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Figure 2. Involvement of <t>ADAM10</t> and γ-secretase complex in N-cadherin cleavage. (A) T24 cells were treated with increasing concentrations of the γ-secretase inhibitor DAPT (5, 10, 20 µM) for 24 h. Total cell lysates were subjected to immunoblotting with the 3B9 antibody raised against the cytoplasmic domain

Figure 2. Involvement of <t>ADAM10</t> and γ-secretase complex in N-cadherin cleavage. (A) T24 cells were treated with increasing concentrations of the γ-secretase inhibitor DAPT (5, 10, 20 µM) for 24 h. Total cell lysates were subjected to immunoblotting with the 3B9 antibody raised against the cytoplasmic domain

Adam10, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 4 article reviews

adam10 - by Bioz Stars, 2026-03

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1) Product Images from "GW501516-Mediated Targeting of Tetraspanin 15 Regulates ADAM10-Dependent N-Cadherin Cleavage in Invasive Bladder Cancer Cells."

Article Title: GW501516-Mediated Targeting of Tetraspanin 15 Regulates ADAM10-Dependent N-Cadherin Cleavage in Invasive Bladder Cancer Cells.

Journal: Cells

doi: 10.3390/cells13080708

Figure 2. Involvement of ADAM10 and γ-secretase complex in N-cadherin cleavage. (A) T24 cells were treated with increasing concentrations of the γ-secretase inhibitor DAPT (5, 10, 20 µM) for 24 h. Total cell lysates were subjected to immunoblotting with the 3B9 antibody raised against the cytoplasmic domain

Figure Legend Snippet: Figure 2. Involvement of ADAM10 and γ-secretase complex in N-cadherin cleavage. (A) T24 cells were treated with increasing concentrations of the γ-secretase inhibitor DAPT (5, 10, 20 µM) for 24 h. Total cell lysates were subjected to immunoblotting with the 3B9 antibody raised against the cytoplasmic domain

Techniques Used: Western Blot

Figure 3. Tspan15 controls ADAM10-mediated cleavage of N-cadherin in T24 cells. (A) Validation of Tspan15 knockdown efficiency at the mRNA level by RTq-PCR analysis in T24 cells transfected with 25 nM TSPAN15 siRNA. Data are means ± SD of three independent experiments performed in triplicates (* p < 0.05). (B) Western blotting analysis of TSPAN15 protein depletion in TSPAN15 siRNA

Figure Legend Snippet: Figure 3. Tspan15 controls ADAM10-mediated cleavage of N-cadherin in T24 cells. (A) Validation of Tspan15 knockdown efficiency at the mRNA level by RTq-PCR analysis in T24 cells transfected with 25 nM TSPAN15 siRNA. Data are means ± SD of three independent experiments performed in triplicates (* p < 0.05). (B) Western blotting analysis of TSPAN15 protein depletion in TSPAN15 siRNA

Techniques Used: Biomarker Discovery, Knockdown, Transfection, Western Blot

Figure 6. ADAM10 is not regulated by PPARβ/δ in T24 cells. Cells were treated with increasing concentrations of GW501516 (1, 15, 25 µM) for 24 h. (A) Adam10 mRNA expression was analyzed by RTq-PCR. Fold inductions represent a comparison with vehicle-treated cells (set at 1) in the absence of GW501516. Data are means ± SD of three independent experiments performed in triplicates. (B) Western blotting analysis of ADAM10 protein (proform and mature form) in control and stimulated cells. β-actin was used as an internal loading control. The graphs depict densitometric analysis results of Western blots by using ImageJ. Data are means ± SD of three independent experiments performed in triplicates. (C) Plin2, a PPARβ target gene, was used as a positive control to validate the efficiency of GW501516. Plin2 mRNA expression was analyzed by RTq-PCR. Fold inductions represent a comparison with vehicle-treated cells (set at 1) in the absence of GW501516. Data are means ± SD of three independent experiments performed in triplicates (* p < 0.05).

Figure Legend Snippet: Figure 6. ADAM10 is not regulated by PPARβ/δ in T24 cells. Cells were treated with increasing concentrations of GW501516 (1, 15, 25 µM) for 24 h. (A) Adam10 mRNA expression was analyzed by RTq-PCR. Fold inductions represent a comparison with vehicle-treated cells (set at 1) in the absence of GW501516. Data are means ± SD of three independent experiments performed in triplicates. (B) Western blotting analysis of ADAM10 protein (proform and mature form) in control and stimulated cells. β-actin was used as an internal loading control. The graphs depict densitometric analysis results of Western blots by using ImageJ. Data are means ± SD of three independent experiments performed in triplicates. (C) Plin2, a PPARβ target gene, was used as a positive control to validate the efficiency of GW501516. Plin2 mRNA expression was analyzed by RTq-PCR. Fold inductions represent a comparison with vehicle-treated cells (set at 1) in the absence of GW501516. Data are means ± SD of three independent experiments performed in triplicates (* p < 0.05).

Techniques Used: Expressing, Comparison, Western Blot, Control, Positive Control

Image Search Results

( A ) Generation of Tie1 whole-body inducible or lymphatic endothelium–specific Tie1 -KO mice. Tie1 whole-body inducible KO ( Tie1 WB–/– ) was generated by crossing Tie1 fl mice with Rosa26 rtTA TetOCre mice and timed induction with Dox water. Tie1 lymphatic-specific Tie1 KO ( Tie1 LEC–/– ) was generated by crossing Tie1 fl mice with Pdpn Cre mice. ( B ) Gross phenotypes of Tie1 WB–/– embryos that were induced and examined at different time points (first number indicates the induction time point; second number indicates the harvest time point). White asterisk shows blood-filled lymphatics at E15.5 (5 of 7 of the Tie1 WB–/– embryos); arrow shows edema at E18.5 (7 of 9 of the Tie1 WB–/– embryos); and arrowhead shows chylous ascites at P2 (6 of 6 of the Tie1 WB–/– pups). ( C ) LEC-specific Tie1 KO resulted in a phenotype similar to that of whole-body KO. ( D ) Workflow of dermal LEC bulk RNA-Seq. E18.5 embryos induced at E13.5 were euthanized. The skin from each embryo was removed and placed in an Eppendorf tube for enzymatic digestion. The single-cell suspension was labeled with antibodies against CD45, CD31, and Lyve1. CD31 + LYVE1 + cells were sorted into lysis buffer for RNA extraction. Bulk RNA-Seq was performed on the Illumina HiSeq 4000 system. ( E ) Number of differentially expressed genes using a P value of less than 0.01 as the cutoff. Down, downregulated; Up, upregulated. ( F ) Volcano plot shows some of the most differentially expressed genes including Ccl21a , valve genes and tip cell–enriched genes. ( G ) Heatmaps of manually selected vascular-relevant genes categorized as labeled. ( H ) Overlap between our data set (blue) and a data set from HUVECs with transcriptionally active FOXO1 (orange). Some commonly regulated genes are listed in the square, including tip cell genes (red), polarization genes (blue), ion channel genes (green), and valve genes (black).

