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

Journal: Scientific reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells.

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: Fig. 3. Inhibitors of AKT and ADAM10 diminish SC79-induced RAGE ectodomain shedding. HAECs were preincubated with or without MK-2206 (1 µM), GI 254023X (2 µM), or DMSO (vehicle) for 60 min. Following this, they were further incubated for 30 min with or without SC79 (10 µM). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and an anti-actin antibody. (n = 3, *p < 0.05 vs. control, #p < 0.05 vs. SC79 treatment alone)

Article Snippet: Rabbit polyclonal antibodies against the amino acids 214–500 of human ADAM10 (A10438; for immunofluorescence staining), AKT1 (A11016), AKT3 (A12909), p-AKT1Ser473 (AP0140), and p-AKT2Ser474 (AP0005) were from ABclonal, Inc. (Woburn, MA, USA).

Techniques: Incubation, Control

Journal: Scientific reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells.

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: Fig. 7. SC79 induces RAGE ectodomain shedding by promoting ADAM10 cell surface translocation. (A) Immunofluorescence staining to evaluate the effect of SC79 on ADAM10 localization. HAECs grown in culture dishes with a coverslip were treated with SC79 (10 µM) for 10–120 min. (a) The cells on the coverslip were fixed for 10 min with 4% paraformaldehyde without permeabilization, then immunostained with an antibody to an extracellular portion of ADAM10 and examined using confocal microscopy. DAPI was used to label the nuclei of the cells. Representative photos and the relative fluorescence intensities are shown (scale bar: 100 μm). (b) Cell lysates from cells that were not on the coverslip in the same culture plate were immunoblotted with antibodies to ADAM10 and actin. (n = 3, *p < 0.05 vs. control). (B) ADAM10 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with ADAM10-siRNA or control siRNA and then incubated for 30 min with or without SC79 (10 µM). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and antibodies to ADAM10 and actin. (n = 3, *p < 0.05 vs. control cells transfected with control siRNA). (C) ADAM10 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with ADAM10-siRNA or control siRNA were treated for 30 min with or without SC79 (10 µM). The cells were then treated with AGE- BSA (100 µg/ml) for 24 h. The cell lysates were immunoblotted with antibodies to ICAM-1, ADAM10, and actin. (n = 3, *p < 0.05 vs. control; #p < 0.05 vs. AGE-BSA; †p < 0.05 vs. control cells transfected with ADAM10- siRNA)

Article Snippet: Rabbit polyclonal antibodies against the amino acids 214–500 of human ADAM10 (A10438; for immunofluorescence staining), AKT1 (A11016), AKT3 (A12909), p-AKT1Ser473 (AP0140), and p-AKT2Ser474 (AP0005) were from ABclonal, Inc. (Woburn, MA, USA).

Techniques: Translocation Assay, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence, Control, Knockdown, Transfection, Incubation

Journal: Scientific reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells.

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: Fig. 8. Depletion of AKT1, AKT2, or AKT3 impairs SC79-induced ADAM10 cell surface translocation. (A) AKT inhibition prevents SC79-induced ADAM10 cell surface translocation. HAECs were preincubated with or without MK-2206 (1 µM) or DMSO (vehicle) for 60 min. Following this, they were further incubated for 20 min with or without SC79 (10 µM). Cells were immunostained with an antibody to an extracellular portion of ADAM10. Representative photos and the relative fluorescence intensities are shown (scale bar: 100 μm). (n = 3, *p < 0.05 vs. control; #p < 0.05 vs. SC79 treatment alone). (B–D) AKT1, AKT2, and AKT3 knockdowns prevent SC79-induced ADAM10 cell surface translocation. HAECs grown in culture dishes with a coverslip were transfected with AKT1-siRNA, AKT2-siRNA, AKT3-siRNA, or control siRNA, and then incubated for 20 min with DMSO or SC79 (10 µM). (a) Cells grown on the coverslip were immunostained for ADAM10. Representative photos and the relative fluorescence intensities are shown (scale bar: 100 μm). (b) Cell lysates from cells that were not on the coverslip in the same culture plate were immunoblotted with antibodies to AKT1, AKT2, AKT3, or actin. (n = 3, *p < 0.05 vs. control cells transfected with control siRNA)

Article Snippet: Rabbit polyclonal antibodies against the amino acids 214–500 of human ADAM10 (A10438; for immunofluorescence staining), AKT1 (A11016), AKT3 (A12909), p-AKT1Ser473 (AP0140), and p-AKT2Ser474 (AP0005) were from ABclonal, Inc. (Woburn, MA, USA).

