspan14 protein expression in permeabilized NCI-H460, A549, and NCI-H661 cancer cells, relative to normal human keratinocytes (HaCaT); ( c ) Representative images of gelatin degradation by HaCaT, NCI-H460, A549, and NCI-H661, attributed to histograms show a degraded gelatin area normalized to the total number of cells (relative degraded area). The scale bar in the figures marks a length of 50 μm. ( d ) Representative images of HaCaT, NCI-H460, A549, and NCI-H661 that migrated through the Matrigel ® matrix to the other side of the porous membrane; the histograms represent an average number of cells per field, in 10 independent fields per membrane, from three independent experiments. The scale bar in the pictures marks a length of 100 μm. All results are from at least three independent experiments. p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) indicate statistically significant differences between cancer cell lines and a normal cell line.

Article Snippet: The cells were washed in PBS, fixed in 4% PFA for 15 min at 4 °C, and blocked in 0.5% BSA for 1 h. Anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, USA) was applied at 1:50 dilution in 0.5% BSA, and the cells were incubated overnight at 4 °C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Control, Flow Cytometry, Membrane

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: Transfection with siRNA TSPAN14 for 24 h reduces TSPAN14 expression in NCI-460. ( a ) Quantitative real-time PCR analysis of the TSPAN14 expression normalized to HPRT as endogenous control, in non-transfected NCI-H460 cells (control), the cells transfected with non-coding siRNA (siRNA NC), and siRNA TSPAN14 transfected NCI-H460 cells; ( b ) Representative micrographics of specific fluorescent labeling of Tspan14 protein (green) with the ascribed histogram quantification in control, siRNA NC and siRNA TSPAN14 NCI-H460 cells. Cell nuclei are labeled with Hoechst 33342 (blue). The scale bar in the pictures marks a length of 100 μm. The histogram shows the Tspan14 expression normalized to the control. All results are from at least three independent experiments. p < 0.05 (*), p < 0.001 (***) and p < 0.0001 (****) indicate statistically significant difference between siRNA NC and siRNA TSPAN14 transfected cells relative to control, while p < 0.001 (###) and p < 0.0001 (####) indicates statistically significant difference between siRNA NC and siRNA TSPAN14 .

Article Snippet: The cells were washed in PBS, fixed in 4% PFA for 15 min at 4 °C, and blocked in 0.5% BSA for 1 h. Anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, USA) was applied at 1:50 dilution in 0.5% BSA, and the cells were incubated overnight at 4 °C.

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Control, Labeling

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: NCI-H460 cells with silenced TSPAN14 expression demonstrate higher migratory and invasive potential. ( a ) Representative images of gelatin degradation by control, siRNA NC, and siRNA TSPAN14 transfected cells, attributed to histograms, show a degraded surface of gelatin under the cell per total surface of the cell. The scale bar in the figures marks a length of 50 μm. ( b ) Representative images of control, siRNA NC, and siRNA TSPAN14 cells that migrated through Matrigel ® matrix to the other side of the porous membrane; the histograms represent an average number of cells per field, in 10 independent fields per membrane, from three independent experiments. The scale bar in the pictures marks a length of 100 μm. All results are from at least three independent experiments. p < 0.05 (*) indicates a statistically significant difference between siRNA TSPAN14 transfected cells and control cells.

Article Snippet: The cells were washed in PBS, fixed in 4% PFA for 15 min at 4 °C, and blocked in 0.5% BSA for 1 h. Anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, USA) was applied at 1:50 dilution in 0.5% BSA, and the cells were incubated overnight at 4 °C.

