Journal: International Journal of Biological Sciences

Article Title: Curcumol Induces Necroptosis of Hepatic Stellate Cells by Targeting KAT8 to Suppress HK2 Lactylation and Promote HUWE1-Dependent Ubiquitination

doi: 10.7150/ijbs.125009

Figure Lengend Snippet: Curcumol promotes HUWE1-dependent ubiquitin-mediated degradation of HK2 by inhibiting KAT8-mediated lactylation at the K346 site. A, D. Western blot analysis of HK2 protein half-life in LX2 cells treated with or without protein synthesis inhibitors in the presence of sodium lactate (10 mM, 24 h), with quantification of band intensity (n = 3). B. Western blot analysis of HK2 protein levels in LX2 cells transfected with LDHA siRNA or control siRNA and treated with chloroquine (CQ, 20 μg/mL), MG132 (10 μM), alone or in combination for 24 h (n = 3). C. Schematic diagram illustrating regulators of HK2 ubiquitination. E. Co-immunoprecipitation (Co-IP) analysis of the interaction between HK2 and the E3 ubiquitin ligase HUWE1. F. Western blot analysis of HK2 protein levels in LX2 cells treated with MG149 (50 μM, 48 h), with or without MG132 (10 μM, 6 h), followed by densitometric quantification (n = 3). G. Western blot analysis of HK2 protein levels in LX2 cells expressing HK2-WT or HK2-K346A after treatment with MG132 (10 μM, 8 h) (n = 3). H. Co-IP analysis of HK2 ubiquitination in LX2 cells with or without HUWE1 siRNA transfection (24 h). I. Co-IP analysis of HK2 ubiquitination in LX2 cells transfected with or without KAT8 siRNA (24 h) and treated with Curcumol (30 μM). J. Co-IP analysis was used to examine the interaction between HK2 and HUWE1 in LX2 cells that were co-transfected with the indicated plasmids and treated with Curcumol (30 μM, 8 h). K. Co-IP analysis of HK2 ubiquitination in LX2 cells expressing HK2-WT or HK2-K346A. L. Immunofluorescence analysis of HK2 (green), KAT8 (red), and HUWE1 (yellow) colocalization in liver tissues from CCl₄-induced fibrotic mice and Curcumol-treated mice (n = 3; scale bar: 50 μm). M. Co-IP analysis was performed on the co-transfected plasmids for 24 hours, and then the interactions between HK2, KAT8, and HUWE1 in LX2 cells treated with different concentrations of Curcumol (0, 20, 30, 45 μM) were examined. N, Q. Western blot analysis of HK2 degradation kinetics in LX2 cells expressing HK2-WT or HK2-K346A following HUWE1 siRNA transfection (24 h) and cycloheximide (CHX, 20 μg/mL) treatment for the indicated times (n = 3). O, P. Western blot analysis of HK2 protein levels in LX2 cells transfected with HUWE1 siRNA (24 h), with densitometric quantification (n = 3). Data are shown as mean ± SD, and statistical differences were analyzed by one-way ANOVA. ns indicates no significance; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The antibodies employed in this study include: RIPK1 (17519-1-AP), RIPK3 (17563-1-AP), P-RIPK1 (66854-1-Ig), MLKL (21066-1-AP), P-MLKL (82090-2-RR), HK2 (22029-1-AP), PKM2 (15822-1-AP), LDHA (19987-1-AP), PFKM (55028-1-AP), HUWE1 (19430-1-AP), Ubiquitin (10201-2-AP), HA (51064-2-AP), His (66005-1-Ig), EP300 (20695-1-AP) and P62 (18420-1-AP) from proteintech; KAT8 (sc-271691) from Santa Cruz Biotechnology; KAT6A from Bioswamp; Collagen I (ab26003), β-actin (ab8226), Tubulin (Ab721), anti-mouse IgG (ab190475), anti-rabbit IgG (ab288151) and LC3B (ab192890) from Abcam; Pan-Kla (AB_2868521) from PTM-Bio laboratory; KAT6B(A17116), P-RIPK3(AP1260), AARS(A15017), and AARS2 (A7826) from Abclonal.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Immunofluorescence

Journal: Journal of Virology

Article Title: Direct Activation of Endothelial Cells by SARS-CoV-2 Nucleocapsid Protein Is Blocked by Simvastatin

doi: 10.1128/JVI.01396-21

Figure Lengend Snippet: SARS-CoV2 nucleocapsid protein (NP) is a potent inducer of human endothelial cell activation. (a) HLMECs were incubated with SARS-CoV2 structural proteins (S, N, and E proteins; 1 μg/ml) and five nonstructural proteins (NSP1, NSP3, NSP5, NSP7, and NSP8; 1 μg/ml) for 8 h. (b) HLMECs were treated with 1 μg/ml of NP or 10 ng/ml of TNF-α for different incubation periods as indicated. (c) HLMECs were incubated with indicated concentrations of NP for 8 h. TNF-α at 10 ng/ml served as a positive control. (d) Different cultured cells, including mouse lung vascular endothelial cells (MECs), A549 cells, 293T cells, HUVECs, HAECs, HCAECs, HDMECs, and HLMECs, were treated with NP (1 μg/ml) for 8 h. The expression of ICAM-1, VCAM-1, and VE-cadherin was detected by Western blotting. β-Actin served as a loading control. (e) HLMECs were treated with PBS, NP (1 μg/ml), TNF-α (10 ng/ml), or lipopolysaccharide (LPS) (1 μg/ml) for 8 h. The total RNA was isolated and qPCR was performed for measuring the mRNA levels of TNF-α, ICAM-1, VCAM-1, MCP-1, and IL-6. (f) HLMECs were treated with PBS, NP (1 μg/ml), or TNF-α (10 ng/ml) for 8 h and cocultured with Zombie Red-labeled THP-1 cells for 1 h. After being washed, the adherent cells were imaged and quantitatively analyzed.

Article Snippet: SARS-CoV-2 NSP1 (97-095), NSP5 (10-116), NSP7 (97-096), and NSP8 (97-097) proteins were obtained from Prosci (Poway, CA).

Techniques: Activation Assay, Incubation, Positive Control, Cell Culture, Expressing, Western Blot, Control, Isolation, Labeling