Forskolin

A mouse neuroblastoma cell line, N2a, was cultured in complete media containing DMEM (11965084, Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS) (C0232, Beyotime, Shanghai, China). Cells were maintained at 37 °C, 5% CO₂ and 95% humidified air. Cells were seeded in a six-well plate and treated with forskolin (S1612, Beyotime, Shanghai, China) for 24 h and then harvested for microRNA extraction or Western blot.

The generation of a hepatocellular carcinoma cell line (LMH cells) with stable expression of OATP1A2 (LMH-1A2) has been previously described (19 (link)). LMH-1A2 cells were grown in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (Gibco) supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO2. The Chinese avian HEV strain (CaHEV, GenBank accession number GU954430), initially isolated from chicken bile samples, was produced using a previously described method and stored at −80°C (19 (link)). The RNA copy numbers of the CaHEV viral stock were quantified with quantitative real-time PCR (qPCR) and used to inoculate LMH-1A2 cells at 20 copies per cell throughout this study. For the chemical agents, CytoD was obtained from Selleck Chemicals (Houston, USA). Forskolin and H-89 were obtained from Beyotime Biotechnology (Beijing, China). ATN-161 was obtained from Sigma–Aldrich (St. Louis MO, USA).

Escherichia coli DH5α was used for plasmid amplification. Agrobacterium tumefaciens strain AGL-1 was used for fungal transformation [42 (link)]. T. reesei QM6a (ATCC 13631) was used throughout the study. E. coli and A. tumefaciens were cultured in Luria broth (LB) medium. All strains of T. reesei were maintained on potato dextrose agar (PDA) plates at 28 °C. Conidia were collected from the PDA plates. All strains were cultured in the dark.
Minimal medium (MM; urea 0.3 g/L; (NH₄)₂SO₄ 5 g/L; KH₂PO₄ 15 g/L; MgSO₄ 0.6 g/L; CaCl₂ 0.6 g/L; FeSO₄·7H₂O 5 mg/L; ZnSO₄·7H₂O 1.4 mg/L; CoCl₂·6H₂O 2 mg/L; pH 5.5) with 2% glucose or 1% Avicel was used to assess hyphal growth and cellulase production. Conidia (2 × 10⁶) were cultivated at 28 °C (200 rpm) in 50 mL of MM (2% glucose as the sole carbon source) for 36–48 h. The mycelia were inoculated into 100 mL of freshly prepared MM containing 1% Avicel as the sole carbon source with no further addition or the addition of Mn²⁺ (final concentration 10 mM), DMF (final concentration 1%), forskolin (final concentration 10 µM; Beyotime, Shanghai, China), or dibutyryl cAMP (dbcAMP, final concentration 5 mM; Sigma-Aldrich, St. Louis, MO, USA) as described previously [19 (link)]. Mycelia were grown for 96 h at 28 °C. One millilitre of culture liquid was collected at 24-h intervals to perform the assays.