human hepatoma cell line huh-7  (Apath LLC)

Journal: bioRxiv

Article Title: The Impact of SARS-CoV-2 nsp14 Proofreading on Nucleoside Antiviral Activity: Insights from Genetic and Pharmacological Investigations

doi: 10.1101/2024.07.24.604948

Figure Lengend Snippet:

Article Snippet: Human lung carcinoma cell line A549 (ATCC), human lung epidermoid carcinoma cell line Calu-1 (ATCC), and human hepatoma cell line Huh-7 (ATCC) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen).

Techniques: Activity Assay, Expressing

Journal: MedComm

Article Title: Lycorine Derivative Inhibits SARS‐CoV‐2 Replication by Reducing −1 Programmed Ribosomal Frameshifting via Targeting ZAP

doi: 10.1002/mco2.70715

Figure Lengend Snippet: Lycorine derivatives exhibit potent antiviral activity against SARS‐CoV‐2 and its variants in vitro. (A) Dose‐dependent inhibition of SARS‐CoV‐2 original strain (GD108) infection by selected lycorine derivatives. Vero cells infected with SARS‐CoV‐2 at MOI of 0.05 were treated with serially diluted compounds for 48 h. Viral RNA copy numbers in culture media were quantified by RT‐qPCR (real‐time quantitative PCR). The dose‐inhibition curve for each compound is shown above the corresponding plot ( n = 3). Cytotoxicity assay of lycorine derivatives. Vero cells were treated with lycorine derivatives at gradient concentrations for 48 h. Cytotoxicity was assayed by CCK‐8 ( n = 3). (B) Vero cells were infected with different strains of SARS‐CoV‐2 (Alpha, Beta, Delta, and Omicron BA.1) at MOI of 0.05 and treated with serially diluted compounds ( 7 , 11 , 14 , and remdesivir) for 48 h. Viral RNA copy numbers in culture media were quantified by RT‐qPCR. The EC 50 value for each compound is shown above the corresponding plot ( n = 3). (C) IF of the inhibition of compound 7 on SARS‐CoV‐2 replication in Vero, Calu‐3, Huh‐7, and Caco‐2. At 48 h, the infected cells (MOI = 0.05) treated with different concentrations of compound 7 were fixed and analyzed by IF using the primary antibody against SARS‐CoV‐2 N protein (Green). Cell nuclei were stained with DAPI (blue). Scale bars = 100 µm. (D) WB analysis of the inhibition of compound 7 on SARS‐CoV‐2 replication in Vero, Calu‐3, Huh‐7, and Caco‐2. The cells were infected with SARS‐CoV‐2 GD108 at MOI of 0.05 and then treated with compound 7 . N protein expression was detected by WB at 48 h post‐infection ( n = 3).

Article Snippet: The human lung adenocarcinoma Calu‐3 cell line (Procell, CL‐0054, Wuhan, China), human hepatoma Huh‐7 cell line (Procell, CL‐0120), human colon Caco‐2 cell line (Procell, CL‐0050), human lung adenocarcinoma H1299 cell line (Procell, CL‐0165), and human embryonic kidney HEK293T cell line (Procell, CL‐0005) were cultured in standard medium at 37°C and 5% CO 2 .

Techniques: Activity Assay, In Vitro, Inhibition, Infection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Cytotoxicity Assay, CCK-8 Assay, Staining, Expressing

Journal: MedComm

Article Title: Lycorine Derivative Inhibits SARS‐CoV‐2 Replication by Reducing −1 Programmed Ribosomal Frameshifting via Targeting ZAP