Journal: The Journal of Clinical Investigation

Article Title: The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development

doi: 10.1172/JCI176577

Figure Lengend Snippet: ( A ) Generation of Tie1 whole-body inducible or lymphatic endothelium–specific Tie1 -KO mice. Tie1 whole-body inducible KO ( Tie1 WB–/– ) was generated by crossing Tie1 fl mice with Rosa26 rtTA TetOCre mice and timed induction with Dox water. Tie1 lymphatic-specific Tie1 KO ( Tie1 LEC–/– ) was generated by crossing Tie1 fl mice with Pdpn Cre mice. ( B ) Gross phenotypes of Tie1 WB–/– embryos that were induced and examined at different time points (first number indicates the induction time point; second number indicates the harvest time point). White asterisk shows blood-filled lymphatics at E15.5 (5 of 7 of the Tie1 WB–/– embryos); arrow shows edema at E18.5 (7 of 9 of the Tie1 WB–/– embryos); and arrowhead shows chylous ascites at P2 (6 of 6 of the Tie1 WB–/– pups). ( C ) LEC-specific Tie1 KO resulted in a phenotype similar to that of whole-body KO. ( D ) Workflow of dermal LEC bulk RNA-Seq. E18.5 embryos induced at E13.5 were euthanized. The skin from each embryo was removed and placed in an Eppendorf tube for enzymatic digestion. The single-cell suspension was labeled with antibodies against CD45, CD31, and Lyve1. CD31 + LYVE1 + cells were sorted into lysis buffer for RNA extraction. Bulk RNA-Seq was performed on the Illumina HiSeq 4000 system. ( E ) Number of differentially expressed genes using a P value of less than 0.01 as the cutoff. Down, downregulated; Up, upregulated. ( F ) Volcano plot shows some of the most differentially expressed genes including Ccl21a , valve genes and tip cell–enriched genes. ( G ) Heatmaps of manually selected vascular-relevant genes categorized as labeled. ( H ) Overlap between our data set (blue) and a data set from HUVECs with transcriptionally active FOXO1 (orange). Some commonly regulated genes are listed in the square, including tip cell genes (red), polarization genes (blue), ion channel genes (green), and valve genes (black).

Article Snippet: Silencer Select negative siCtr (s4390843) and siRNA targeting TIE1 (s14141), TIE2 (s13983), ANGPT2 (s1359), ADAM10 (s1004), and ADAM17 (s13719) were purchased from Thermo Fisher Scientific. siRNAs targeting FKHR/FOXO1 (sc-35382) and PIEZO1 (sc-93227) were purchased from Santa Cruz Biotechnology.

Techniques: Generated, RNA Sequencing Assay, Suspension, Labeling, Lysis, RNA Extraction

( A ) Whole mounts of E16.5 skin with lymphatic vessels stained for PROX1 and FOXO1. Nuclei within areas of LECs from WT control, Tie1 WB–/–E13.5 , and Tie2 WB–/–E13.5 mice are indicated by arrowheads (branching points) and arrows (nonbranching points). Scale bars: 20 μm. ( B ) Quantification of FOXO1 localizations in PROX1 + LECs. Averaged values calculated from 3 mice in each group are presented. ( C ) P1 Tie1 WB–/–E18.5 pups and their littermate controls were intraperitoneally injected with 1 μg/g BW Hepta-ANG1 or PBS. After a 30-minute period, pups were euthanized, and mesentery specimens were harvested and stained for PROX1 and FOXO1. Scale bars: 50 μm. ( D ) Quantification of FOXO1 localizations from 4 mice in each group. ( E ) FOXO1 staining of HDLECs transfected with siCtr, si TIE1 , or si TIE2 for 48 hours and subsequently treated with Hepta-ANG1 (1 μg/mL) or PBS for 30 minutes. This experiment was replicated 3 times. Scale bar: 50 μm. ( F ) Quantification of FOXO1 localization in 3 fields of view selected from each group. ( G ) Western blot analysis of p-AKT levels in HDLECs transfected with siCtr, si TIE1 , or si TIE2 and treated with either vehicle or Hepta-ANG1. Each band represents a biological replicate sample ( n = 3). Data are expressed as the mean ± SD. ** P < 0.01 and *** P < 0.001, by 2-tailed, unpaired Student’s t test ( D ) and 2-way ANOVA followed by Tukey’s multiple-comparison test ( F and G ).

Journal: The Journal of Clinical Investigation

Article Title: The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development

doi: 10.1172/JCI176577

Figure Lengend Snippet: ( A ) Whole mounts of E16.5 skin with lymphatic vessels stained for PROX1 and FOXO1. Nuclei within areas of LECs from WT control, Tie1 WB–/–E13.5 , and Tie2 WB–/–E13.5 mice are indicated by arrowheads (branching points) and arrows (nonbranching points). Scale bars: 20 μm. ( B ) Quantification of FOXO1 localizations in PROX1 + LECs. Averaged values calculated from 3 mice in each group are presented. ( C ) P1 Tie1 WB–/–E18.5 pups and their littermate controls were intraperitoneally injected with 1 μg/g BW Hepta-ANG1 or PBS. After a 30-minute period, pups were euthanized, and mesentery specimens were harvested and stained for PROX1 and FOXO1. Scale bars: 50 μm. ( D ) Quantification of FOXO1 localizations from 4 mice in each group. ( E ) FOXO1 staining of HDLECs transfected with siCtr, si TIE1 , or si TIE2 for 48 hours and subsequently treated with Hepta-ANG1 (1 μg/mL) or PBS for 30 minutes. This experiment was replicated 3 times. Scale bar: 50 μm. ( F ) Quantification of FOXO1 localization in 3 fields of view selected from each group. ( G ) Western blot analysis of p-AKT levels in HDLECs transfected with siCtr, si TIE1 , or si TIE2 and treated with either vehicle or Hepta-ANG1. Each band represents a biological replicate sample ( n = 3). Data are expressed as the mean ± SD. ** P < 0.01 and *** P < 0.001, by 2-tailed, unpaired Student’s t test ( D ) and 2-way ANOVA followed by Tukey’s multiple-comparison test ( F and G ).