Techniques: Expressing, Control, Transfection, Membrane

Journal: Life

Article Title: Decreased TSPAN14 Expression Contributes to NSCLC Progression

doi: 10.3390/life12091291

Figure Lengend Snippet: The qPCR analysis of matrix metalloproteinases MMP2 and MMP9 gene expression in control, the siRNA NC, and siRNA TSPAN14 transfected NCI-H460 cells. The gene expression of MMP2 and MMP9 was normalized to ACTB as endogenous control. All results are from four independent experiments. p < 0.01 (**) and p < 0.001 (***) indicate statistically significant differences between siRNA TSPAN14 transfected cells and control cells, while p < 0.001 (###) indicates a statistically significant difference between siRNA NC and siRNA TSPAN14 transfected cells.

Article Snippet: The cells were washed in PBS, fixed in 4% PFA for 15 min at 4 °C, and blocked in 0.5% BSA for 1 h. Anti-Tspan14 antibody (PA5-37979, Thermo Fisher Scientific, USA) was applied at 1:50 dilution in 0.5% BSA, and the cells were incubated overnight at 4 °C.

Techniques: Gene Expression, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: GPVI cleavage is dependent on Tspan15 and Tspan33 in transfected HEK-293T cells. ( Ai ) Wild-type (WT), ADAM10-knockout (A10 KO), Tspan14-knockout (T14 KO), Tspan15-knockout (T15 KO), Tspan33-knockout (T33 KO) and Tspan15/33 double knockout (T15/33 dKO) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged human GPVI and FcRγ (+), or empty vector (–). Cells were treated with 2 mM NEM (+), or vehicle control (–) for 30 min prior to harvest. Cells were then lysed in 1% Triton X-100 and lysates subjected to Western blotting with an anti-Myc antibody. ( Aii ) The percentage of cleaved GPVI from Ai was calculated. Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a two-way ANOVA followed by a Dunnett’s multiple comparison test, compared to their respective WT controls in each stimulation condition (***, p < 0.001; ****, p < 0.0001). ( B ) ADAM10 surface expression on the cell types described in panel A were analyzed by flow cytometry. Geometric mean fluorescence intensity was used to assess surface ADAM10 levels and data were made relative to WT values. Error bars represent the standard error of the mean from three independent experiments. Average geometric mean intensities were arcsine transformed and then statistically analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparison test, compared to WT (****, p < 0.0001). The data shown are from single CRISPR/Cas9 knockout clones, but similar data were generated using a second set of clones generated using different guide RNA sequences (data not shown).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Transfection, Knock-Out, Double Knockout, Expressing, Construct, Plasmid Preparation, Control, Western Blot, Transformation Assay, Comparison, Flow Cytometry, Fluorescence, CRISPR, Clone Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Tspan15 and Tspan33 are required for cleavage of endogenous GPVI in HEL cells. ( Ai ) Wild-type (WT), ADAM10-knockout (A10 KO), Tspan14-knockout (T14 KO), Tspan15-knockout (T15 KO), Tspan33-knockout (T33 KO) and GPVI-knockout (GPVI KO) HEL cells were treated with 6.2 ng/mL PMA for 72 h. Cells were stimulated with 2 mM NEM (+) for 1 h before harvest. Cells were lysed in 1% Triton X-100 and separated by SDS-PAGE. Lysates were probed with a mouse anti-human GPVI antibody (11A7), which recognises the extracellular region of GPVI and therefore only the full-length protein (top panel) and a rabbit anti-human GPVI antibody, which recognises the cytoplasmic tail of GPVI (bottom panel). ( Aii ) Relative GPVI cleavage was calculated by normalising the cleaved fragment signal to the full-length GPVI; the NEM-treated WT was arbitrarily set at 100. Error bars represent the standard error of the mean from three independent experiments. Data were normalised by arcsine transformation and statistically analyzed using a two-way ANOVA followed by a Bonferroni multiple comparison test, compared to WT (***, p < 0.001; ****, p < 0.0001). ( Bi ) WT, T33 KO and Tspan15/33 double knockout (T15/33 dKO) HEL cells were treated with PMA and subjected to lysis and Western blotting as described in panel ( Ai ). ( Bii ) Relative GPVI cleaved was quantitated and statistically analyzed as described in panel ( Aii ). ( C ) ADAM10 surface expression on PMA-differentiated cells used in panels A and B were analyzed by flow cytometry and quantitated as described in B. The data shown are from single CRISPR/Cas9 knockout clones, but similar data were generated using a second set of clones generated using different guide RNA sequences (data not shown).