Journal: bioRxiv

Article Title: MODULATION OF LIPID METABOLISM BY THAPSIGARGIN INHIBITS HEPATITIS C VIRUS INFECTION

doi: 10.64898/2026.03.02.709032

Figure Lengend Snippet: Huh7.5 cells were treated with DMSO or 10 µM IXA4 for 72 hours. (A) Cellular RNA was harvested and XBP1s (IRE1 pathway), CHOP (PERK pathway) and HERPUD1 (ATF6 pathway) mRNA expression was assessed by RT qPCR. (B) Expression of genes related to LD biogenesis ( FASN , DGAT1 and DGAT2 ) was assessed by RT-qPCR. (C) Huh7 cells were electroporated with 5 µg of HCV SGR RNA. Four hours-post electroporation, cells were treated with DMSO or 10 µM IXA4 for 72h and RNA replication was measured by luciferase expression. Data are expressed as a percentage of HCV replication in DMSO-treated samples. (D-E) Huh7.5 cells were pre-treated with DMSO or 0.05 µM Tg for 30 minutes, and subsequently infected with HCV at MOI 0.2 for 72h. (D) Cells were fixed and stained for cell nuclei (DAPI, blue) and LDs (BODIPY-493; green), HCV core (red), and assessed by confocal microscopy under 63x magnification. (E) Cellular supernatant was collected, and viral extracellular titer was assessed by focus forming assay. Graphs shown mean +/− SEM from 3 independent experiments. RT-qPCR was performed in technical triplicate per biological replicate. Confocal microscopy data are representative of at least three biological replicates with at least three technical replicates per condition. *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001.

Article Snippet: Primary antibodies against ATF6 (Cell Signaling Technology (CST) 8089, dilution 1:1000), IRE1a (CST 3294, dilution 1:1000), PERK (CST 5683, dilution 1:1000), and beta-actin (Abcam ab8226, dilution 1:5000), and secondary antibodies Licor IRDye-680 goat anti-mouse and Licor IRDye-800 were used for western blot analysis.

Techniques: Expressing, Quantitative RT-PCR, Electroporation, Luciferase, Infection, Staining, Confocal Microscopy, Focus Forming Assay

Journal: bioRxiv

Article Title: MODULATION OF LIPID METABOLISM BY THAPSIGARGIN INHIBITS HEPATITIS C VIRUS INFECTION

doi: 10.64898/2026.03.02.709032

Figure Lengend Snippet: (A-C) Huh7.5 cells were transduced with shCTRL, shATF6, shIRE1 or shPERK lentivirus to generate stable knockdowns of ATF6, IRE1 and PERK. (A) Successful silencing was confirmed by western blot analysis. (B-C) Cells were treated with DMSO or 0.1 µM Tg for 30 minutes and subsequently infected with HCVcc (MOI 0.2) for 72 hours. (B) Cells were fixed and stained for cell nuclei (DAPI; blue), lipid droplets (BODIPY-493; green) or HCV core (red) and imaged using a confocal microscope at 63x magnification. (C) Cellular supernatants were collected, and viral titer was assessed by focus-forming assay. (D) Huh7.5 cells were treated with DMSO or 1 µg/mL tunicamycin (Tm) for 4 hours. Cellular RNA was harvested and XBP1s and HERPUD1 mRNA expression was assessed by RT-qPCR. (E-G) Huh7.5 cells were pre-treated with DMSO or 1 µg/mL Tm for four hours, and subsequently infected with HCVcc (MOI 0.2) for 72 hours. (E) intracellular HCV RNA was assessed by RT-qPCR, (F) Cells were fixed and stained as described in (B) . (G) Viral titer was assessed by focus forming assay. Graphs shown mean +/− SEM from 3 independent experiments. RT-qPCR was performed in technical triplicate per biological replicate. Confocal microscopy data are representative of at least three biological replicates with at least three technical replicates per condition. *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001.

Article Snippet: Primary antibodies against ATF6 (Cell Signaling Technology (CST) 8089, dilution 1:1000), IRE1a (CST 3294, dilution 1:1000), PERK (CST 5683, dilution 1:1000), and beta-actin (Abcam ab8226, dilution 1:5000), and secondary antibodies Licor IRDye-680 goat anti-mouse and Licor IRDye-800 were used for western blot analysis.

Techniques: Transduction, Western Blot, Infection, Staining, Microscopy, Focus Forming Assay, Expressing, Quantitative RT-PCR, Confocal Microscopy