Xhoi r0146s

RPMI 1640 medium (61870-036) and fetal bovine serum [FBS] (10099141) were purchased from Gibco (USA, NY). CORT and neferine (HY-N0441) were purchased from MedChem Express (NJ, USA). The GenElute Gel Extraction Kit (NA1111) was purchased from Sigma-Aldrich (Darmstadt, Germany). BamHI (R0136S) and XhoI (R0146S) were purchased from NEB (NY, USA). Lipofectamine 3000 Transfection Reagent (L3000015) was purchased from Invitrogen (CA, USA). G418 (G8161) was purchased from Solarbio (Beijing, China). RNApure Tissue and Cell Kit (CW0584), HiFiScript cDNA Synthesis Kit (CW2569), and Super TaqMan Mixture (CW2698) were obtained from Cwbiotech (Beijing, China). Antibodies against Bcl-2 (ab32124), Bax (ab32503), Bad (ab32445), p53 (ab26), Bak (ab32371), succinate-CoA ligase GDP-forming beta subunit [SUCLG2] (ab96172), aconitase 2 [ACO2] (ab110321), malate dehydrogenase 1 [MDH1] (ab180152), citrate synthase [CS] (ab96600), isocitrate dehydrogenase [IDH] (ab172964), NF-κB p65 (ab16502), and glyceraldehyde 3-phosphate dehydrogenase [GAPDH] (ab8245) were purchased from Abcam (Massachusetts, US). ELISA kits for IL-1β (ab100562), IL-2 (ab174444), IL-6 (ab46027), TNF-α (ab181421), interferon-γ [IFN-γ] (ab46025), and granulocyte colony-stimulating factor [G-CSF] (ab100524) were purchased from Abcam. Rabbit and mouse secondary antibodies were purchased from ECL.

Individual flies were homogenized in 100 μL solution A (0.1M Tris, 0.1M EDTA, 0.1%SDS, pH 9.0) and incubated at 70℃ for 25 min for cell lysis and DNA denaturation. 14 μL KAc (8M) was added for renaturation on ice for 30 min. DNA extracts were separated at 4℃ by centrifugation for 15 min at 14,000 rpm. After that, total DNA was precipitated with 60 μL isopropyl alcohol and washed with 70% ethanol, then finally diluted in 10 μl ultra-pure water. 2 μL DNA extract was used for each PCR reaction (Taq DNA polymerase, M0273L, NEB).
For enzyme treatment in Supplementary Fig. 1b and c, 25 μL PCR product was purified and treated with XhoI restriction enzyme (XhoI, R0146S, NEB) at 37℃ overnight.
The separation of mitochondria and nucleus in Fig. 2c was conducted following the protocol from the Qproteome Mitochondria Isolation Kit (QIAGEN, 37612). About 30 1-day-old flies were homogenized and subjected to the mitochondria isolation process. The pellets containing mitochondria and nuclei were later subjected to the DNA extraction process. Resultant pellets containing mtDNA or nuclear DNA were suspended in 30 μL water. PCR was carried out using 2 μL mtDNA and nuclear DNA extraction.