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Knock-Out, SDS Page, Transformation Assay, Comparison, Double Knockout, Lysis, Western Blot, Expressing, Flow Cytometry, CRISPR, Clone Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Tspan15/ADAM10 is the most efficient scissor for GPVI in HEK-293T cells. ( Ai ) Tspan15/33 double knockout (T15/33 dKO) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged human GPVI and FcRγ (+) or empty vector (–), and FLAG-tagged human Tspan14, Tspan15, Tspan33 or empty vector control (–). Cells were treated with 2 mM NEM (+), or vehicle control (–) for 30 min prior to harvest, and then lysed in 1% Triton X-100 followed by anti-Myc and anti-FLAG Western blotting. ( Aii ) The percentage of cleaved GPVI from Ai was calculated. Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a two-way ANOVA followed by a Dunnett’s multiple comparison test, compared to their respective non-transfected controls in each stimulation condition (*, p < 0.05, **, p < 0.01; ****, p < 0.0001).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Double Knockout, Transfection, Expressing, Construct, Plasmid Preparation, Control, Western Blot, Transformation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: The extracellular region of Tspan15 is required for efficient GPVI cleavage and the C-terminus is inhibitory. ( A ) Schematic of N-terminally FLAG-tagged Tspan14 (grey) and Tspan15 (black) chimeras, where the entire extracellular region (EC), cytoplasmic domain (Cyto) or C-terminal tail (C) were exchanged. Truncation of both the N- and C-terminal tails is indicated by ΔNC. Ovals represent N-glycosylation sites. ( B ) Wild-type (WT) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged GPVI and FcRγ (+) or empty vector (–). In addition to GPVI and FcRγ, Tspan14/15/33 triple knockout (T14/15/33 tKO) HEK-293T cells were co-transfected with HA-tagged ADAM10 alone, or in combination with FLAG-tagged Tspan14 and Tspan15 constructs described in panel A. Cells were lysed in 1% Triton X-100 followed by Western blotting with anti-Myc, anti-FLAG and anti-HA antibodies (top panels). The percentage of cleaved GPVI was calculated (lower panel). Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparison test, compared to T14/15/33 tKO cells transfected with wild-type Tspan15 and ADAM10 (***, p < 0.001; ****, p < 0.0001).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Glycoproteomics, Transfection, Expressing, Construct, Plasmid Preparation, Triple Knockout, Western Blot, Transformation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: The capacity of Tspan15 wild-type and mutant forms to promote GPVI cleavage is unrelated to the extent to which they co-localise with GPVI. Tspan14/15/33 triple knockout HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged GPVI, FcRγ, ADAM10 and FLAG-tagged Tspan14 or 15 chimeras that are described in detail in A. Cells were fixed and stained with anti-Myc (GPVI–Myc, magenta) and anti-FLAG (FLAG-Tspan, green) antibodies. ( Ai ) The middle planes of the cells were imaged using Airyscan confocal microscopy in super-resolution mode (scale bar 5 μm). No signal was detected in either channel in the empty vector-transfected cells (data not shown). ( Aii ) The degree of co-localization between GPVI–Myc and FLAG-TspanC8s is presented as the percentage of co-localizing pixels in the GPVI–Myc (magenta) channel. Data are representative of 15 fields of view from three independent experiments and are normalized by arcsine transformation before being statistically analyzed by a one-way ANOVA, followed by Dunnett’s multiple comparison tests, compared to cells transfected with wild-type Tspan15 (**, p < 0.01; ****, p < 0.0001). ( B ) Scatter plot summarising the relationship between degree of co-localization, expressed as the average of data presented in panel Aii , and percentage of GPVI cleaved, expressed as mean of the quantitation presented in B.

