5 azadc 5 aza 2 deoxycytidine

The drought-tolerant potato variety used was Q, and the drought-sensitive variety was A. We can use the paper bridge method to inoculate the stems of two varieties of potato tissue cultured seedlings in MS liquid medium (without agar); each stem has an axillary bud, and the test-tube seedling culture environment condition is 22 (±2) °C. The light intensity is 25–37.5 μmol m⁻² s⁻¹ and the light duration is 16 h every day. After 24 days of cultivation, the potato test-tube plantlets were treated with three treatment methods; namely, 200 mmolL-1 mannitol treatment (simulated drought treatment and referred to as M in the following), 60 μmol L-1 5-azadC treatment (demethylation and referred to as A in the following), and the two reagents were treated simultaneously (referred to as D in the following). After the treatment, the stems and leaves of the test tube seedlings were treated for 2 h, 6 h, 12 h, and untreated (control, 0 h), quickly frozen with liquid nitrogen, and stored in an ultra-low temperature refrigerator at −80 °C for later use. Three biological replicates were used for each treatment. 5-azadC (5-aza-2′-deoxycytidine) was purchased from Sigma-Aldrich.

For DNA methylation inhibitor treatment in silkworm pupae, 1 mg 5-aza-dC (5-aza-2′-deoxycytidine) (Sigma, California, USA) was dissolved in deionized distilled water (ddH₂O) with a 10 μg/μL final concentration. Newly molted pupae (P0 stage) were injected into the abdomen with freshly prepared 5-aza-dC (20 μg per pupa) or ddH₂O as control. Three replicates of about 30-40 pupae per replicate were carried out. In addition, for inhibitor 5-aza-dC treatment in the cultured Bm12 cells, the freshly prepared 5-aza-dC was diluted 10 times to 1 μg/μL final concentration, and 1 μg 5-aza-dC was applied to cells.

The antibodies against β-actin and LMP1 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and DAKO (Glostrup, Denmark), respectively. The antibodies to detect DNMT1, VDAC, and total OXPHOS were from Abcam (Cambridge, UK). Anti-p-AKT and PTEN were obtained from Cell Signaling Technologies (Danvers, MA, USA).
Grifolin (2-trans,trans-farnesyl-5-methylresorcinol) was provided by Kunming Institute of Botany, the Chinese Academy of Sciences (purity >99%, High performance liquid chromatography (HPLC) analysis).
Dimethyl sulphoxide (DMSO, Sigma) was used to dissolve grifolin. The final concentration of DMSO in the culture media was kept at <0.1% v/v, which had no significant effect on cell growth.
5-aza-dC (5-aza-2-deoxycytidine) was purchased from Sigma-Aldrich and DMSO was used to dissolve 5-aza-dC.