Sang m3 insect medium

First, we selected an 826-bp dCNDP2 gene fragment, which is present in both transcript isoforms of the gene, as a template for the synthesis of double-stranded RNA (dsRNA). We amplified the DNA fragment using primers 5′-TAATACGACTCACTATAGGGAGGcgagatcggtcg-3′ and 5′-TAATACGACTCACTATAGGGAGGatagcgccacctgg-3′ that contain the T7 polymerase promoter sequence at their 5′ ends (shown in capital letters). The PCR product was purified using the GeneJET PCR Purification Kit (Thermo Scientific, K0702) and then used as a template to synthesize dsRNA as described earlier [28 ], with minor modifications. Treatment with DNaseI was done after heating the synthesized dsRNA to 65 °C and its slow cooling to room temperature; in addition, the phenol/chloroform extraction was omitted.
S2 cells were free from mycoplasma contamination and were cultured in 39.4 g/L Shields and Sang M3 Insect medium (Sigma, S8398) supplemented with 0.5 g/L KHCO₃ and 20% heat-inactivated fetal bovine serum (FBS; Thermo Scientific, 10270106) at 25 °C. RNAi treatments were carried out as described previously [29 (link)], with the following modifications. Twenty-five μg of purified dsRNA was added to the cells three times (on the first, the third and the fifth days of incubation) and cells were harvested for analyses after 7 days of RNAi. Control S2 cell samples were prepared in the same way, but without addition of dsRNA.

Shields and Sang M3 Insect Medium, Dispase II, vinblastine, thapsigargin, EGTA and CaCl₂ were purchased from Sigma-Aldrich. CPA, and ionomycin were purchased from Abcam, Collagenase A was purchased from Roche. Fura-2/AM was purchased from Thermo Fisher. All other materials were analytical or of the highest available grade.

OSS cells were grown in prepared from Shields and Sang M3 Insect Medium (Sigma) supplemented with 0.6 mg/ml glutathione, 10 mU/ml insulin, 10% fetal bovine serum and 10% fly extract.