sirna of human ca3 sc-60309  (Santa Cruz Biotechnology)

Journal: Gastroenterology

Article Title: Squalene Epoxidase Induces Nonalcoholic Steatohepatitis Via Binding to Carbonic Anhydrase III and is a Therapeutic Target.

doi: 10.1053/j.gastro.2021.02.051

Figure Lengend Snippet: Figure 4. Sqle activates de novo lipogenesis via CA3 in NASH. (A) Silver staining results of co-IP by using Sqle tg mice liver protein. (B) The interaction between SQLE and CA3 was further confirmed by using Sqle tg mice liver protein and commercial recombinant human SQLE and CA3 protein. (C) Direct binding of SQLE and CA3 were confirmed using enzyme-linked immunosorbent assay. (D–E) Western blot analysis showed that SQLE stabilizes CA3 protein expression by inhibiting auto- phagy-lysosome–dependent protein degradation pathway. (F) CA3 overexpression increase intracellular Triglyceride level and protein expression of SREBP1C, FASN, and p- IkBa and p-P65 NF-kB subunit in LO2 and HKCI10 cells. (G) Silence CA3 expression reverse SQLE induced TG level and protein expression of FASN and p-P65. (H) Western blot analysis showed Acetazolamide reduced liver TG and protein expression of CA3 and reduced SQLE-induced protein expression of ACC, FASN, p-P65, and p- IkBa in SQLE-tg mice. (I) Western blot analysis showed that cholesterol treatment cannot increase Car3 protein expression. *P < .05; **P < .01, ***P < .001

Article Snippet: For purified human SQLE protein (H00006713, NOVUS Centennial)) and CA3 (2185-CA, R&D Systems Minneapolis, MN) protein (Supplementary Table 2), 1 mg of human SQLE protein and 2 mg human CA3 protein were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4oC.

Techniques: Silver Staining, Co-Immunoprecipitation Assay, Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Over Expression

Journal: Gastroenterology

Article Title: Squalene Epoxidase Induces Nonalcoholic Steatohepatitis Via Binding to Carbonic Anhydrase III and is a Therapeutic Target.

doi: 10.1053/j.gastro.2021.02.051

Figure Lengend Snippet: Figure 6. Pharmacologic inhibition of SQLE and CA3 synergistically ameliorated NASH development. (A) Schematic diagram of combined drug treatment in HFHC diet–induced mouse NASH model. Combined terbinafine and acetazolamide treatment significantly reduced liver weight and liver-to-body weight ratio. (B) Terbinafine plus Acetazolamide abolished HFHC diet– induced accumulation of liver triglyceride, FFA, and TBARS, (C) and accumulation of serum triglyceride, ALT, and AST and improved glucose tolerance test and ITT. (D) Liver histology showed that combined terbinafine plus acetazolamide syner- gistically attenuated steatohepatitis, lipid accumulation, and liver fibrosis (**P < .01; ***P < .001 vs PBS group). (E) Serum IL1a, IL1b, IL6, IL17, MCP-1, MIP-1b, and TNF-a concentration of 4 groups. (F) Western blot analysis showed that combined drug treatment decreased p-P65 and p-IkBa in HFHC-fed WT mice. (G) qPCR analysis indicated that terbinafine plus acetazolamide synergistically suppressed mRNA expression of genes involved in lipogenesis, triglyceride biosynthesis, and fibrosis. Scale bars, 100 mm. *P < .05; **P < .01; ***P < .001.

Article Snippet: For purified human SQLE protein (H00006713, NOVUS Centennial)) and CA3 (2185-CA, R&D Systems Minneapolis, MN) protein (Supplementary Table 2), 1 mg of human SQLE protein and 2 mg human CA3 protein were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4oC.

Techniques: Inhibition, Concentration Assay, Western Blot, Expressing

Journal: Gastroenterology

Article Title: Squalene Epoxidase Induces Nonalcoholic Steatohepatitis Via Binding to Carbonic Anhydrase III and is a Therapeutic Target.

doi: 10.1053/j.gastro.2021.02.051

Figure Lengend Snippet: Figure 7. SQLE is up-regulated in human NASH and serum SQLE/CA3 are novel biomarkers for the clinical diagnosis of NASH. (A) SQLE mRNA and (B) protein expression was up-regulated in patients with NAFLD. (C) Serum SQLE concentration in 72 healthy people, 65 patients with steatosis, and 80 patients with NASH. (D) Correlation analysis between serum SQLE and clinical information of patients with NAFLD. (E) Serum CA3 concentration in 72 healthy people, 65 patients with steatosis, and 80 patients with NASH. (F) Correlation analysis between serum CA3 and clinical information of patients with NAFLD. (G) Correlation analysis between serum SQLE and serum CA3 concentration in patients with NAFLD. (H) AUROC of combined serum SQLE and CA3 in discriminating NAFLD and NASH in all patients.

