Meta imaging series

HFFs were grown on coverslips and used for infection with HCMV strain AD169. At three days post-infection, cells were fixed with 4% paraformaldehyde solution (10 min, room temperature) and permeabilized by incubation with 0.2% Triton X-100 solution (15 min, 4 °C). Non-specific staining was blocked by incubation with 2 mg/mL human γ-globulin (cohn fraction II, Sigma Aldrich; 40 min, 37 °C). Indirect immunofluorescence staining was performed by stepwise incubation with primary antibodies for 90 min each at 37 °C, followed by incubation with dye-conjugated secondary antibodies (Alexa, Molecular Probes, Inc., Eugene, OR, USA) for 30 min at 37 °C. Cell samples were mounted with Vectashield Mounting Medium containing DAPI and analyzed using a DMI6000 B microscope and a 63× HCX PL APO CS oil immersion objective lens (Leica Microsystems, Wetzlar, Germany). Confocal laser-scanning microscopy was performed with a TCS SP5 microscope (Leica Microsystems). Images were processed using the Meta-Imaging series (Molecular Devices, Sunnyvale, CA, USA) and LAS AF software (version 1.8.2 build 1465; Leica Microsystems).

IHC detection was performed using the HRP/DAB IHC detection kit (ABC kit, #ab64261) and an anti-p53 antibody (Thermo MA5-16387), following the manufacturer’s protocol. Images were taken with AxioVision software, and cell staining intensity was quantified using the Meta Imaging Series software from Molecular Devices.

Liver tissues (all lobes) were fixed in 10% formalin overnight, and then dehydrated and embedded in paraffin blocks, which were sectioned at a thickness of 5 μm. For Sirius red staining, sections were stained with hematoxylin (Weigert’s) for 8 min, washed with running tap water for 10 min, followed by incubation with 0.1% (wt/vol) Sirius red diluted in picric acid solution for 1 h. The slides were then rinsed in two quick changes of 0.5% (vol/vol) acetic acid to remove unbound dye. For immunostaining, sections were first heated in sodium citrate buffer to retrieve antigen. Sections were then blocked in 10% FBS blocking buffer followed by incubation with the following primary antibodies: anti-c-Jun (60A8; Cell Signaling), anti-F4/80 (SP115; Thermo Fisher Scientific), and anti-Ki67 (15580; Abcam), before quenching endogenous peroxidase using 3% hydrogen peroxide. TUNEL staining was performed as per manufacturer’s recommendations using the apoptosis detection kit (206386; Abcam). To quantify staining, 20 randomly taken images of 10× fields per section were evaluated by Meta Imaging Series (Molecular Devices) software.
A small portion of the liver was homogenized in ice cold NaCl buffer. The supernatant was collected, normalized before subjected to AST/ALT measurements using an AST/ALT Assay kit (Nanjing Jiancheng Bioengineering Institute) according to the supplier’s protocol.