Journal: PLOS One

Article Title: Insulin enhances RAGE ectodomain shedding by inducing Rab14-dependent ADAM10 cell surface trafficking in human aortic endothelial cells

doi: 10.1371/journal.pone.0358445

Figure Lengend Snippet: (A) Insulin promotes ADAM10 cell surface translocation. HAECs were cultured on glass coverslips placed in cell culture dishes and treated with 100 nM insulin for up to 120 min. (a) Cells grown on coverslips were subjected to immunofluorescence staining to assess cell surface ADAM10. Representative images and the relative fluorescence intensities are shown (scale bar, 100 μm). (b) Cells cultured in the same dishes but outside the coverslips were analyzed by Western blot to determine total ADAM10 expression. (n = 3 independent experiments, * p < 0.05 vs. control). (B) ADAM10 depletion abolishes insulin-induced RAGE ectodomain shedding. HAECs transfected with control siRNA or ADAM10 siRNA were treated with or without 100 nM insulin for 30 min. Cell lysates and culture supernatants were analyzed by Western blot using antibodies against the RAGE extracellular domain, ADAM10, and actin. (n = 3 independent experiments, * p < 0.05 vs. control cells transfected with control-siRNA; # p < 0.05 vs. insulin-treated cells following control siRNA transfection). (C) ADAM10 depletion abolishes the inhibitory effect of insulin on AGE-BSA-induced ICAM-1 expression. HAECs transfected with control siRNA or ADAM10 siRNA were pretreated with or without 100 nM insulin for 30 min, followed by incubation with AGE-BSA (100 μg/mL) for 24 h. Cell lysates were analyzed by Western blot using antibodies against ICAM-1, ADAM10, and actin. (n = 4 independent experiments, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA; † p < 0.05 vs. control cells transfected with ADAM10-siRNA).

Article Snippet: The cells were then incubated overnight at 4°C with an antibody against the extracellular domain of ADAM10 (ABclonal, A10438; rabbit antibody recognizing amino acids 214–500 of ADAM10).

Techniques: Translocation Assay, Cell Culture, Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing, Control, Transfection, Incubation

Journal: PLOS One

Article Title: Insulin enhances RAGE ectodomain shedding by inducing Rab14-dependent ADAM10 cell surface trafficking in human aortic endothelial cells

doi: 10.1371/journal.pone.0358445

Figure Lengend Snippet: HAECs were cultured on glass coverslips placed in cell culture dishes. (A) Cells were pretreated with MK-2206 (1 μM) or DMSO (vehicle) for 60 min, followed by treatment with or without insulin (100 nM) for 20 min. Cell surface ADAM10 was assessed by immunofluorescence staining. Representative images and the relative fluorescence intensities are shown (scale bar, 100 μm). (n = 3 independent experiments, * p < 0.05 vs. control; # p < 0.05 vs. insulin). (B-D) HAECs were transfected with control siRNA, AKT1 siRNA, AKT2 siRNA, or AKT3 siRNA and then treated with or without insulin (100 nM) for 20 min. (a) Cells grown on coverslips were subjected to immunofluorescence staining to assess cell surface ADAM10. Representative images and the relative fluorescence intensities are shown (scale bar, 100 μm).(b) Cells cultured in the same dishes but outside the coverslips were analyzed by Western blot to confirm knockdown of AKT1, AKT2, or AKT3. (n = 3 independent experiments, * p < 0.05 vs. control cells transfected with control siRNA; # p < 0.05 vs. insulin-treated cells following control siRNA transfection).

Article Snippet: The cells were then incubated overnight at 4°C with an antibody against the extracellular domain of ADAM10 (ABclonal, A10438; rabbit antibody recognizing amino acids 214–500 of ADAM10).

Techniques: Cell Culture, Immunofluorescence, Staining, Fluorescence, Control, Transfection, Western Blot, Knockdown

Journal: PLOS One

Article Title: Insulin enhances RAGE ectodomain shedding by inducing Rab14-dependent ADAM10 cell surface trafficking in human aortic endothelial cells

doi: 10.1371/journal.pone.0358445

Figure Lengend Snippet: (A) Insulin enhances the interaction between Rab14 and ADAM10. HAECs were treated with or without insulin (100 nM) for 20 min. Cell lysates were incubated overnight at 4°C with control IgG or an anti-ADAM10 antibody, followed by incubation with protein A-agarose. Input lysates and immunoprecipitates were analyzed by Western blot using antibodies against ADAM10 and Rab14. (n = 6 independent experiments, * p < 0.05 vs. control). (B) Insulin promotes the translocation of Rab14 and ADAM10 to the cell surface. HAECs were treated with or without insulin (100 nM) for 20 min. Cell surface proteins were labeled with biotin and isolated from intracellular proteins using avidin-coated agarose beads. Whole-cell lysates, intracellular protein fractions, and cell surface protein fractions were analyzed by Western blot using antibodies against ADAM10, Rab14, and actin. (n = 6 independent experiments, * p < 0.05 vs. control).