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Mutagenesis, Triple Knockout, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Plasmid Preparation, Transformation Assay, Comparison, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Co-localization between ADAM10 and GPVI is similar in Tspan14-knockout and Tspan15/33 double knockout HEL cells. Wild-type (WT), Tspan14-knockout (T14 KO) and Tspan15/33 double knockout (T15/33 dKO) HEL cells were treated with 6.2 ng/mL PMA for 72 h. Cells were fixed and stained with anti-ADAM10 mAb (11G2, magenta) and an antibody against the extracellular region of GPVI (336A9, green). Images of the ( A ) basal membrane and ( B ) middle section of the cells were captured using Airyscan confocal microscopy in super-resolution mode (top panels; scale bar 5 μm). No ADAM10 signal was detected in control ADAM10-knockout cells and no GPVI signal was detected in control GPVI-knockout cells (data not shown). The degree of co-localization between ADAM10 and GPVI is presented as the percentage of co-localizing pixels in the ADAM10 (magenta) channel (lower panels). Data are representative of 15 fields of view from three independent experiments and are normalized by arcsine transformation and statistically analyzed by a one-way ANOVA, followed by Bonferroni’s multiple comparison tests (n.s., not significant; *, p < 0.05).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Knock-Out, Double Knockout, Staining, Membrane, Confocal Microscopy, Control, Transformation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: GPVI cleavage is dependent on Tspan15 and Tspan33 in transfected HEK-293T cells. ( Ai ) Wild-type (WT), ADAM10-knockout (A10 KO), Tspan14-knockout (T14 KO), Tspan15-knockout (T15 KO), Tspan33-knockout (T33 KO) and Tspan15/33 double knockout (T15/33 dKO) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged human GPVI and FcRγ (+), or empty vector (–). Cells were treated with 2 mM NEM (+), or vehicle control (–) for 30 min prior to harvest. Cells were then lysed in 1% Triton X-100 and lysates subjected to Western blotting with an anti-Myc antibody. ( Aii ) The percentage of cleaved GPVI from Ai was calculated. Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a two-way ANOVA followed by a Dunnett’s multiple comparison test, compared to their respective WT controls in each stimulation condition (***, p < 0.001; ****, p < 0.0001). ( B ) ADAM10 surface expression on the cell types described in panel A were analyzed by flow cytometry. Geometric mean fluorescence intensity was used to assess surface ADAM10 levels and data were made relative to WT values. Error bars represent the standard error of the mean from three independent experiments. Average geometric mean intensities were arcsine transformed and then statistically analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparison test, compared to WT (****, p < 0.0001). The data shown are from single CRISPR/Cas9 knockout clones, but similar data were generated using a second set of clones generated using different guide RNA sequences (data not shown).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Transfection, Knock-Out, Double Knockout, Expressing, Construct, Plasmid Preparation, Control, Western Blot, Transformation Assay, Comparison, Flow Cytometry, Fluorescence, CRISPR, Clone Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Tspan15 and Tspan33 are required for cleavage of endogenous GPVI in HEL cells. ( Ai ) Wild-type (WT), ADAM10-knockout (A10 KO), Tspan14-knockout (T14 KO), Tspan15-knockout (T15 KO), Tspan33-knockout (T33 KO) and GPVI-knockout (GPVI KO) HEL cells were treated with 6.2 ng/mL PMA for 72 h. Cells were stimulated with 2 mM NEM (+) for 1 h before harvest. Cells were lysed in 1% Triton X-100 and separated by SDS-PAGE. Lysates were probed with a mouse anti-human GPVI antibody (11A7), which recognises the extracellular region of GPVI and therefore only the full-length protein (top panel) and a rabbit anti-human GPVI antibody, which recognises the cytoplasmic tail of GPVI (bottom panel). ( Aii ) Relative GPVI cleavage was calculated by normalising the cleaved fragment signal to the full-length GPVI; the NEM-treated WT was arbitrarily set at 100. Error bars represent the standard error of the mean from three independent experiments. Data were normalised by arcsine transformation and statistically analyzed using a two-way ANOVA followed by a Bonferroni multiple comparison test, compared to WT (***, p < 0.001; ****, p < 0.0001). ( Bi ) WT, T33 KO and Tspan15/33 double knockout (T15/33 dKO) HEL cells were treated with PMA and subjected to lysis and Western blotting as described in panel ( Ai ). ( Bii ) Relative GPVI cleaved was quantitated and statistically analyzed as described in panel ( Aii ). ( C ) ADAM10 surface expression on PMA-differentiated cells used in panels A and B were analyzed by flow cytometry and quantitated as described in B. The data shown are from single CRISPR/Cas9 knockout clones, but similar data were generated using a second set of clones generated using different guide RNA sequences (data not shown).