Techniques: Staining, Injection, Transfection, Western Blot, Comparison

( A ) HDLECs were transfected with siCtr, si TIE1 , si TIE2 , or si TIE1 /si TIE2 for 48 hours and then exposed to either vehicle or 250 nM Yoda1 for 30 minutes. After fixation, cells were stained for FOXO1. This experiment was carried out concurrently with the one depicted in . The samples used in the siCtr plus DMSO and siCtr plus Yoda1 groups were identical to those in . Scale bar: 50 μm. ( B ) Quantification of cells exhibiting nuclear FOXO1 staining. This experiment was repeated 3 times. ( C ) HDLECs were transfected with the specified siRNAs and treated with vehicle or Yoda1 as described above. Cell lysates were subjected to Western blot analysis to assess AKT phosphorylation. TIE2 was isolated from cell lysates via immunoprecipitation and subsequently analyzed by Western blotting to evaluate its phosphorylation status. Each band represents a biological replicate sample ( n = 3). ( D ) qPCR analysis of HDLECs transfected with siCtr or si TIE1 and subsequently treated with Yoda1 (250 nM, 24 hours) or vehicle. Expression levels of TIE1 , FOXC2 , GATA2 , GJA4 , and ITGA9 genes were measured. Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-way ANOVA followed by Tukey’s multiple-comparison test ( B and C ) and 2-tailed, unpaired Student’s t test ( D ).

Journal: The Journal of Clinical Investigation

Article Title: The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development

doi: 10.1172/JCI176577

Figure Lengend Snippet: ( A ) HDLECs were transfected with siCtr, si TIE1 , si TIE2 , or si TIE1 /si TIE2 for 48 hours and then exposed to either vehicle or 250 nM Yoda1 for 30 minutes. After fixation, cells were stained for FOXO1. This experiment was carried out concurrently with the one depicted in . The samples used in the siCtr plus DMSO and siCtr plus Yoda1 groups were identical to those in . Scale bar: 50 μm. ( B ) Quantification of cells exhibiting nuclear FOXO1 staining. This experiment was repeated 3 times. ( C ) HDLECs were transfected with the specified siRNAs and treated with vehicle or Yoda1 as described above. Cell lysates were subjected to Western blot analysis to assess AKT phosphorylation. TIE2 was isolated from cell lysates via immunoprecipitation and subsequently analyzed by Western blotting to evaluate its phosphorylation status. Each band represents a biological replicate sample ( n = 3). ( D ) qPCR analysis of HDLECs transfected with siCtr or si TIE1 and subsequently treated with Yoda1 (250 nM, 24 hours) or vehicle. Expression levels of TIE1 , FOXC2 , GATA2 , GJA4 , and ITGA9 genes were measured. Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-way ANOVA followed by Tukey’s multiple-comparison test ( B and C ) and 2-tailed, unpaired Student’s t test ( D ).

Techniques: Transfection, Staining, Western Blot, Isolation, Immunoprecipitation, Expressing, Comparison

( A ) Following a 30-minute treatment with Yoda1, HDLECs displayed a reduced distribution of TIE1 at cell-cell junctions (indicated by arrows), as observed in TIE1 and tight-junction protein 1 (ZO-1) immunostaining. Scale bar: 20 μm. ( B ) Yoda1-treated HDLECs exhibited increased TIE1 shedding, as confirmed by Western blot analysis (left panel: cell lysate protein samples; right panel: protein samples obtained from TCA precipitation of the culture medium). Each band represents a biological replicate sample ( n = 3). ( C ) HDLECs treated with either siCtr or si ADAM17 were stimulated with Yoda1 or vehicle control, followed by staining for TIE1 and ZO-1. Arrows highlight the areas at cell-cell junctions where TIE1 shedding occurred. Scale bars: 50 μm. ( D ) TIE1 shedding was assessed by Western blot analysis using medium samples subjected to TCA precipitation from siCtr-, si ADAM17 -, or si ADAM10 -treated HDLECs after vehicle or Yoda1 treatment. sTIE1, soluble TIE1. Each band represents a biological replicate sample ( n = 3). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed, unpaired Student’s t test ( B ) or 2-way ANOVA followed by Tukey’s multiple-comparison test ( D ).

Journal: The Journal of Clinical Investigation

Article Title: The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development

doi: 10.1172/JCI176577

Figure Lengend Snippet: ( A ) Following a 30-minute treatment with Yoda1, HDLECs displayed a reduced distribution of TIE1 at cell-cell junctions (indicated by arrows), as observed in TIE1 and tight-junction protein 1 (ZO-1) immunostaining. Scale bar: 20 μm. ( B ) Yoda1-treated HDLECs exhibited increased TIE1 shedding, as confirmed by Western blot analysis (left panel: cell lysate protein samples; right panel: protein samples obtained from TCA precipitation of the culture medium). Each band represents a biological replicate sample ( n = 3). ( C ) HDLECs treated with either siCtr or si ADAM17 were stimulated with Yoda1 or vehicle control, followed by staining for TIE1 and ZO-1. Arrows highlight the areas at cell-cell junctions where TIE1 shedding occurred. Scale bars: 50 μm. ( D ) TIE1 shedding was assessed by Western blot analysis using medium samples subjected to TCA precipitation from siCtr-, si ADAM17 -, or si ADAM10 -treated HDLECs after vehicle or Yoda1 treatment. sTIE1, soluble TIE1. Each band represents a biological replicate sample ( n = 3). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed, unpaired Student’s t test ( B ) or 2-way ANOVA followed by Tukey’s multiple-comparison test ( D ).