Article Snippet: For purified human SQLE protein (H00006713, NOVUS Centennial)) and CA3 (2185-CA, R&D Systems Minneapolis, MN) protein (Supplementary Table 2), 1 mg of human SQLE protein and 2 mg human CA3 protein were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4oC.

Techniques: Biomarker Discovery, Expressing, Concentration Assay

Journal: Gastroenterology

Article Title: Squalene Epoxidase Induces Nonalcoholic Steatohepatitis Via Binding to Carbonic Anhydrase III and is a Therapeutic Target.

doi: 10.1053/j.gastro.2021.02.051

Figure Lengend Snippet: Figure 4. Sqle activates de novo lipogenesis via CA3 in NASH. (A) Silver staining results of co-IP by using Sqle tg mice liver protein. (B) The interaction between SQLE and CA3 was further confirmed by using Sqle tg mice liver protein and commercial recombinant human SQLE and CA3 protein. (C) Direct binding of SQLE and CA3 were confirmed using enzyme-linked immunosorbent assay. (D–E) Western blot analysis showed that SQLE stabilizes CA3 protein expression by inhibiting auto- phagy-lysosome–dependent protein degradation pathway. (F) CA3 overexpression increase intracellular Triglyceride level and protein expression of SREBP1C, FASN, and p- IkBa and p-P65 NF-kB subunit in LO2 and HKCI10 cells. (G) Silence CA3 expression reverse SQLE induced TG level and protein expression of FASN and p-P65. (H) Western blot analysis showed Acetazolamide reduced liver TG and protein expression of CA3 and reduced SQLE-induced protein expression of ACC, FASN, p-P65, and p- IkBa in SQLE-tg mice. (I) Western blot analysis showed that cholesterol treatment cannot increase Car3 protein expression. *P < .05; **P < .01, ***P < .001

Article Snippet: SiRNA of human CA3 (sc-60309) and control (siCTL) were ordered from Santa Cruz (Dallas, USA).

Techniques: Silver Staining, Co-Immunoprecipitation Assay, Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Over Expression

Journal: Gastroenterology

Article Title: Squalene Epoxidase Induces Nonalcoholic Steatohepatitis Via Binding to Carbonic Anhydrase III and is a Therapeutic Target.

doi: 10.1053/j.gastro.2021.02.051

Figure Lengend Snippet: Figure 6. Pharmacologic inhibition of SQLE and CA3 synergistically ameliorated NASH development. (A) Schematic diagram of combined drug treatment in HFHC diet–induced mouse NASH model. Combined terbinafine and acetazolamide treatment significantly reduced liver weight and liver-to-body weight ratio. (B) Terbinafine plus Acetazolamide abolished HFHC diet– induced accumulation of liver triglyceride, FFA, and TBARS, (C) and accumulation of serum triglyceride, ALT, and AST and improved glucose tolerance test and ITT. (D) Liver histology showed that combined terbinafine plus acetazolamide syner- gistically attenuated steatohepatitis, lipid accumulation, and liver fibrosis (**P < .01; ***P < .001 vs PBS group). (E) Serum IL1a, IL1b, IL6, IL17, MCP-1, MIP-1b, and TNF-a concentration of 4 groups. (F) Western blot analysis showed that combined drug treatment decreased p-P65 and p-IkBa in HFHC-fed WT mice. (G) qPCR analysis indicated that terbinafine plus acetazolamide synergistically suppressed mRNA expression of genes involved in lipogenesis, triglyceride biosynthesis, and fibrosis. Scale bars, 100 mm. *P < .05; **P < .01; ***P < .001.

Article Snippet: SiRNA of human CA3 (sc-60309) and control (siCTL) were ordered from Santa Cruz (Dallas, USA).

Techniques: Inhibition, Concentration Assay, Western Blot, Expressing

Journal: Gastroenterology

Article Title: Squalene Epoxidase Induces Nonalcoholic Steatohepatitis Via Binding to Carbonic Anhydrase III and is a Therapeutic Target.

doi: 10.1053/j.gastro.2021.02.051

Figure Lengend Snippet: Figure 7. SQLE is up-regulated in human NASH and serum SQLE/CA3 are novel biomarkers for the clinical diagnosis of NASH. (A) SQLE mRNA and (B) protein expression was up-regulated in patients with NAFLD. (C) Serum SQLE concentration in 72 healthy people, 65 patients with steatosis, and 80 patients with NASH. (D) Correlation analysis between serum SQLE and clinical information of patients with NAFLD. (E) Serum CA3 concentration in 72 healthy people, 65 patients with steatosis, and 80 patients with NASH. (F) Correlation analysis between serum CA3 and clinical information of patients with NAFLD. (G) Correlation analysis between serum SQLE and serum CA3 concentration in patients with NAFLD. (H) AUROC of combined serum SQLE and CA3 in discriminating NAFLD and NASH in all patients.

Article Snippet: SiRNA of human CA3 (sc-60309) and control (siCTL) were ordered from Santa Cruz (Dallas, USA).

Techniques: Biomarker Discovery, Expressing, Concentration Assay