Journal: World Journal of Gastroenterology

Article Title: Fas counterattack in cholangiocarcinoma: A mechanism for immune evasion in human hilar cholangiocarcinomas

doi: 10.3748/wjg.v7.i6.860

Figure Lengend Snippet: FasL positive in human hilar cholangiocarcinom as (brown). SABC × 200

Article Snippet: Immunohistochemistry for FasL and CD45 Detection of FasL expression and CD45 positive cells was performed using a rabbit polyclonal anti-human FasL specific IgG and a mouse anti-human monoclonal antibody on paraffin sections of human cholangiocarcinoma respectively (Boster Biological Technology Company, Wuhan, China).

Techniques:

Journal: World Journal of Gastroenterology

Article Title: Fas counterattack in cholangiocarcinoma: A mechanism for immune evasion in human hilar cholangiocarcinomas

doi: 10.3748/wjg.v7.i6.860

Figure Lengend Snippet: Expression of FasL in cholangiocarcinoma cell line. QBC939 × 200

Article Snippet: Immunohistochemistry for FasL and CD45 Detection of FasL expression and CD45 positive cells was performed using a rabbit polyclonal anti-human FasL specific IgG and a mouse anti-human monoclonal antibody on paraffin sections of human cholangiocarcinoma respectively (Boster Biological Technology Company, Wuhan, China).

Techniques: Expressing

Journal: World Journal of Gastroenterology

Article Title: Fas counterattack in cholangiocarcinoma: A mechanism for immune evasion in human hilar cholangiocarcinomas

doi: 10.3748/wjg.v7.i6.860

Figure Lengend Snippet: Expression of FasL mRNA in human cholangiocarcinoma cells QBC939. M: DL 2000 Marker; 1: FasL; 2: FasL+β-actin

Article Snippet: Immunohistochemistry for FasL and CD45 Detection of FasL expression and CD45 positive cells was performed using a rabbit polyclonal anti-human FasL specific IgG and a mouse anti-human monoclonal antibody on paraffin sections of human cholangiocarcinoma respectively (Boster Biological Technology Company, Wuhan, China).

Techniques: Expressing, Marker

Journal: World Journal of Gastroenterology

Article Title: Fas counterattack in cholangiocarcinoma: A mechanism for immune evasion in human hilar cholangiocarcinomas

doi: 10.3748/wjg.v7.i6.860

Figure Lengend Snippet: Western blotting of FasL protein with mAb from QBC939 cell cultures clone 33 from QBC939 cell cultures

Article Snippet: Immunohistochemistry for FasL and CD45 Detection of FasL expression and CD45 positive cells was performed using a rabbit polyclonal anti-human FasL specific IgG and a mouse anti-human monoclonal antibody on paraffin sections of human cholangiocarcinoma respectively (Boster Biological Technology Company, Wuhan, China).

Techniques: Western Blot

Journal: International Journal of Molecular Medicine

Article Title: ADAM10 promotes the proliferation of ligamentum flavum cells by activating the PI3K/AKT pathway

doi: 10.3892/ijmm.2020.4809

Figure Lengend Snippet: ADAM10 was enriched in the DEGs. The top 10 key genes were screened through the PPI network map. The different colors in the image merely represent different genes and have no other substantial meaning. ADAM10, A disintegrin and metalloproteinase 10; DEGs, differentially expressed genes; PPI, protein-protein interaction.

Article Snippet: The sequences of the target gene A disintegrin and metalloproteinase 10 (ADAM10) and a corresponding empty plasmid were obtained from Sangon Biotech (Shanghai) Co., Ltd.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: ADAM10 promotes the proliferation of ligamentum flavum cells by activating the PI3K/AKT pathway

doi: 10.3892/ijmm.2020.4809

Figure Lengend Snippet: Silencing of ADAM10 inhibits PI3K/AKT pathways (A) Western blot analysis of ADAM10 protein in the 3 groups of cells. (B) RT-qPCR analysis of ADAM10 mRNA in the 3 groups of cells. (C) Western blot analysis of ADAM10 protein in the 3 groups of cells. (D) Western blot analysis of p-PI3K and total PI3K protein in the 3 groups of cells. (E) Western blot analysis of normalized p-PI3K expression (p-PI3K/total PI3K) in the 3 groups of cells. (F) Western blot analysis of p-AKT and total AKT protein in the 3 groups of cells. (G) Western blot analysis of normalized p-AKT expression (p-AKT/total AKT) in the 3 groups of cells. All data are expressed as the means ± SD, * P<0.05 vs. the control1 and sham1 groups. ADAM10, A disintegrin and metalloproteinase 10.