Techniques: Immunostaining, Western Blot, TCA Precipitation, Staining, Comparison

( A ) Intracellular calcium levels in HDLECs treated with Yoda1 or vehicle were visualized using confocal microscopy with the cell-permeable Ca 2+ indicator Fluo-8 AM. Scale bar: 50 μm. ( B ) Quantification of Yoda1-induced calcium influx in HDLECs pretreated with varying concentrations of the calcium chelator BAPTA. RFU, relative fluorescence units; T–T 0 , difference between the value measured at time point (T) and the value measured immediately prior to the treatment (T 0 ). ( C ) Immunostaining for FOXO1, ANGPT2, and TIE1 in HDLECs treated with either vehicle or the calcium ionophore A23187 for 30 minutes. Arrows indicate the areas at cell-cell junctions where TIE1 shedding occurred. Scale bar: 50 μm. ( D ) Western blot analysis of TIE1 shedding and ANGPT2 exocytosis in HDLECs treated with vehicle or A23187. Each band represents a biological replicate sample ( n = 3). ( E ) Western blot analysis of Yoda1-triggered TIE1 shedding and ANGPT2 exocytosis in the presence or absence of BAPTA. Each band represents a biological replicate sample ( n = 3). Data are expressed as the mean ± SD. ** P < 0.01 and *** P < 0.001, by 2-tailed, unpaired Student’s t test ( D ) and 2-way ANOVA followed by Tukey’s multiple-comparison test ( E ).

Journal: The Journal of Clinical Investigation

Article Title: The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development

doi: 10.1172/JCI176577

Figure Lengend Snippet: ( A ) Intracellular calcium levels in HDLECs treated with Yoda1 or vehicle were visualized using confocal microscopy with the cell-permeable Ca 2+ indicator Fluo-8 AM. Scale bar: 50 μm. ( B ) Quantification of Yoda1-induced calcium influx in HDLECs pretreated with varying concentrations of the calcium chelator BAPTA. RFU, relative fluorescence units; T–T 0 , difference between the value measured at time point (T) and the value measured immediately prior to the treatment (T 0 ). ( C ) Immunostaining for FOXO1, ANGPT2, and TIE1 in HDLECs treated with either vehicle or the calcium ionophore A23187 for 30 minutes. Arrows indicate the areas at cell-cell junctions where TIE1 shedding occurred. Scale bar: 50 μm. ( D ) Western blot analysis of TIE1 shedding and ANGPT2 exocytosis in HDLECs treated with vehicle or A23187. Each band represents a biological replicate sample ( n = 3). ( E ) Western blot analysis of Yoda1-triggered TIE1 shedding and ANGPT2 exocytosis in the presence or absence of BAPTA. Each band represents a biological replicate sample ( n = 3). Data are expressed as the mean ± SD. ** P < 0.01 and *** P < 0.001, by 2-tailed, unpaired Student’s t test ( D ) and 2-way ANOVA followed by Tukey’s multiple-comparison test ( E ).

Techniques: Confocal Microscopy, Fluorescence, Immunostaining, Western Blot, Comparison

Activation of the mechanosensory cation channel PIEZO1 initiates a cascade of events crucial for lymphatic development. This activation leads to an increase in intracellular calcium levels, subsequently triggering the release of ANGPT2 from intracellular vesicles and activation of the protease ADAM17. ADAM17 cleaves cell membrane–anchored TIE1, facilitating the binding and activation of TIE2 by the released ANGPT2. This activation, in turn, initiates downstream signaling through the PI3K/AKT/FOXO1 pathways. The translocation of FOXO1 from the nucleus to the cytoplasm alleviates its repression of lymphatic valve– and other lymphatic-associated genes that are crucial for lymphatic development. This finely orchestrated axis plays a pivotal role in governing the intricate processes involved in the formation and maturation of the lymphatic system.

Journal: The Journal of Clinical Investigation

Article Title: The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development

doi: 10.1172/JCI176577

Figure Lengend Snippet: Activation of the mechanosensory cation channel PIEZO1 initiates a cascade of events crucial for lymphatic development. This activation leads to an increase in intracellular calcium levels, subsequently triggering the release of ANGPT2 from intracellular vesicles and activation of the protease ADAM17. ADAM17 cleaves cell membrane–anchored TIE1, facilitating the binding and activation of TIE2 by the released ANGPT2. This activation, in turn, initiates downstream signaling through the PI3K/AKT/FOXO1 pathways. The translocation of FOXO1 from the nucleus to the cytoplasm alleviates its repression of lymphatic valve– and other lymphatic-associated genes that are crucial for lymphatic development. This finely orchestrated axis plays a pivotal role in governing the intricate processes involved in the formation and maturation of the lymphatic system.

Techniques: Activation Assay, Membrane, Binding Assay, Translocation Assay

Journal: Cells

Article Title: GW501516-Mediated Targeting of Tetraspanin 15 Regulates ADAM10-Dependent N-Cadherin Cleavage in Invasive Bladder Cancer Cells.

doi: 10.3390/cells13080708

Figure Lengend Snippet: Figure 2. Involvement of ADAM10 and γ-secretase complex in N-cadherin cleavage. (A) T24 cells were treated with increasing concentrations of the γ-secretase inhibitor DAPT (5, 10, 20 µM) for 24 h. Total cell lysates were subjected to immunoblotting with the 3B9 antibody raised against the cytoplasmic domain

Article Snippet: Control siRNA (negative control for evaluating RNAi off-target effects, sc-37007), PPARβ/δ (sc-36305), Adam10 (sc-41410), and Tspan15 (sc-90664) specific siRNA (pool of 3 target-specific 19–25 nucleotide siRNAs) were from Santa Cruz Biotechnology.

Techniques: Western Blot

Journal: Cells

Article Title: GW501516-Mediated Targeting of Tetraspanin 15 Regulates ADAM10-Dependent N-Cadherin Cleavage in Invasive Bladder Cancer Cells.

doi: 10.3390/cells13080708

Figure Lengend Snippet: Figure 3. Tspan15 controls ADAM10-mediated cleavage of N-cadherin in T24 cells. (A) Validation of Tspan15 knockdown efficiency at the mRNA level by RTq-PCR analysis in T24 cells transfected with 25 nM TSPAN15 siRNA. Data are means ± SD of three independent experiments performed in triplicates (* p < 0.05). (B) Western blotting analysis of TSPAN15 protein depletion in TSPAN15 siRNA

Techniques: Biomarker Discovery, Knockdown, Transfection, Western Blot

Journal: Cells

Article Title: GW501516-Mediated Targeting of Tetraspanin 15 Regulates ADAM10-Dependent N-Cadherin Cleavage in Invasive Bladder Cancer Cells.

doi: 10.3390/cells13080708

Figure Lengend Snippet: Figure 6. ADAM10 is not regulated by PPARβ/δ in T24 cells. Cells were treated with increasing concentrations of GW501516 (1, 15, 25 µM) for 24 h. (A) Adam10 mRNA expression was analyzed by RTq-PCR. Fold inductions represent a comparison with vehicle-treated cells (set at 1) in the absence of GW501516. Data are means ± SD of three independent experiments performed in triplicates. (B) Western blotting analysis of ADAM10 protein (proform and mature form) in control and stimulated cells. β-actin was used as an internal loading control. The graphs depict densitometric analysis results of Western blots by using ImageJ. Data are means ± SD of three independent experiments performed in triplicates. (C) Plin2, a PPARβ target gene, was used as a positive control to validate the efficiency of GW501516. Plin2 mRNA expression was analyzed by RTq-PCR. Fold inductions represent a comparison with vehicle-treated cells (set at 1) in the absence of GW501516. Data are means ± SD of three independent experiments performed in triplicates (* p < 0.05).