doi: 10.1002/mco2.70715

Figure Lengend Snippet: Compound 7 directly binds to ZAP and leads to a decrease in –1PRF. (A) Schematic representation of the dual‐luciferase frameshift reporter construct. The coding sequences for Renilla luciferase and Firefly luciferase were separated by the SARS‐CoV‐2 –1FSE sequence (13460‐13548). (B) Fold change in the Firefly/Renilla (F/R) luciferase ratio after treatment with the indicated drugs (lycorine at 10 µM, compound 7 at 10 µM, merafloxacin at 40 µM). Huh‐7 and H1299 cells transfected with the pHRF‐FSE (–1) luciferase reporter vector were treated with DMSO (Control) or the indicated drugs for 48 h ( n = 6 per group). (C) The frameshift Reporter mRNA containing a 3×FLAG‐tag followed by nucleotides 12686–14190 of the SARS‐CoV‐2 genome was translated in a RRL translation system in the presence of compound 7 or merafloxacin. The 3×FLAG‐tag was introduced at the N‐terminus to facilitate detection. WB analysis of the compound 7 effect on the –1PRF frameshift efficiency (FE) using anti‐flag antibody (Sigma, F3165). BC (blank control), NC (negative control). (D) Relative abundance of Nsp9, Nsp12, and Nsp15 in compound 7 (0, 2.5, and 5 µM) or Mer (merafloxacin, 40 µM)‐treated Vero cells after SARS‐CoV‐2 infection (MOI = 0.05). (E) The cellular target of compound 7 was identified using DARTS technology coupled with LC–MS/MS in H1299 cells. M, marker. (F) Venn diagram between the target proteins of compound 7 and the in vitro RNA antisense purification of the SARS‐CoV‐2 frameshift site from the literature. (G) ZAP protein stability was increased upon compound 7 (10 µM) treatment in H1299 cell lysates. (H) H1299 cells were transfected with the pcDNA3.1‐3×Flag‐Nsp12 plasmid and cultured for 48 h. Cells were then lysed and treated with 10 µM compound 7 (+) or DMSO (–). Recombinant Nsp12 (rNsp12) protein was detected by WB analysis, which was performed using anti‐Flag and anti‐Nsp12 antibodies for detection. (I) CETSA confirmed the binding of compound 7 (50 µM) to ZAP in 293T cells, with GAPDH serving as the internal control. (J) The binding of compound 7 to ZAP was depicted through BLI.

Article Snippet: The human lung adenocarcinoma Calu‐3 cell line (Procell, CL‐0054, Wuhan, China), human hepatoma Huh‐7 cell line (Procell, CL‐0120), human colon Caco‐2 cell line (Procell, CL‐0050), human lung adenocarcinoma H1299 cell line (Procell, CL‐0165), and human embryonic kidney HEK293T cell line (Procell, CL‐0005) were cultured in standard medium at 37°C and 5% CO 2 .

Techniques: Luciferase, Construct, Sequencing, Transfection, Plasmid Preparation, Control, Negative Control, Infection, Liquid Chromatography with Mass Spectroscopy, Marker, In Vitro, Purification, Cell Culture, Recombinant, Binding Assay

Journal: MedComm

Article Title: Lycorine Derivative Inhibits SARS‐CoV‐2 Replication by Reducing −1 Programmed Ribosomal Frameshifting via Targeting ZAP

doi: 10.1002/mco2.70715

Figure Lengend Snippet: Compound 7 exerts antiviral efficacy dependent upon ZAP‐S. (A) Effects of ZAP‐S knockdown on –1PRF followed by DMSO or compound 7 (10 µM) treatment in Huh‐7 and H1299 cells ( n = 3). (B) Compound 7 (10 µM) in combination with ZAP overexpression synergistically decreased –1PRF in H1299 and Huh‐7 cells ( n = 4); (C and D) Antiviral activity of compound 7 (2.5 µM) following ZAP‐S knockdown, or overexpression of ZAP‐S and mutant ZAP‐S. IF visualization of SARS‐CoV‐2 N protein (green) and cell nuclei (blue) in infected Huh‐7 cells at 48 h posttreatment (left). Scale bar: 100 µm. WB of N protein expression in Huh‐7 cells infected with SARS‐CoV‐2 (right).

Article Snippet: The human lung adenocarcinoma Calu‐3 cell line (Procell, CL‐0054, Wuhan, China), human hepatoma Huh‐7 cell line (Procell, CL‐0120), human colon Caco‐2 cell line (Procell, CL‐0050), human lung adenocarcinoma H1299 cell line (Procell, CL‐0165), and human embryonic kidney HEK293T cell line (Procell, CL‐0005) were cultured in standard medium at 37°C and 5% CO 2 .

Techniques: Knockdown, Over Expression, Activity Assay, Mutagenesis, Infection, Expressing