Article Snippet: The sequences of the target gene A disintegrin and metalloproteinase 10 (ADAM10) and a corresponding empty plasmid were obtained from Sangon Biotech (Shanghai) Co., Ltd.

Techniques: Western Blot, Quantitative RT-PCR, Expressing

Journal: International Journal of Molecular Medicine

Article Title: ADAM10 promotes the proliferation of ligamentum flavum cells by activating the PI3K/AKT pathway

doi: 10.3892/ijmm.2020.4809

Figure Lengend Snippet: Silencing of ADAM10 inhibit ligamentum cell proliferation via the PI3K/AKT pathway. (A) Results of the EdU incorporation assay illustrating that the proliferative capacity of ligamentum flavum cells was significantly lower in the ADAM10(−) group than the control group and ADAM10(−) + 740Y-P group. However, the proliferative ability did not differ between the control group and the ADAM10(−) + 740Y-P group. (B) Box plot of the EdU results. (C) Results of the CCK-8 assay showing that the OD value was significantly lower in the ADAM10(−) group than the control group and ADAM10(−) + 740Y-P group. However, the OD value did not differ between the control group and ADAM10(−) + 740Y-P group at any time point. All data are expressed as the means ± SD, * P<0.05 vs. the control and ADAM10(−) + 740Y-P groups. ADAM10, A disintegrin and metalloproteinase 10.

Article Snippet: The sequences of the target gene A disintegrin and metalloproteinase 10 (ADAM10) and a corresponding empty plasmid were obtained from Sangon Biotech (Shanghai) Co., Ltd.

Techniques: Control, CCK-8 Assay

Journal: International Journal of Molecular Medicine

Article Title: ADAM10 promotes the proliferation of ligamentum flavum cells by activating the PI3K/AKT pathway

doi: 10.3892/ijmm.2020.4809

Figure Lengend Snippet: ADAM10 overexpression activates the PI3K/AKT pathway. (A) Western blot band of the ADAM10 protein in the three groups of cells. (B) PCR analysis of the ADAM10 mRNA in the three groups of cells. (C) Western blot analysis of ADAM10 protein in the 3 groups of cells. (D) Western blot analysis of p-PI3K and total PI3K protein in the 3 groups of cells. (E) Western blot analysis of normalized p-PI3K expression (p-PI3K/total PI3K) in the 3 groups of cells. (F) Western blot analysis of the p-AKT and total AKT protein in the 3 groups of cells. (G) Western blot analysis of normalized p-AKT expression (p-AKT/total AKT) in the 3 groups of cells. All data are expressed as the means ± SD, * P<0.05 vs. the control2 and sham2 groups. ADAM10, A disintegrin and metalloproteinase 10.

Article Snippet: The sequences of the target gene A disintegrin and metalloproteinase 10 (ADAM10) and a corresponding empty plasmid were obtained from Sangon Biotech (Shanghai) Co., Ltd.

Techniques: Over Expression, Western Blot, Expressing

Journal: International Journal of Molecular Medicine

Article Title: ADAM10 promotes the proliferation of ligamentum flavum cells by activating the PI3K/AKT pathway

doi: 10.3892/ijmm.2020.4809

Figure Lengend Snippet: ADAM10 overexpression promotes ligamentum flavum cell proliferation via the PI3K/AKT pathway. (A) Results of the EdU incorporation assay illustrating that the proliferative capacity of ligamentum flavum cells was significantly higher in the ADAM10(+) group than the control group and ADAM10(+) + LY294002 group. However, the proliferative ability did not differ between the control group and ADAM10(+) + LY294002 group. (B) Box plot of the EdU results. (C) Results of the CCK-8 assay showing that the OD value was significantly higher in the ADAM10(+) group than the control group and ADAM10(+) + LY294002 group. However, the OD value did not differ between the control group and ADAM10(+) + LY294002 group at any time. All data are expressed as the means ± SD, * P<0.05 vs. the control and ADAM10(+) + LY294002 group. ADAM10, A disintegrin and metalloproteinase 10.