Techniques: Expressing, Comparison, Western Blot, Control, Positive Control

( A ) Experimental design of affinity enrichment mass spectrometry for the identification of the CD81 protein complex in human hepatoma cell lines (IntAct entry https://www.ebi.ac.uk/intact/search?query=IM-25678 ). (B) Results of two-sample t-tests comparing protein label free quantification (LFQ) intensities of CD81 co-IPS from Lunet N hCD81 cells (CD81) with those of Lunet N (CTRL) displayed as a volcano plot. (C) Same as in (B), comparing protein LFQ intensities of HA co-IPS from Lunet N hCD81-HA (CD81) with those of Lunet N (CTRL). (D) Ranked protein abundances in whole cell proteomes of human hepatoma cells. (E) Protein enrichment in CD81 co-IPs from primary human hepatocytes (PHH) of two independent donors. Isotype control antibodies were used as control (CTRL). (F) Transcript expression of ADAM10 , HCV entry factors, receptors transactivated by ADAM10 ( EGFR , ERBB2 , ERBB3 , TNFRSF1A ) and negative controls ( CD8A , CLEC4M ) across cells in healthy human liver tissue from nine donors using the cell type annotation from Aizarani et al. . The size of the dots represents the percentage of transcript positive cells per cell type with at least one detected transcript for the respective gene, and the colour the average expression in log 2 scale. Mass spectrometry datasets show median values of four independent experiments for the hepatoma cells. Proteomics data derived from and described in (Bruening et al. 2018) .

Journal: PLOS Pathogens

Article Title: The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity

doi: 10.1371/journal.ppat.1011759

Figure Lengend Snippet: ( A ) Experimental design of affinity enrichment mass spectrometry for the identification of the CD81 protein complex in human hepatoma cell lines (IntAct entry https://www.ebi.ac.uk/intact/search?query=IM-25678 ). (B) Results of two-sample t-tests comparing protein label free quantification (LFQ) intensities of CD81 co-IPS from Lunet N hCD81 cells (CD81) with those of Lunet N (CTRL) displayed as a volcano plot. (C) Same as in (B), comparing protein LFQ intensities of HA co-IPS from Lunet N hCD81-HA (CD81) with those of Lunet N (CTRL). (D) Ranked protein abundances in whole cell proteomes of human hepatoma cells. (E) Protein enrichment in CD81 co-IPs from primary human hepatocytes (PHH) of two independent donors. Isotype control antibodies were used as control (CTRL). (F) Transcript expression of ADAM10 , HCV entry factors, receptors transactivated by ADAM10 ( EGFR , ERBB2 , ERBB3 , TNFRSF1A ) and negative controls ( CD8A , CLEC4M ) across cells in healthy human liver tissue from nine donors using the cell type annotation from Aizarani et al. . The size of the dots represents the percentage of transcript positive cells per cell type with at least one detected transcript for the respective gene, and the colour the average expression in log 2 scale. Mass spectrometry datasets show median values of four independent experiments for the hepatoma cells. Proteomics data derived from and described in (Bruening et al. 2018) .

Article Snippet: Huh-7.5-Fluc cells were seeded in 24 well plates at a density of 2x10 5 cells/well and cultured for 24 h before transfection with 7.5 pmol of a pool of three different siRNAs targeting ADAM10 mRNA (Thermo Fisher scientific; siRNA IDs: s1004, s1005, s1006) or ADAM17 mRNA (Thermo Fisher scientific; siRNA IDs: s13719, s13718, s13720) using Lipofectamine RNAiMAX (Thermo Fisher scientific) according to manufacturer´s instructions.

Techniques: Mass Spectrometry, Protein Enrichment, Expressing, Derivative Assay

(A,B) ADAM10 (A) and CD81 (B) surface expression levels measured by antibody staining and flow cytometry on Huh-7.5-Fluc cells treated with a pool of 3 siRNAs targeting ADAM10 , CD81 , or a non-targeting siRNA control. Mean fluorescence intensity (MFI) values normalized to control siRNA are shown. (C,D) ADAM10 (C) and CD81 (D) protein expression levels determined by immunoblot of lysates from Huh7.5-Fluc cells treated as in (A). (E) Huh-7.5-FLuc cells were treated as in (A) and (B) for 48 h and infected with a Jc1-based renilla luciferase reporter virus (JcR2A). Infectivity was quantified 72 hpi as renilla luciferase activity. Infectivity values are normalized to non-targeting siRNA control. (F) Cell viability of cells treated as in (E) was assessed by MTT assay. (G) . ADAM10 surface expression in cells treated with the ADAM10 inhibitor GI254023X or DMSO was measured as in (A). (H) HCV infection of cells pre-treated with GI254023X or SR-BI inhibitor ITX5061 for 16h. Virus inoculum was supplemented with inhibitors, removed 4 hpi and replaced with medium supplemented with inhibitors. Infectivity was measured and values were normalized as in (E). (I) Cell viability upon inhibitor treatment as in (H) was assessed by MTT assay and values were normalized to DMSO control. (J) Huh-7.5-FLuc cells were treated with a sgRNA targeting ADAM10 exon 11 and a non-targeting sgRNA control. ADAM10 expression was measured as in (A). (K) ADAM10 KO and control cells were infected as in (E). Infectivity was measured as described in E and values were normalized to non-targeting control. (L) Huh-7.5-Fluc cells were transduced with lentiviruses encoding ADAM10 (ADAM10 OE) or control lentiviruses (EV). Surface expression levels of ADAM10 overexpressing cells were quantified by antibody staining and flow cytometry. (M) Huh-7.5-Fluc cells were transduced as in (L). 72 h post transduction, cells were infected with JcR2a. 72 h post infection, infection was quantified as in (E). (N) ADAM10 KO and control cells were transduced as in (L). Cells were then infected with JcR2a 72 h post transduction. Infectivity was measured 72 hpi as in (E). Data show the mean +/- SD of three biological replicates. One-way ANOVA with Dunnett´s multiple comparison test (E and H). Paired t-test (K and M). * P < 0.05; ** P < 0.01; *** P ≤ 0.001.

