hepad38 cells  (Thermo Fisher)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mechanism verification of first-in-class hydrophobic tagging-based degraders of HBV core protein

doi: 10.1016/j.apsb.2025.02.033

Figure Lengend Snippet: Degradation test for HBC with target compounds HyT-(S1–S15) . HepAD38 cells were cultured in the presence of tetracycline (tet+) or in the absence of tetracycline and mock treated (tet–) or treated with HyT-(S1–S15) (maximum non-toxic concentration by CPE method, HyT-S1 : 4 μmol/L; HyT-(S2–S6) : 5 μmol/L; HyT-(S7–S12) : 6 μmol/L; HyT-(S13–S14) : 2 μmol/L; HyT-S15 : 20 μmol/L; SP-5 (20 μmol/L) or NVR 3-778 (6 μmol/L) for 72 h. (A, B) HBV core protein (HBC) expression was detected by Western blot assay with a rabbit polyclonal antibody. GAPDH served as the loading control. (C) The quantification results of HBC/GAPDH (or β -actin) ratio from two independent immunoblots are shown.

Article Snippet: HepAD38 cells were cultured in a tetracycline-free complete medium for 96 h. After digestion with trypsin, the cell pellet was resuspended with PBS containing protease inhibitors (Thermo).

Techniques: Cell Culture, Concentration Assay, Expressing, Western Blot, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mechanism verification of first-in-class hydrophobic tagging-based degraders of HBV core protein

doi: 10.1016/j.apsb.2025.02.033

Figure Lengend Snippet: Degradation test for HBC with target compounds HyT-(S16–S29) . HepAD38 cells were cultured in the presence of tetracycline (tet+) or in the absence of tetracycline and mock treated (tet–) or treated with HyT-(S16–S29) (maximum non-toxic concentration by CPE method, HyT-S16 : 60 μmol/L; HyT-S17 : 2 μmol/L; HyT-S18 : 10 μmol/L; HyT-S19 : 10 μmol/L; HyT-S20 : 30 μmol/L; HyT-S21 : 30 μmol/L; HyT-S22 : 15 μmol/L; HyT-S23 : 30 μmol/L; HyT-S24 : 60 μmol/L; HyT-S25 : 15 μmol/L; HyT-S26 : 30 μmol/L; HyT-S27 : 60 μmol/L; HyT-S28 : 30 μmol/L; HyT-S29 : 30 μmol/L; SP-5 (20 μmol/L) or NVR 3-778 (6 μmol/L) for 72 h. (A, B) HBC expression was detected by Western blot assay. GAPDH served as the loading control. (C) The quantification results of HBC/GAPDH ratio from two independent immunoblots are shown.

Article Snippet: HepAD38 cells were cultured in a tetracycline-free complete medium for 96 h. After digestion with trypsin, the cell pellet was resuspended with PBS containing protease inhibitors (Thermo).

Techniques: Cell Culture, Concentration Assay, Expressing, Western Blot, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mechanism verification of first-in-class hydrophobic tagging-based degraders of HBV core protein

doi: 10.1016/j.apsb.2025.02.033

Figure Lengend Snippet: HyT-S7 degraded HBC in concentration-and time-dependent manner. (A, B) HepAD38 cells cultured in the absence of tetracycline were treated with the indicated concentrations of the compound HyT-S7 , SP-O-7 (20 μmol/L) or NVR 3-778 (6 μmol/L) for 24 h. HBC expression was detected by Western blot assay. GAPDH served as the loading control. The quantification results of HBC/GAPDH ratio from three independent immunoblots are shown. (C) HepAD38 cells were cultured in tetracycline-free complete medium for 24 h and then incubated with treated with 6 μmol/L HyT-S7 or DMSO in the presence of 50 μg/mL cycloheximide (CHX) for the indicated time. After washing with pre-chilled PBS, the cells were lysed and HBV core protein levels were determined by Western blot assay.

Article Snippet: HepAD38 cells were cultured in a tetracycline-free complete medium for 96 h. After digestion with trypsin, the cell pellet was resuspended with PBS containing protease inhibitors (Thermo).

Techniques: Concentration Assay, Cell Culture, Expressing, Western Blot, Control, Incubation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mechanism verification of first-in-class hydrophobic tagging-based degraders of HBV core protein

doi: 10.1016/j.apsb.2025.02.033

Figure Lengend Snippet: Degradation mechanism analysis of HyT-S7 . (A) HepAD38 cells cultured in the absence of tetracycline were treated with the indicated concentrations of HyT-S7 with or without proteasome inhibitor MG132 (5 μmol/L) or bortezomib (0.5 μmol/L); (B) Hsp70 inducer 17-AAG (2 μmol/L); (C) autophagy inhibitor 3-MA (5 mmol/L) or hydroxychloroquine (HCQ) sulfate (50 μmol/L) for 8 h. HBC and GAPDH proteins were detected by Western blot assay. (D, E) The data-independent acquisition (DIA)-based proteomic analysis of HepAD38 cells treated with DMSO or 6 μmol/L HyT-S7 for 24 h. (D) Volcano plots demonstrating global changes in protein abundance. The x axis displays the relative abundance of all identified proteins (8013) in compound HyT-S7 -treated cells (log2 FC) vs . DMSO-treated cells. The y axis displays P values (–log10) from triplicate experiments. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis of differentially expressed proteins. a: Metabolism; b: Organismal systems; c: Environmental information processing; d: Genetic information processing; e: Human diseases; f: Cellular processes.

Article Snippet: HepAD38 cells were cultured in a tetracycline-free complete medium for 96 h. After digestion with trypsin, the cell pellet was resuspended with PBS containing protease inhibitors (Thermo).

Techniques: Cell Culture, Western Blot, Data-independent acquisition, Quantitative Proteomics

Journal: Acta Pharmaceutica Sinica. B

Article Title: Discovery and mechanism verification of first-in-class hydrophobic tagging-based degraders of HBV core protein

doi: 10.1016/j.apsb.2025.02.033

Figure Lengend Snippet: Compound HyT-S7 binds to HBC. (A, B) Cellular thermal shift assay (CETSA). Incubate the cell lysate from HepAD38 cells cultured in complete culture medium without tetracycline for 96 h with DMSO, HyT-S7 (6 μmol/L), NVR 3-778 (6 μmol/L), SP-5 (20 μmol/L), or SP-O-7 (20 μmol/L) for 30 min at room temperature. Then the mixtures were heated on a gradient from 37 to 72 °C for 3 min. The supernatant was collected and loaded onto a 10% SDS-PAGE gel. The levels of HBC were detected by Western blot assay. The quantitative results of HBC of group DMSO and HyT-S7 are shown. Data are shown as mean ± SD from three independent experiments. (C–F) The interactions between HBC protein, and the compound HyT-S7 , NVR 3-778, SP-5, or SP-O-7 were analyzed by a SPR assay performed on a CM5 chip integrated in the Reichert4 SPR system.

Article Snippet: HepAD38 cells were cultured in a tetracycline-free complete medium for 96 h. After digestion with trypsin, the cell pellet was resuspended with PBS containing protease inhibitors (Thermo).

Techniques: Thermal Shift Assay, Cell Culture, SDS Page, Western Blot, SPR Assay