Article Snippet: The sequences of the target gene A disintegrin and metalloproteinase 10 (ADAM10) and a corresponding empty plasmid were obtained from Sangon Biotech (Shanghai) Co., Ltd.

Techniques: Over Expression, Control, CCK-8 Assay

Journal: Redox Biology

Article Title: Role of redox-sensitive catalytic interaction with ADAM10 in mutant-selective extracellular shedding of prion protein

doi: 10.1016/j.redox.2022.102456

Figure Lengend Snippet: Analysis of the proliferation-promoting effect of inhibitory Q212P shedding (A) The effect of inhibitory Q212P shedding on cell proliferation was determined by the colony-forming assay. Wild-type and ADAM10-deficient (ΔADAM10) cells expressing Q212P-RFP were cultured for 3 weeks in the presence of Dox, and allowed to form colonies, that were visualized by staining with crystal violet (left panel). The relative number of large colonies (>2 mm diameter) out of total colonies from three independent experiments (three times in an independent experiment) are plotted as a percentage, and significance was considered with p -values (left panel). (B) The potential proliferation-promoting effect of inhibitory Q212P shedding is illustrated (C) The protocol shows the sequential centrifugation of CMs containing wtPrP-RFP and Q212P-RFP. (D) The solubility of extracellular Q212P was examined by sequential centrifugation. Cells expressing wtPrP-RFP and Q212P-RFP were induced by Dox treatment for 24 h and subjected to centrifugal fractionation as described in (C). PrPs-RFP concentrated in indicated fractions were detected by immunoblotting with an RFP-specific antibody. The plots represent the changes of surface and extracellular Q212P-RFP in indicated fractions by ADAM10 depletion ( n = 3), and significance was considered with p -values as described in “Materials and Methods”. (E) CMs of wild-type and ΔADAM10 cells expressing Q212P-RFP were centrifuged at 200,000× g for 1 h. The supernatant (S200) and pellet (P200) were subjected to immunoblotting with an RFP-specific antibody. The plots represent the changes of Q212P and N1 fragments in CMs by ADAM10 depletion ( n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Purchased antibodies included anti-FLAG M1 antibody (Merck, SF3040), anti-ADAM10 (Merck, AB19026), anti-ADAM17 (Abcam, ab2051), anti-GAPDH (Santa Cruz, SC-20357), anti-HA high affinity (Merck, 11867431001), anti-CD230 (prion) clone 3F4 (BioLegend, 800,307) and HRP-conjugated streptavidin (ThermoFisher, N100).

Techniques: Expressing, Cell Culture, Staining, Centrifugation, Solubility, Fractionation, Western Blot

Journal: Redox Biology

Article Title: Role of redox-sensitive catalytic interaction with ADAM10 in mutant-selective extracellular shedding of prion protein