Journal: PLOS Pathogens

Article Title: The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity

doi: 10.1371/journal.ppat.1011759

Figure Lengend Snippet: (A,B) ADAM10 (A) and CD81 (B) surface expression levels measured by antibody staining and flow cytometry on Huh-7.5-Fluc cells treated with a pool of 3 siRNAs targeting ADAM10 , CD81 , or a non-targeting siRNA control. Mean fluorescence intensity (MFI) values normalized to control siRNA are shown. (C,D) ADAM10 (C) and CD81 (D) protein expression levels determined by immunoblot of lysates from Huh7.5-Fluc cells treated as in (A). (E) Huh-7.5-FLuc cells were treated as in (A) and (B) for 48 h and infected with a Jc1-based renilla luciferase reporter virus (JcR2A). Infectivity was quantified 72 hpi as renilla luciferase activity. Infectivity values are normalized to non-targeting siRNA control. (F) Cell viability of cells treated as in (E) was assessed by MTT assay. (G) . ADAM10 surface expression in cells treated with the ADAM10 inhibitor GI254023X or DMSO was measured as in (A). (H) HCV infection of cells pre-treated with GI254023X or SR-BI inhibitor ITX5061 for 16h. Virus inoculum was supplemented with inhibitors, removed 4 hpi and replaced with medium supplemented with inhibitors. Infectivity was measured and values were normalized as in (E). (I) Cell viability upon inhibitor treatment as in (H) was assessed by MTT assay and values were normalized to DMSO control. (J) Huh-7.5-FLuc cells were treated with a sgRNA targeting ADAM10 exon 11 and a non-targeting sgRNA control. ADAM10 expression was measured as in (A). (K) ADAM10 KO and control cells were infected as in (E). Infectivity was measured as described in E and values were normalized to non-targeting control. (L) Huh-7.5-Fluc cells were transduced with lentiviruses encoding ADAM10 (ADAM10 OE) or control lentiviruses (EV). Surface expression levels of ADAM10 overexpressing cells were quantified by antibody staining and flow cytometry. (M) Huh-7.5-Fluc cells were transduced as in (L). 72 h post transduction, cells were infected with JcR2a. 72 h post infection, infection was quantified as in (E). (N) ADAM10 KO and control cells were transduced as in (L). Cells were then infected with JcR2a 72 h post transduction. Infectivity was measured 72 hpi as in (E). Data show the mean +/- SD of three biological replicates. One-way ANOVA with Dunnett´s multiple comparison test (E and H). Paired t-test (K and M). * P < 0.05; ** P < 0.01; *** P ≤ 0.001.

Techniques: Expressing, Staining, Flow Cytometry, Fluorescence, Western Blot, Infection, Luciferase, Virus, Activity Assay, MTT Assay, Transduction, Comparison

(A) Scheme of the generation of E1/E2 pseudotyped lentivirus. Pseudoparticles encode a firefly luciferase for quantification of transduction efficiency. (B) Huh-7.5 cells treated for 48 h with pools of 3 siRNAs targeting ADAM10 or CD81 or a non-targeting control (CTRL) were transduced with lentiviruses pseudotyped with HCV E1/E2 glycoproteins. 72 h post transduction, transduction efficiency was measured as firefly luciferase activity. Values represented relative to non-targeting control (CTRL). (C) Huh-7.5 cells were treated with the ADAM10 inhibitor GI254023X and the SR-BI inhibitor ITX 5061 for 16 h and transduced with the lentiviruses described in (A). Infectivity was measured 72 h post transduction as in (B). (D,E) Huh-7.5-Fluc cells were electroporated with a wildtype (D) or replication deficient ΔGDD mutant (E) HCV subgenomic replicon (SGR) encoding firefly luciferase and treated with ADAM10 inhibitor GI254023X or the NS3/4A inhibitor telaprevir during and after electroporation. HCV subgenome replication was assessed as firefly luciferase activity. Data show the fold change over 4 h luciferase activity values (F) ADAM10 surface expression was measured by antibody staining and flow cytometry at the indicated time points post 12 h inhibitor treatment and washout. Mean fluorescence intensity (MFI) values are normalized to DMSO at each time point. (G) Scheme of the ADAM10 inhibitor time of addition assay. Huh-7.5-Fluc cells were treated with ADAM10 inhibitor GI254023X or DMSO prior, during or after HCV virus inoculation. (H) HCV infection of cells treated with inhibitor as shown in (G). Infectivity was measured by renilla luciferase assay at 72 hpi. Infectivity values are normalized to DMSO controls for each time point. (I) Scheme showing the HCV cell-to-cell spread assay. Huh-7.5-Fluc cells were infected with GFP-tagged HCV (MOI 0.1). 7 dpi, Huh-7.5-Fluc GFP+ cells were mixed with uninfected Huh-7.5-Fluc cells pretreated with the ADAM10 inhibitor GI254023X, the matrix metalloprotease inhibitor BB-94, or DMSO at a 1:5 ratio. 4 h later, cells were overlayed with Avicel-containing medium supplemented with inhibitors or DMSO. Infectivity was measured 72 hpi using flow cytometry. (J) Percentage of GFP positive cells 7 dpi. (K) Cell-to-cell spread of HCV measured as percentage of GFP positive cells in the conditions described in (I) was normalized to DMSO controls. Data shows the mean +/- SD of three biological replicates. Two- way ANOVA with Dunnett´s multiple comparison test (D). Unpaired t-test (H). One-way ANOVA with Dunnett´s multiple comparison test (K). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Journal: PLOS Pathogens