Techniques: Translocation Assay, Inhibition, Incubation, Fluorescence, Control, Transfection

Journal: Scientific reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells.

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: Fig. 9. Rab14 interacts with ADAM10 and, upon SC79 treatment, moves to the cell surface. (A) Rab14 co- immunoprecipitates with ADAM10. HAECs were treated with DMSO or SC79 (10 µM) for 20 min before being lysed with NP-40 buffer. Lysates were incubated with rabbit anti-ADAM10 antibody or rabbit IgG as a control overnight at 4 °C, followed by incubation with Protein A agarose. The input and immunoprecipitation fractions were analyzed using anti-ADAM10 and anti-Rab14 antibodies. (n = 4, *p < 0.05 vs. cells treated with DMSO and immunoprecipitated with ADAM10 antibody, ns: not significant). (B) SC79 translocates ADAM10 and Rab14 from intracellular compartments to the cell surface. HAECs were treated with DMSO or SC79 (10 µM) for 20 min. The cell surface proteins were biotin-labeled and isolated with avidin-coated agarose beads. Whole cell lysates, biotin-bound cell surface proteins, and biotin-unbound intracellular proteins were examined by Western blotting using anti-ADAM10, Anti-Rab14, and anti-actin antibodies. (n = 5, *p < 0.05)

Article Snippet: Rabbit polyclonal antibodies against the amino acids 214–500 of human ADAM10 (A10438; for immunofluorescence staining), AKT1 (A11016), AKT3 (A12909), p-AKT1Ser473 (AP0140), and p-AKT2Ser474 (AP0005) were from ABclonal, Inc. (Woburn, MA, USA).

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

Journal: Scientific reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells.

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: Fig. 10. Rab14 is required for SC79-induced ADAM10 cell surface translocation. (A) Rab14 knockdown prevents SC79-induced ADAM10 cell surface translocation. HAECs grown in culture dishes with a coverslip were transfected with Rab14-siRNA or control siRNA and then incubated for 20 min with DMSO or SC79 (10 µM). (a) Cells grown on the coverslip were immunostained with an antibody to an extracellular portion of ADAM10. Representative photos and the relative fluorescence intensities are shown (scale bar: 100 μm). (b) Cell lysates from cells that were not on the coverslip in the same culture plate were immunoblotted with antibodies to Rab14 and actin. (n = 3, *p < 0.05 vs. control cells transfected with control siRNA). (B) Rab14 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with Rab14-siRNA or control siRNA and then incubated for 30 min with or without SC79 (10 µM). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and antibodies to Rab14 and actin. (n = 3, *p < 0.05 vs. control cells transfected with control siRNA). (C) Rab14 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with Rab14-siRNA or control siRNA were treated for 30 min with or without SC79 (10 µM). The cells were then treated with AGE- BSA (100 µg/ml) for 24 h. The cell lysates were immunoblotted with antibodies to ICAM-1, Rab14, and actin. (n = 4, *p < 0.05 vs. control; #p < 0.05 vs. AGE-BSA; †p < 0.05 vs. control cells transfected with Rab14-siRNA)

Article Snippet: Rabbit polyclonal antibodies against the amino acids 214–500 of human ADAM10 (A10438; for immunofluorescence staining), AKT1 (A11016), AKT3 (A12909), p-AKT1Ser473 (AP0140), and p-AKT2Ser474 (AP0005) were from ABclonal, Inc. (Woburn, MA, USA).

Techniques: Translocation Assay, Knockdown, Transfection, Control, Incubation, Fluorescence

