Schneider s medium

Tissues were dissected and total RNA was extracted from embryos, nonsedated 6th instar larvae or 1-wk-old mature adults under a freshly prepared mixture of Schneider’s medium (Invitrogen, CA, US) and Tribolium saline (1:1, v/v) as described in SI Appendix, Materials and Methods.

Drosophila S2 cells are maintained in Schneider’s medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen). S2 cells stably expressing miR-34 were generated by transfection with pRmHa-3-miR-34 and the selection marker plasmid pHS-neo using the calcium phosphate method, followed by selection in medium containing 400 μg/mL G418 (Calbiochem). dsRNA treatment was performed as described previously [87 (link)–89 (link)]. Briefly, ~2 × 10⁶ S2 cells were seeded in 6-well plates for 24 h and then transfected with 3 μg of the appropriate dsRNA using the calcium phosphate protocol. Two days later, the cells were harvested, replated in 6-cm plates for 24 h, and then transfected again with another 9 μg of dsRNA. Three days later, the cells were harvested and used in assays. DNA transfection was preformed in the same manner as dsRNA transfections, except that only a single round of transfection was performed.

Mira (Flag-Mira WT and Flag-Mira L529E) and Stau (Flag-Stau WT and Flag-Stau H994E) variants were subcloned into UASt.attB vector (A gift from Konrad Basler).
Drosophila S2 cells were grown at 25 °C in Schneider's medium (Invitrogen) supplemented with 10% fetal bovine serum. All transfections were performed using Effectene Reagent (Qiagen) according to the manufacturer's instructions. Briefly, S2 cells were cotransfected with 0.5 μg plasmids of interest (Mira or Stau) together with act-gal4 plasmid. Cells were collected at 48 h later and lysed in Nonidet P-40 lysis buffer containing 50 mM Tris, pH 8.0, 250 mM NaCl, 0.5% Nonidet P-40, 0.2 mM EDTA, protease inhibitor cocktail (complete, Roche) and phosphatase inhibitor. The lysate was collected and cleared by centrifugation at 13,000 r.p.m. for 5 min at 4 °C. The samples were separated by 10% polyacrylamide SDS–PAGE gels followed by transferring to PVDF membranes (Millipore). Mouse anti-Flag antibody (Sigma, 1/2,000), Rabbit anti-Mira antibody (Generated in our lab, 1/1,000) and Rabbit anti-Stau antibody (Daniel St Johnston, 1/1,000) were diluted in TBST with 5% non-fat dry milk.

Cells were cultured in Schneider’s medium (Invitrogen) containing penicillin (50 u/mL), streptomycin (50 μg/mL) and 10% fetal bovine serum (FBS), at 28°C.

S2 cells were obtained from the University of California, Berkeley Cell Culture Facility and cultured using standard methods at 25°C in Schneider's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Transfections were performed using the Effectene kit (Qiagen) according to the manufacturer's instructions. 2×10⁶ cells per well of a six-well plate were transfected with 500 ng of DNA per plasmid. Cells were incubated in transfection complexes for 48 h and then switched into fresh medium containing 0.5 mM CuSO₄ to induce expression of the metallothionein promoter for 48 h before experiments. The induced polarity assay was performed essentially as described previously (Johnston, 2020 (link)). After 48 h of induction, transfected S2 cells were resuspended in 3 ml of fresh medium containing 0.5 mM CuSO₄. The cell suspensions were agitated in an orbital shaker at 150 rpm in six-well plates for 2 h to induce cell clusters. 1 ml per condition of the clustered cell suspension was then allowed to settle on poly-D-lysine-coated coverslips and adhere for 30 min. The cells were then fixed for 20 min in 4% paraformaldehyde (PFA) and processed for immunofluorescence as described below.