Article Title: The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity

doi: 10.1371/journal.ppat.1011759

Figure Lengend Snippet: (A) Scheme of the generation of E1/E2 pseudotyped lentivirus. Pseudoparticles encode a firefly luciferase for quantification of transduction efficiency. (B) Huh-7.5 cells treated for 48 h with pools of 3 siRNAs targeting ADAM10 or CD81 or a non-targeting control (CTRL) were transduced with lentiviruses pseudotyped with HCV E1/E2 glycoproteins. 72 h post transduction, transduction efficiency was measured as firefly luciferase activity. Values represented relative to non-targeting control (CTRL). (C) Huh-7.5 cells were treated with the ADAM10 inhibitor GI254023X and the SR-BI inhibitor ITX 5061 for 16 h and transduced with the lentiviruses described in (A). Infectivity was measured 72 h post transduction as in (B). (D,E) Huh-7.5-Fluc cells were electroporated with a wildtype (D) or replication deficient ΔGDD mutant (E) HCV subgenomic replicon (SGR) encoding firefly luciferase and treated with ADAM10 inhibitor GI254023X or the NS3/4A inhibitor telaprevir during and after electroporation. HCV subgenome replication was assessed as firefly luciferase activity. Data show the fold change over 4 h luciferase activity values (F) ADAM10 surface expression was measured by antibody staining and flow cytometry at the indicated time points post 12 h inhibitor treatment and washout. Mean fluorescence intensity (MFI) values are normalized to DMSO at each time point. (G) Scheme of the ADAM10 inhibitor time of addition assay. Huh-7.5-Fluc cells were treated with ADAM10 inhibitor GI254023X or DMSO prior, during or after HCV virus inoculation. (H) HCV infection of cells treated with inhibitor as shown in (G). Infectivity was measured by renilla luciferase assay at 72 hpi. Infectivity values are normalized to DMSO controls for each time point. (I) Scheme showing the HCV cell-to-cell spread assay. Huh-7.5-Fluc cells were infected with GFP-tagged HCV (MOI 0.1). 7 dpi, Huh-7.5-Fluc GFP+ cells were mixed with uninfected Huh-7.5-Fluc cells pretreated with the ADAM10 inhibitor GI254023X, the matrix metalloprotease inhibitor BB-94, or DMSO at a 1:5 ratio. 4 h later, cells were overlayed with Avicel-containing medium supplemented with inhibitors or DMSO. Infectivity was measured 72 hpi using flow cytometry. (J) Percentage of GFP positive cells 7 dpi. (K) Cell-to-cell spread of HCV measured as percentage of GFP positive cells in the conditions described in (I) was normalized to DMSO controls. Data shows the mean +/- SD of three biological replicates. Two- way ANOVA with Dunnett´s multiple comparison test (D). Unpaired t-test (H). One-way ANOVA with Dunnett´s multiple comparison test (K). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Techniques: Luciferase, Transduction, Activity Assay, Infection, Mutagenesis, Electroporation, Expressing, Staining, Flow Cytometry, Fluorescence, Virus, Comparison

(A) Surface expression of HCV entry factors on Huh-7.5-Fluc cells treated with the ADAM10 inhibitor GI254023X or DMSO for 72 h. Values represent mean and SD of the mean fluorescence intensity of three independent experiments and are presented as percentage of DMSO control values. (B) Cell-to-cell spread of HCV in Huh-7.5-Fluc cells treated with the matrix metalloprotease inhibitor BB-94 was quantified as in . (C) ADAM10 surface expression upon BB-94 or DMSO treatment quantified as in (A). (D) HCV infection of Huh-7.5-Fluc cells treated with non-targeting siRNA control or pools of 3 siRNAs targeting ADAM17 or CD81 . 48 h post siRNA transfection, cells were infected with JcR2a for 72 h. Infection levels were measured by renilla luciferase assay and normalized to the non-targeting control. (E) Huh-7.5-Fluc cells were treated as in (D). 48 h post siRNA transfection, ADAM17 surface expression levels were analyzed by antibody staining and flow cytometry (F) Scheme of receptors and downstream kinases activated by ADAM10 sheddase activity. (G) Representative histogram showing increased ERK1/2 phosphorylation upon EGF stimulation in DMSO-treated, serum-starved cells. (H) ERK1/2 phosphorylation in Huh-7.5-Fluc cells treated with ADAM10 inhibitor GI254023X or DMSO for 48 h, serum starved for 12 h or left in medium containing serum and stimulated with 1 μg/mL EGF for 15 min. Values represent mean and SD of the mean fluorescence intensity of four independent experiments and presented as percentage of DMSO control values. (I,J) pERK1/2 (I) and total ERK1/2 (J) protein levels analyzed by immunoblot from Huh-7.5-Fluc cell lysates after serum starvation and EGF stimulation for 15 min as in (G). (K,L) pEGFR levels measured by flow cytometry (K) or immunoblot (L) in Huh-7.5-Fluc cells treated with ADAM10 inhibitor GI254023X or DMSO for 48 h, serum starved for 12 h and stimulated with EGF for 15 min. Mean fluorescence intensity analysis as in (A). (M) Rescue of ADAM10 KO cell infection phenotype by pretreatment 16 h prior to and during HCV infection with the ADAM10 substrates TNF-α, EGF, or E-cadherin. Infection measured as renilla luciferase activity as in (D) and values normalized to the respective scrambled guide RNA control. (N) Relative cell number of ADAM10 KO and control cells upon treatment with ADAM10 substrates as in (M). Values normalized to scrambled guide RNA control cells. Data show the mean +/- SD of three biological replicates unless stated otherwise. One-way ANOVA with Dunnett´s multiple comparison test (B, D). Paired t-test (, E, H, M). * P < 0.05; ** P < 0.01; *** P < 0.001.