doi: 10.1016/j.redox.2022.102456

Figure Lengend Snippet: Analysis of ADAM10-mediated Q212P shedding (A) ADAM10-deficient cells expressing Q212P-RFP were established using CRISPR/Cas9-mediated genome editing (ΔADAM10) and verified by RT-PCR (left panel) and immunoblotting with ADAM10-specific antibody (right panel). Note, X: non-specific band (B) The effect of ADAM10 on Q212P shedding was analyzed by pulse-chase experiments. Wild type (WT) and ΔADAM10 cells expressing Q212P-RFP were pulse-labeled for 30 min and allowed to recover for 1.5 h (“C1”), followed by the chase for an additional 4.5 h (“C2”) in the absence or presence of NEM as indicated. The fully solubilized cells and CM were subjected to immunoprecipitation with an RFP-specific antibody. Plotted are the changes of intact Q212P-RFP (left panel) and free RFPs (middle panel) in cells and CMs (right panel) by NEM (5 mM) treatment ( n = 3), and significance was considered with p -values as described in “Materials and Methods”. Note, “N·S.” means “Not Significant” (C) The importance of surface expression of ADAM10 in Q212P shedding was analyzed. ΔADAM10 cells expressing Q212P-RFP were transfected with constructs expressing wild-type or mutant (AXXA) ADAM10 fused with HA epitope and subjected to pulse-chase experiments as described in the upper panel. At the indicated time points, the cells and CM were harvested and immunoprecipitated with an RFP-specific antibody (1:500). The expression of transfected ADAM10 was detected in the fully solubilized cells by immunoblotting with an anti-HA antibody (1:5000). The plots represent the relative amount of Q212P-RFP in the CM of cells expressing wild-type versus mutant ADAM10 harvested at 5 h after the chase was plotted ( n = 3; p = 0.017) (D) Surface expression of wtPrP-RFP and Q212P-RFP was visualized by direct RFP fluorescence in wild-type and ΔADAM10 cells following treatment with cycloheximide (100 μg/mL) for 1 h. Scale bar: 50 μm.

Article Snippet: Purchased antibodies included anti-FLAG M1 antibody (Merck, SF3040), anti-ADAM10 (Merck, AB19026), anti-ADAM17 (Abcam, ab2051), anti-GAPDH (Santa Cruz, SC-20357), anti-HA high affinity (Merck, 11867431001), anti-CD230 (prion) clone 3F4 (BioLegend, 800,307) and HRP-conjugated streptavidin (ThermoFisher, N100).

Techniques: Expressing, CRISPR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Pulse Chase, Labeling, Immunoprecipitation, Transfection, Construct, Mutagenesis, Fluorescence

Journal: Redox Biology

Article Title: Role of redox-sensitive catalytic interaction with ADAM10 in mutant-selective extracellular shedding of prion protein

doi: 10.1016/j.redox.2022.102456

Figure Lengend Snippet: Analysis of catalytic interaction between ADAM10 and Q212P (A) Experimental strategy of surface-restricted Nb labeling to confirm the dynamic interactions between ADAM10 and PrPs is described. (B) Surface interaction of PrPs and ADAM10 was assessed by the surface-restricted Nb labeling as in (A). Cells expressing wtPrP and Q212P (PrPs) inserted by GFP were crosslinked with DSP (2 mM). Complexes containing PrPs-GFP labeled with Nb were recovered by immobilized anti-FLAG antibody (Nb-PD), eluted by FLAG peptides (1 mg/mL), and analyzed by immunoblotting with ADAM10 (1:1000), ADAM17 (1:1000), GFP (1:2000), and FLAG (1:1000)-specific antibodies. (C) Effects of ADAM17 on PrP shedding were analyzed by pulse-chase experiments. WT, ΔADAM10, and ΔADAM17 cells expressing wtPrP-RFP and Q212P-RFP were pulse-labeled for 30 min and allowed to recover for 1.5 h, followed by the chase for an additional 4.5 h in the presence of NEM as in B. The fully solubilized cells and CM were subjected to immunoprecipitation with an RFP-specific antibody (1:500). (D) The catalytic interaction of ADAM10 with Q212P at the cell surface is described.

Article Snippet: Purchased antibodies included anti-FLAG M1 antibody (Merck, SF3040), anti-ADAM10 (Merck, AB19026), anti-ADAM17 (Abcam, ab2051), anti-GAPDH (Santa Cruz, SC-20357), anti-HA high affinity (Merck, 11867431001), anti-CD230 (prion) clone 3F4 (BioLegend, 800,307) and HRP-conjugated streptavidin (ThermoFisher, N100).

