Schneider s drosophila media

We grew D. melanogaster S2 cells (Invitrogen) at room temperature in Schneider’s Drosophila media (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. To induce ER stress, we treated cells with dithiothreitol (DTT, 2 mM, 5 hr) or tunicamycin (Tm, 5 μg/mL, 16 hr) unless otherwise stated.
To deplete cells of Atf4 by RNAi, we amplified a 527-nucleotide region from the coding sequence of Atf4 (also known as cryptocephal/crc, CG8669) using primers with T7 RNA polymerase sites at the 5′ ends. This amplicon has no predicted off-target 21 nt siRNA sequences, as determined using the Drosophila RNAi Screening Center (http://www.flyrnai.org). We used this polymerase chain reaction (PCR) product to generate double-stranded RNA (dsRNA) by in vitro transcription (Megascript T7 kit; Ambion). We then incubated S2 cells with 15 μg of dsRNA in serum-free media for 45 min, replaced the serum, and allowed the cells to recover for 5 d. We retreated cells with 45 μg of dsRNA and induced ER stress 1 d after the second dsRNA treatment.

Ae. aegypti A20 and Aag2 cells were maintained in Leibovitz’s L-15 media (Gibco) supplemented with 10% FBS (Atlanta Biologicals) and 1% Pen-strep (Corning) at 28 °C. Aag2 cells were also maintained in Schneider’s Drosophila Media (Thermo Fisher) with 10% FBS (Atlanta Biologicals) and 1% Pen-strep (Corning) at 28 °C. Both cell lines are available upon request.

S2 cells, wild-type S2R+ cells, and tsc2 KO S2R+ cells [44 (link), 45 (link)] were grown in Schneider’s Drosophila Media (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and penicillin–streptomycin. For insulin treatment, 25 µg/ml of insulin was treated for the indicated time lengths. For rapamycin (LC Laboratories) treatment, cells were incubated with 20 nM rapamycin for the indicated time lengths.
For RNAi experiments, PCR templates for dsRNA against CCT1 through CCT8 were prepared using primers designed by SnapDragon-dsRNA design (http://www.flyrnai.org/snapdragon). dsRNAs for CCT1-8 were generated by PCR using MEGAscript T7 (Ambion) and purified using MEGAClear (Ambion). Thirty micrograms of dsRNA was treated in S2R+ cells in six-well plates for 3 days using bathing method [46 (link)].
For immunoprecipitation between Myc-CCT4 and Rheb-V5 proteins, coding sequences of CCT4 and Rheb were cloned into pAc5.1-V5/His (Invitrogen) with N-terminal Myc tag with and without C-terminal stop codon, respectively. The cloned constructs were transfected in S2 cells using Effectene reagent (Qiagen).

Drosophila S2 cells (R69007, Thermo Fisher Scientific) were cultured in Schneider’s Drosophila Media (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and penicillin-streptomycin-glutamine (Thermo Fisher Scientific) in 75 cm² flasks. The actin-Gal4 [77 (link)] and tubulin-Gal4 [78 (link)] plasmids were used as in previous publications. Cells were transfected with 1 μg of each plasmid DNA with the Effectence transfection kit (Qiagen). After 48 hours transfection, cells were fixed in 4% paraformaldehyde and mounted on coverslips coated in 0.5mg/mL Concanavalin A and ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific).
Fly adult brains and third instar wing discs were dissected, fixed, and immunostained as previously described [4 (link), 77 (link)]. The LacZ primary antibody was used at 1:500 dilution (A-11132, Thermo Fisher Scientific). Repo (8D12) and Elav (7E8A10) co-staining were performed using a 1:10 dilution (Developmental Studies Hybridoma Bank). DyLight 405, Alexa Fluor 488, and Alexa Fluor 647 conjugated secondary antibodies were used at 1:100 dilution against Repo and Elav antibodies and at 1:500 against LacZ (Jackson ImmunoResearch). All S2 cell and fly brain images were acquired on a Zeiss LSM 800 confocal microscope and processed in the Zeiss ZEN software package.

Drosophila S2 cells were purchased from Thermo Fisher Scientific and were grown at 25°C in Schneider's Drosophila Media (Thermo Fisher Scientific), supplemented with 10% heat‐inactivated FBS, 1% Pen‐Strep and 1/500 MycoZap Plus‐CL. For circadian entrainment protocol, S2 cells were subjected to temperature cycles (12 h at 23°C, 12 h at 28°C) for at least 1 week, with media changes occurring every 3–4 days at the transition between 23 and 28°C. The last medium change was performed at t = 0 h, and cells were plated into six‐well plates. Cells were kept at 25°C for the remaining of the experiment, with sample collection occurring at 3‐h intervals between 24 and 81 h. For the RNA‐Seq experiment at 28°C, S2 cells were grown at 28°C in Schneider's Drosophila Media, supplemented with 10% heat‐inactivated FBS and 1% Pen‐Strep. Cells were plated into six‐well plates and were synchronised with 2 days of temperature cycles (12 h at 28°C, 12 h at 23°C) followed by 24 h at 28°C, before the start of sample collection at t = 24 h. To measure the change in cell density over the time course, we used the Countess Automated Cell Counter (Invitrogen) system following manufacturer's instructions.

Drosophila S2 cells were maintained in Schneider's Drosophila media (Thermo Fisher Scientific) supplemented with 10% FBS in a 25°C incubator. S2 cells were transfected with actin-GAL4 and UAS-wfs1-HA plasmids using HilyMax (Dojindo) following the manufacturer’s protocol. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 48 h after transfection and stained with anti-HA (Santa Cruz Biotechnology) or anti-KDEL (Enzo Life Sciences) antibody. Antibodies were detected by anti-rabbit IgG conjugated Alexa 488 (Abcam) or ant-mouse IgG conjugated Alexa 647 (Abcam). ER and mitochondria were labeled by Concanavalin A (ConA) conjugated Alexa 647 (Thermo Fisher Scientific) and Mitotracker (Thermo Fisher Scientific) respectively. The stained cells were analyzed using a confocal microscope (Carl Zeiss, LSM 780).