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Knock-Out, SDS Page, Transformation Assay, Comparison, Double Knockout, Lysis, Western Blot, Expressing, Flow Cytometry, CRISPR, Clone Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Tspan15/ADAM10 is the most efficient scissor for GPVI in HEK-293T cells. ( Ai ) Tspan15/33 double knockout (T15/33 dKO) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged human GPVI and FcRγ (+) or empty vector (–), and FLAG-tagged human Tspan14, Tspan15, Tspan33 or empty vector control (–). Cells were treated with 2 mM NEM (+), or vehicle control (–) for 30 min prior to harvest, and then lysed in 1% Triton X-100 followed by anti-Myc and anti-FLAG Western blotting. ( Aii ) The percentage of cleaved GPVI from Ai was calculated. Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a two-way ANOVA followed by a Dunnett’s multiple comparison test, compared to their respective non-transfected controls in each stimulation condition (*, p < 0.05, **, p < 0.01; ****, p < 0.0001).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Double Knockout, Transfection, Expressing, Construct, Plasmid Preparation, Control, Western Blot, Transformation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: The extracellular region of Tspan15 is required for efficient GPVI cleavage and the C-terminus is inhibitory. ( A ) Schematic of N-terminally FLAG-tagged Tspan14 (grey) and Tspan15 (black) chimeras, where the entire extracellular region (EC), cytoplasmic domain (Cyto) or C-terminal tail (C) were exchanged. Truncation of both the N- and C-terminal tails is indicated by ΔNC. Ovals represent N-glycosylation sites. ( B ) Wild-type (WT) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged GPVI and FcRγ (+) or empty vector (–). In addition to GPVI and FcRγ, Tspan14/15/33 triple knockout (T14/15/33 tKO) HEK-293T cells were co-transfected with HA-tagged ADAM10 alone, or in combination with FLAG-tagged Tspan14 and Tspan15 constructs described in panel A. Cells were lysed in 1% Triton X-100 followed by Western blotting with anti-Myc, anti-FLAG and anti-HA antibodies (top panels). The percentage of cleaved GPVI was calculated (lower panel). Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparison test, compared to T14/15/33 tKO cells transfected with wild-type Tspan15 and ADAM10 (***, p < 0.001; ****, p < 0.0001).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Glycoproteomics, Transfection, Expressing, Construct, Plasmid Preparation, Triple Knockout, Western Blot, Transformation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: The capacity of Tspan15 wild-type and mutant forms to promote GPVI cleavage is unrelated to the extent to which they co-localise with GPVI. Tspan14/15/33 triple knockout HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged GPVI, FcRγ, ADAM10 and FLAG-tagged Tspan14 or 15 chimeras that are described in detail in A. Cells were fixed and stained with anti-Myc (GPVI–Myc, magenta) and anti-FLAG (FLAG-Tspan, green) antibodies. ( Ai ) The middle planes of the cells were imaged using Airyscan confocal microscopy in super-resolution mode (scale bar 5 μm). No signal was detected in either channel in the empty vector-transfected cells (data not shown). ( Aii ) The degree of co-localization between GPVI–Myc and FLAG-TspanC8s is presented as the percentage of co-localizing pixels in the GPVI–Myc (magenta) channel. Data are representative of 15 fields of view from three independent experiments and are normalized by arcsine transformation before being statistically analyzed by a one-way ANOVA, followed by Dunnett’s multiple comparison tests, compared to cells transfected with wild-type Tspan15 (**, p < 0.01; ****, p < 0.0001). ( B ) Scatter plot summarising the relationship between degree of co-localization, expressed as the average of data presented in panel Aii , and percentage of GPVI cleaved, expressed as mean of the quantitation presented in B.