Techniques: Labeling, Expressing, Western Blot, Pulse Chase, Immunoprecipitation

Journal: Redox Biology

Article Title: Role of redox-sensitive catalytic interaction with ADAM10 in mutant-selective extracellular shedding of prion protein

doi: 10.1016/j.redox.2022.102456

Figure Lengend Snippet: Analysis of redox-sensitive Q212P shedding (A) Q212P* synthesis induced by DTT was analyzed by pulse-chase experiments. Cells expressing wtPrP-RFP and Q212P-RFP were pulse-labeled followed by the chase for 1 h in the absence or presence of DTT as indicated. The fully solubilized cells were subjected to immunoprecipitation with an RFP-specific antibody. Equal loading was confirmed by the detection of total proteins newly synthesized. Note, *: Q212P* (B) A higher oligomerization propensity of Q212P* was analyzed by the density gradient centrifugation. Cells expressing Q212P-RFP were treated with DTT (10 mM) for 1 h, solubilized in IP buffer containing 1% CHAPS, and separated on the sucrose gradient (5–25%) for 3 h in TLS-55 rotor at 50,000 rpm. Each fraction from the top to the bottom of the sucrose gradient was blotted with an RFP-specific antibody (1:5000). Note, T: unfractionated protein samples (0.1 vol) (C) Q212P* shedding was analyzed by pulse-chase experiments. Cells expressing Q212P-RFP were pulse-labeled for 30 min, chased in the absence (“-DTT”) or presence of DTT (10 mM) (“+DTT”) for 1 h, and recovered for the indicated time after DTT withdrawal (upper panel). Cells were fully solubilized and subjected to immunoprecipitation with an RFP-specific antibody (D) The effect of ADAM10 on Q212P* shedding was determined in ΔADAM10 cells in the same method as described in (C) . The plots represent the changes of surface Q212P-RFP (left panel) and free RFPs in cells (middle panel) and CMs (right panel) by ADAM10 depletion ( n = 3). The significance was analyzed through p -values calculated using the student's-t test as described in “Materials and Methods”. (E) Redox-sensitive Q212P shedding is described.

Article Snippet: Purchased antibodies included anti-FLAG M1 antibody (Merck, SF3040), anti-ADAM10 (Merck, AB19026), anti-ADAM17 (Abcam, ab2051), anti-GAPDH (Santa Cruz, SC-20357), anti-HA high affinity (Merck, 11867431001), anti-CD230 (prion) clone 3F4 (BioLegend, 800,307) and HRP-conjugated streptavidin (ThermoFisher, N100).

Techniques: Pulse Chase, Expressing, Labeling, Immunoprecipitation, Synthesized, Gradient Centrifugation

Journal: Redox Biology

Article Title: Role of redox-sensitive catalytic interaction with ADAM10 in mutant-selective extracellular shedding of prion protein

doi: 10.1016/j.redox.2022.102456

Figure Lengend Snippet: A working model depicting the role of ADAM10 in promoting Q212P shedding On the cell surface, the PrP can interact with the cysteine-rich region of ADAM10, either catalytically or non-catalytically. Q212P mutation provides catalytic interaction of PrP with ADAM10, switching the conformation of the cysteine-rich region, allowing PrP to access the ADAM10 active site, and inducing its extracellular shedding. Under redox perturbation, inhibitory Q212P shedding induces the surface accumulation of oligomeric Q212P but is restored, albeit somewhat slowly, upon redox recovery as long as ADAM10 is activated. Nevertheless, cells prefer ADAM10-independent endo-lysosomal degradation to eliminate surface Q212P because it is more efficient than ADAM10-dependent extracellular shedding. This strategy renders cells more capable of fully digesting misfolded surface Q212P that may exert unexpected protein toxicity when released into the extracellular space.

Article Snippet: Purchased antibodies included anti-FLAG M1 antibody (Merck, SF3040), anti-ADAM10 (Merck, AB19026), anti-ADAM17 (Abcam, ab2051), anti-GAPDH (Santa Cruz, SC-20357), anti-HA high affinity (Merck, 11867431001), anti-CD230 (prion) clone 3F4 (BioLegend, 800,307) and HRP-conjugated streptavidin (ThermoFisher, N100).

Techniques: Mutagenesis