Journal: PLOS Pathogens

Article Title: Oxidative stress-driven enhanced iron production and scavenging through Ferroportin reorientation worsens anemia in antimony-resistant Leishmania donovani infection

doi: 10.1371/journal.ppat.1012858

Figure Lengend Snippet: (A.i.) Western blot of whole cell lysate of uninfected-MФs, LD-R, and LD-S infected-MФs at 24 hrs pi showing the expression of precursor-ADAM10 (p-ADAM10, ~90 kDa) and mature-ADAM10 (m-ADAM10, ~64 kDa) keeping β-actin acts as a housekeeping control. (A.ii.) Bar graph representing the relative intensity of mature-ADAM10 (m-ADAM10) vs precursor-ADAM10 (p-ADAM10). Significant upregulation of relative intensity of m-ADAM10 with respect to p-ADAM10 in LD-R 24 hrs pi as compared to both UI and LD-S 24hrs pi (****, P ≤ 0.0001) whereas no change is observed between LD-R 24 hrs pi and UI (ns, P > 0.05). (B.i.a.) Western blot of whole cell lysate showing furin expression of DFO-treated-MФs, LD-S, and LD-R infected-MФs at 24 hrs pi. (B.i.b.) Bar graph representing the relative intensity of Furin, i.e., fold change with respect to β-actin for whole cell lysate. Significant enrichment of Furin observed in LD-S as compared to both DFO-treated control and LD-R 24 hrs pi (****, P ≤ 0.0001) whereas no change is observed between LD-R 24 hrs pi and DFO-treated control (ns, P > 0.05). Each densitometry analysis is presented as a bar graph of Mean ± SEM for 3 biological replicates. (B.ii.) 40X Confocal images showing the localization of furin (red) in uninfected-MФs, LD-S, and LD-R infected-MФs. (B.iii.) Western blot of furin expression in the supernatant fraction of same experiments sets as B.i. (C.) Confocal images showing Furin (red, 3 rd panel and edges, 6 th panel), ADAM10 (purple), and CD11b (green) colocalization in LD-S and LD-R infected-MФs at 24 hrs pi. One representative small nucleus of LD has been marked in ( * ) to show the infected-MФs. Scale bars indicate 20µm. (D.) Scheme showing Furin maturation starting from Endoplasmic reticulum (ER) where it remains inactivated (observed in LD-S-infected-MФs at 24 hrs pi) to getting shed as an active enzyme (observed in LD-R-infected-MФs at 24 hrs pi) to cleave prodomain of p-ADAM10 rendering it activated (m-ADAM10), which in turn cleaves extracellular domain of SIRPα in LD-R-infected-MФs at 24 hrs pi.

Article Snippet: Antibody against ADAM10 was purchased from Abclonal (A10438) and Invitrogen (MA5-23867).

Techniques: Western Blot, Infection, Expressing, Control

Journal: PLOS Pathogens

Article Title: Oxidative stress-driven enhanced iron production and scavenging through Ferroportin reorientation worsens anemia in antimony-resistant Leishmania donovani infection

doi: 10.1371/journal.ppat.1012858

Figure Lengend Snippet: At 4 hrs pi (left panel), LD-R triggers ROS production which promotes nuclear translocation of p50 and c-Rel. p50/c-Rel binds to the promoter of Heme-oxygenase 1 and activates it. HO-1 protein catalyzes the breakdown of heme into iron (Fe 2+ ). LD-R reshuffles Ferroportin (Fpn) around LD-R-PV while Fpn remains in MФ surface in LD-S infection. NRAMP1 is downregulated in LD-R-PV as compared to LD-S-PV while there is a uniform expression of Transferrin receptor 1 (CD71) in both cases. All these factors drive excess iron produced from HO-1 inside LD-R-PV as compared to LD-S-PV. At 24 hrs pi (right panel), LD-R proliferates at a rapid rate utilizing this excess iron inside LD-R-PV resulting in a drop of the iron level. This iron depletion condition promotes Furin shedding as an active enzyme which then binds to the prodomain of p-ADAM10 present in the macrophage membrane and cleaves it, rendering it activated (m-ADAM10). m-ADAM10 binds to the extracellular domain of SIRPα and cleaves it, breaking the discriminatory signal of CD47-enriched live RBCs or CD47-less senescent RBCs, resulting in aggravated erythrophagocytosis of both live and senescent RBCs. Contrarily, in LD-S infection, Furin remains mostly around perinuclear space which is the inactive form and cannot interact with membrane-associated p-ADAM10. Thus, SIRPα remains intact and elicits a ‘Don’t eat me’ signal upon interacting with CD47-enriched live RBCs resulting in controlled erythrophagocytosis.

Article Snippet: Antibody against ADAM10 was purchased from Abclonal (A10438) and Invitrogen (MA5-23867).

Techniques: Translocation Assay, Infection, Expressing, Produced, Membrane