Journal: PLOS Pathogens

Article Title: The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity

doi: 10.1371/journal.ppat.1011759

Figure Lengend Snippet: (A) Surface expression of HCV entry factors on Huh-7.5-Fluc cells treated with the ADAM10 inhibitor GI254023X or DMSO for 72 h. Values represent mean and SD of the mean fluorescence intensity of three independent experiments and are presented as percentage of DMSO control values. (B) Cell-to-cell spread of HCV in Huh-7.5-Fluc cells treated with the matrix metalloprotease inhibitor BB-94 was quantified as in . (C) ADAM10 surface expression upon BB-94 or DMSO treatment quantified as in (A). (D) HCV infection of Huh-7.5-Fluc cells treated with non-targeting siRNA control or pools of 3 siRNAs targeting ADAM17 or CD81 . 48 h post siRNA transfection, cells were infected with JcR2a for 72 h. Infection levels were measured by renilla luciferase assay and normalized to the non-targeting control. (E) Huh-7.5-Fluc cells were treated as in (D). 48 h post siRNA transfection, ADAM17 surface expression levels were analyzed by antibody staining and flow cytometry (F) Scheme of receptors and downstream kinases activated by ADAM10 sheddase activity. (G) Representative histogram showing increased ERK1/2 phosphorylation upon EGF stimulation in DMSO-treated, serum-starved cells. (H) ERK1/2 phosphorylation in Huh-7.5-Fluc cells treated with ADAM10 inhibitor GI254023X or DMSO for 48 h, serum starved for 12 h or left in medium containing serum and stimulated with 1 μg/mL EGF for 15 min. Values represent mean and SD of the mean fluorescence intensity of four independent experiments and presented as percentage of DMSO control values. (I,J) pERK1/2 (I) and total ERK1/2 (J) protein levels analyzed by immunoblot from Huh-7.5-Fluc cell lysates after serum starvation and EGF stimulation for 15 min as in (G). (K,L) pEGFR levels measured by flow cytometry (K) or immunoblot (L) in Huh-7.5-Fluc cells treated with ADAM10 inhibitor GI254023X or DMSO for 48 h, serum starved for 12 h and stimulated with EGF for 15 min. Mean fluorescence intensity analysis as in (A). (M) Rescue of ADAM10 KO cell infection phenotype by pretreatment 16 h prior to and during HCV infection with the ADAM10 substrates TNF-α, EGF, or E-cadherin. Infection measured as renilla luciferase activity as in (D) and values normalized to the respective scrambled guide RNA control. (N) Relative cell number of ADAM10 KO and control cells upon treatment with ADAM10 substrates as in (M). Values normalized to scrambled guide RNA control cells. Data show the mean +/- SD of three biological replicates unless stated otherwise. One-way ANOVA with Dunnett´s multiple comparison test (B, D). Paired t-test (, E, H, M). * P < 0.05; ** P < 0.01; *** P < 0.001.

Techniques: Expressing, Fluorescence, Infection, Transfection, Luciferase, Staining, Flow Cytometry, Activity Assay, Western Blot, Comparison

(A) CHIKV-GFP (ECSA LR2006) infection of Huh-7.5-Fluc cells treated with the ADAM10 inhibitor GI254023X or DMSO (MOI 1). Infection levels at 24 h were measured by quantification of GFP signal and normalization to DMSO control. (B) VEEV-GFP (TC-83) infection (MOI 0.1) of Huh-7.5-Fluc cells treated with ADAM10 inhibitor or solvent control as in (A). (C) Renilla luciferase reporter hCoV-229E infection of Huh-7.5-Fluc cells treated as in (A). 72 h post infection, renilla luciferase activity was determined as a measure of infection. Data show the mean +/- SD of three biological replicates.

Journal: PLOS Pathogens

Article Title: The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity

doi: 10.1371/journal.ppat.1011759

Figure Lengend Snippet: (A) CHIKV-GFP (ECSA LR2006) infection of Huh-7.5-Fluc cells treated with the ADAM10 inhibitor GI254023X or DMSO (MOI 1). Infection levels at 24 h were measured by quantification of GFP signal and normalization to DMSO control. (B) VEEV-GFP (TC-83) infection (MOI 0.1) of Huh-7.5-Fluc cells treated with ADAM10 inhibitor or solvent control as in (A). (C) Renilla luciferase reporter hCoV-229E infection of Huh-7.5-Fluc cells treated as in (A). 72 h post infection, renilla luciferase activity was determined as a measure of infection. Data show the mean +/- SD of three biological replicates.

Techniques: Infection, Solvent, Luciferase, Activity Assay

(A) ADAM10 surface expression on primary human hepatocytes (PHH) upon ADAM10 inhibitor GI254023X treatment in presence or absence of JAK-STAT inhibitor ruxolitinib at 96 h post inhibitor treatment. (B) Quantification of ADAM10 surface staining as in (A) for PHH from three independent donors measured as mean fluorescence intensity (MFI). (C) Effect of ADAM10 inhibition on PHH infection with HCV upon pre-treatment with the Jak-STAT inhibitor ruxolitinib with and without ADAM10 inhibitor GI254023X for 24h. Cells were then infected with cell-culture adapted HCV P100 (supplemented with ruxolitinib and GI254023X as indicated) at MOI 1. Virus inoculum was removed 5h post infection and replaced with medium containing ruxolitinib and GI254023X as indicated. Virus supernatants were collected 24, 48 and 72 hpi and titrated on Huh-7.5 cells by TCID50. (C). Data show the mean +/- SEM of three biological replicates, i.e. PHH from three independent donors (B,C) or a representative experiment (A). Two-way ANOVA with Sidak´s multiple comparison test (ns = non-significant).

Journal: PLOS Pathogens

Article Title: The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity

doi: 10.1371/journal.ppat.1011759

Figure Lengend Snippet: (A) ADAM10 surface expression on primary human hepatocytes (PHH) upon ADAM10 inhibitor GI254023X treatment in presence or absence of JAK-STAT inhibitor ruxolitinib at 96 h post inhibitor treatment. (B) Quantification of ADAM10 surface staining as in (A) for PHH from three independent donors measured as mean fluorescence intensity (MFI). (C) Effect of ADAM10 inhibition on PHH infection with HCV upon pre-treatment with the Jak-STAT inhibitor ruxolitinib with and without ADAM10 inhibitor GI254023X for 24h. Cells were then infected with cell-culture adapted HCV P100 (supplemented with ruxolitinib and GI254023X as indicated) at MOI 1. Virus inoculum was removed 5h post infection and replaced with medium containing ruxolitinib and GI254023X as indicated. Virus supernatants were collected 24, 48 and 72 hpi and titrated on Huh-7.5 cells by TCID50. (C). Data show the mean +/- SEM of three biological replicates, i.e. PHH from three independent donors (B,C) or a representative experiment (A). Two-way ANOVA with Sidak´s multiple comparison test (ns = non-significant).

Techniques: Expressing, Staining, Fluorescence, Inhibition, Infection, Cell Culture, Virus, Comparison

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1) Product Images from "GW501516-Mediated Targeting of Tetraspanin 15 Regulates ADAM10-Dependent N-Cadherin Cleavage in Invasive Bladder Cancer Cells."

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