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Mutagenesis, Triple Knockout, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Plasmid Preparation, Transformation Assay, Comparison, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Co-localization between ADAM10 and GPVI is similar in Tspan14-knockout and Tspan15/33 double knockout HEL cells. Wild-type (WT), Tspan14-knockout (T14 KO) and Tspan15/33 double knockout (T15/33 dKO) HEL cells were treated with 6.2 ng/mL PMA for 72 h. Cells were fixed and stained with anti-ADAM10 mAb (11G2, magenta) and an antibody against the extracellular region of GPVI (336A9, green). Images of the ( A ) basal membrane and ( B ) middle section of the cells were captured using Airyscan confocal microscopy in super-resolution mode (top panels; scale bar 5 μm). No ADAM10 signal was detected in control ADAM10-knockout cells and no GPVI signal was detected in control GPVI-knockout cells (data not shown). The degree of co-localization between ADAM10 and GPVI is presented as the percentage of co-localizing pixels in the ADAM10 (magenta) channel (lower panels). Data are representative of 15 fields of view from three independent experiments and are normalized by arcsine transformation and statistically analyzed by a one-way ANOVA, followed by Bonferroni’s multiple comparison tests (n.s., not significant; *, p < 0.05).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Knock-Out, Double Knockout, Staining, Membrane, Confocal Microscopy, Control, Transformation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Evidence that cut site position on GPVI contributes to specific cleavage by Tspan15/ADAM10 and Tspan33/ADAM10. ( A ) Amino acid alignment of wild-type GPVI and the stalk extension mutants, GPVI+5 and GPVI+10: ADAM10 cut site is denoted by ^; residues inserted are highlighted in grey; transmembrane region is underlined. ( B ) Wild-type (WT) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged GPVI and FcRγ (+) or empty vector (–). ADAM10/17 double knockout (A10/17 dKO) HEK-293T cells were co-transfected with GPVI and FcRγ, HA-tagged ADAM10 alone, or in combination with FLAG-tagged TspanC8s (Tspan5, 10, 14, 15, 17 or 33). Cells were lysed in 1% Triton X-100 followed by Western blotting with anti-Myc, anti-FLAG and anti-HA antibodies (data not shown). The percentage of cleaved GPVI was calculated. Cleavage assays for ( C ) GPVI+5 and ( D ) GPVI+10 was performed as described in panel B. Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparison test, compared to A10/17 dKO cells transfected with ADAM10 alone (*, p < 0.05; **, p < 0.01, ***, p < 0.001; ****, p < 0.0001).

Article Snippet: Synthesized fragments of extracellular domain chimeras between Tspan14 and Tspan15 (Thermo Fisher Scientific, Horsham, UK) and Tspan15 with the cytoplasmic domain replaced by that of Tspan14 (Twist Bioscience, South San Francisco, CA, USA) were subcloned into the pEF6/Myc-His-FLAG vector.

Techniques: Transfection, Expressing, Construct, Plasmid Preparation, Double Knockout, Western Blot, Transformation Assay, Comparison