Article Snippet: The cells were then incubated overnight at 4°C with an antibody against the extracellular domain of ADAM10 (ABclonal, A10438; rabbit antibody recognizing amino acids 214–500 of ADAM10).

Techniques: Incubation, Control, Western Blot, Translocation Assay, Labeling, Isolation, Avidin-Biotin Assay

Journal: PLOS One

Article Title: Insulin enhances RAGE ectodomain shedding by inducing Rab14-dependent ADAM10 cell surface trafficking in human aortic endothelial cells

doi: 10.1371/journal.pone.0358445

Figure Lengend Snippet: (A) Rab14 depletion abolishes insulin-induced ADAM10 cell surface translocation. HAECs were cultured on glass coverslips placed in cell culture dishes. Cells were transfected with control siRNA or Rab14 siRNA and then treated with or without 100 nM insulin for 20 min. (a) Cells grown on coverslips were subjected to immunofluorescence staining to assess cell surface ADAM10. Representative images and the relative fluorescence intensities are shown (scale bar, 100 μm). (b) Cells cultured in the same dishes but outside the coverslips were analyzed by Western blot to confirm Rab14 knockdown. (n = 3 independent experiments, * p < 0.05 vs. control cells transfected with control siRNA; # p < 0.05 vs. insulin-treated cells following control siRNA transfection). (B) Rab14 depletion abolishes insulin-induced RAGE ectodomain shedding. HAECs transfected with control siRNA or Rab14 siRNA were treated with or without 100 nM insulin for 30 min. Cell lysates and culture supernatants were analyzed by Western blot using antibodies against the RAGE extracellular domain, Rab14, and actin. (n = 3 independent experiments, * p < 0.05 vs. control cells transfected with control siRNA; # p < 0.05 vs. insulin-treated cells following control siRNA transfection). (C) Rab14 depletion abolishes the inhibitory effect of insulin on AGE-BSA-induced ICAM-1 expression. HAECs transfected with control siRNA or Rab14 siRNA were pretreated with or without 100 nM insulin for 30 min, followed by incubation with AGE-BSA (100 μg/mL) for 24 h. Cell lysates were analyzed by Western blot using antibodies against ICAM-1, Rab14, and actin. (n = 4 independent experiments, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA; † p < 0.05 vs. control cells transfected with Rab14-siRNA).

Article Snippet: The cells were then incubated overnight at 4°C with an antibody against the extracellular domain of ADAM10 (ABclonal, A10438; rabbit antibody recognizing amino acids 214–500 of ADAM10).

Techniques: Translocation Assay, Cell Culture, Transfection, Control, Immunofluorescence, Staining, Fluorescence, Western Blot, Knockdown, Expressing, Incubation

Journal: PLOS One

Article Title: Insulin enhances RAGE ectodomain shedding by inducing Rab14-dependent ADAM10 cell surface trafficking in human aortic endothelial cells

doi: 10.1371/journal.pone.0358445

Figure Lengend Snippet: Based on existing literature, we propose that TBC1D1 and TBC1D4 may function as Rab GTPase-activating proteins that negatively regulate Rab14, thereby controlling the trafficking of ADAM10-containing vesicles originating from the trans-Golgi network. Upon insulin stimulation, AKT is activated and subsequently phosphorylates TBC1D1 and TBC1D4, thereby reducing their inhibitory effect on Rab14. Rab14 can then be activated by its guanine nucleotide exchange factors, such as DENND6A and DENND6B, promoting the translocation of ADAM10-containing vesicles to the plasma membrane, where ADAM10 cleaves multiple substrates, including RAGE.

Article Snippet: The cells were then incubated overnight at 4°C with an antibody against the extracellular domain of ADAM10 (ABclonal, A10438; rabbit antibody recognizing amino acids 214–500 of ADAM10).

Techniques: Translocation Assay, Clinical Proteomics, Membrane