Journal: iScience

Article Title: Direct interaction of HMGB1 with SARS-CoV-2 facilitates its infection via RAGE-dependent endocytosis

doi: 10.1016/j.isci.2025.113063

Figure Lengend Snippet: HMGB1 induces SARS-CoV-2 infection in an ACE2-independent RAGE-dependent manner (A) Western blot analysis of receptors responsible for SARS-CoV-2 infection and HMGB1 binding. S.E., short exposure; L.E., long exposure. (B) Flow cytometry analysis of ectodomain ACE2 in A549 and NCI-H1975 cells used in this study. Vero E6 cells were used as control. (C) A549 cells were transfected with shRNA-ACE2 for 48 h prior to infection with 1 MOI SARS-CoV-2, which was preincubated with HMGB1 for 1 h at 37°C, cultured further for 3 h, and subjected to western blotting. (D and E) A549 cells were infected for 1 h with 1 MOI SARS-CoV-2, which was preincubated with HMGB1 for 1 h, in the presence of 40 μg/mL sRAGE at 37°C as indicated. Cells were harvested at 3 hpi and subjected to western blotting (D) n = 3, and qRT-PCR for viral RNA measurement (E) n = 3. (F) NCI-H1975 cells were infected with SARS-CoV-2 preincubated with 20 μg/mL HMGB1 in the presence of azeliragon. Cells were harvested at 3 hpi and subjected to western blotting. (G) A549 cells were infected with 5 MOI SARS-CoV-2, which was treated as above to observe NP. Representative confocal images are shown. The percentage of infected cells and NP intensity were measured by counting at least 700 visible cells. n = 4. (H) Cycloheximide pretreated NCI-H1975 cells were infected with 5 MOI SARS-CoV-2 (preincubated with HMGB1). Cells were stained for NP before permeabilization (green; external) and post-permeabilization (red; external and internal). Representative images and their magnifications are shown. The percentage of intracellular spots was measured by counting at least 200 visible cells. n = 4. (I and J) SARS2pp was preincubated with or without HMGB1 in the presence of sRAGE before transduction in NCI-H1975 cells. NanoLuc luciferase activity was measured 72 h post-transduction. n = 3 Scale bars represent 5 μm. Data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant, using one-way ANOVA with Tukey’s multiple comparison test and Student’s unpaired t-test. HMGB1, high-mobility group box 1; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; MOI, multiplicity of infection; qRT-PCR, quantitative reverse transcription polymerase chain reaction; NP, nucleocapsid protein; SEM, standard error of the mean; ANOVA, analysis of variance; hpi, hours post-infection; RAGE, receptor for advanced glycation end-products; ACE2, angiotensin-converting enzyme 2; SARS2pp, SARS-CoV-2 spike protein (S)-pseudotyped retrovirus.

Article Snippet: 40 μg/mL sRAGE (11629-HCCH, Sino Biological, Oklahoma City, OK, USA), azeliragon (S6415, Selleckchem, Houston, TX, USA), dynasore (D7693, Sigma-Aldrich, St. Louis, MO, USA) and chloroquine (C6628, Sigma-Aldrich) were pretreated for 2 h at 37°C, prior to the infection procedure.

Techniques: Infection, Western Blot, Binding Assay, Flow Cytometry, Control, Transfection, shRNA, Cell Culture, Quantitative RT-PCR, Staining, Transduction, Luciferase, Activity Assay, Comparison, Reverse Transcription, Polymerase Chain Reaction

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: SC79 induces the shedding of the RAGE ectodomain. HAECs were incubated with 10 µM SC79 for various times (5, 10, 30, and 60 min) ( n = 4) ( A ) or different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min ( n = 3) ( B ). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and an anti-actin antibody. To compare the size of RAGE in cell lysate and culture supernatant, untreated cell lysate (a) and conditioned media from cells treated with 10 µM SC79 for 30 min (b) were run on the same gel and immunoblotted with the RAGE antibody ( C ). The cell lysates of HAECs treated with different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min were immunoblotted with an antibody to the C-terminal domain of human RAGE and an anti-actin antibody (n = 4) ( D ). ( * p < 0.05 vs. control)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, 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: 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: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, 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: AKT1 activation is required for SC79-induced RAGE ectodomain shedding. ( A ) HAECs express all three AKT isoforms, and AKT1-, AKT2-, and AKT3-siRNAs selectively deplete each AKT isoform. HAECs were transfected with AKT1-, AKT2-, AKT3-siRNAs, or control siRNAs, and the cell lysates were immunoblotted with antibodies to AKT1, AKT2, AKT3, or actin. ( n = 3, * p < 0.05 vs. control). ( B ) SC79 activates AKT1. HAECs were incubated with 10 µM SC79 for various times (1, 5, 10, and 30 min) (upper panel) or different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min (lower panel). The cell lysates were immunoblotted with antibodies to p-AKT1 (Ser473) and AKT1. ( n = 3, * p < 0.05 vs. control). ( C ) AKT1 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with AKT1-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 AKT1 and actin. ( n = 3, * p < 0.05 vs. control cells transfected with control siRNA). ( D ) AKT1 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with AKT1-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, AKT1, and actin. ( n = 3, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA; † p < 0.05 vs. control cells transfected with AKT1-siRNA)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Transfection, Control, Incubation, Knockdown

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: AKT2 activation is required for SC79-induced RAGE ectodomain shedding. ( A ) SC79 activates AKT2. HAECs were incubated with 10 µM SC79 for various times (1, 5, 10, and 30 min) (upper panel) or different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min (lower panel). The cell lysates were immunoblotted with antibodies to p-AKT2 (Ser474) and AKT2. ( n = 4, * p < 0.05 vs. control). ( B ) AKT2 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with AKT2-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 AKT2 and actin. ( n = 4, * p < 0.05 vs. control cells transfected with control siRNA). ( C ) AKT2 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with AKT2-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, AKT2, and actin. ( n = 3, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA; † p < 0.05 vs. control cells transfected with AKT2-siRNA)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Incubation, Control, Knockdown, 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: AKT3 activation is required for SC79-induced RAGE ectodomain shedding. ( A ) SC79 activates AKT3. HAECs were incubated with 10 µM SC79 for various times (1, 5, 10, and 30 min) (upper panel) or different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min (lower panel). The cell lysates were immunoblotted with antibodies to p-AKT3 (Ser472) and AKT3. ( n = 3, * p < 0.05 vs. control). ( B ) AKT3 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with AKT3-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 AKT3 and actin. ( n = 3, * p < 0.05 vs. control cells transfected with control siRNA). ( C ) AKT3 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with AKT3-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, AKT3, and actin. ( n = 3, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA; † p < 0.05 vs. control cells transfected with AKT3-siRNA)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Incubation, Control, Knockdown, 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: 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: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, 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: 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: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

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