Phenformin

Wild-type (Bristol N2), CB4088 him-5(e1490) males, DH1390 rme-2(b1008), LIU1 ldrIs1 [dhs-3p::dhs-3::GF+unc-76(+)] and a transgenic strain bIs1(vit-2p::vit-2::GFP) expressing a fusion of YP170 were used in this study. For the measurements of different developmental stages, synchronized worms were prepared via a two-generation egg lay method. For the first generation, ~10–20 adult worms were placed on the NGM OP50 plate and incubated at 20 °C for 2 h, and then the worms were removed. After an 80-h incubation at 20 °C, the laid eggs/embryos grew to gravid worms. Approximately 10–20 synchronized gravid worms were then picked and placed on the NGM OP50 plate and incubated at 20 °C for 2 h. After that, the gravid worms were removed and the synchronized embryos were incubated at 20 °C until appropriate stages for observation. This two-generation egg lay method can minimize the time error of synchronization, ensure more reproducible measurement results and avoid the starvation effect such as synchronization by L1 starvation arrest. The fixed LipidTOX staining of worms was carried out as previously described^{22 (link)}. Phenformin was purchased from Sigma-Aldrich. Sodium azide was purchased from Sigma-Aldrich and used to anesthetize worms for microscopy.

OvCa cells were maintained in standard DMEM supplemented with 10% FB-Essence (VWR Life Science SeraDigm, Radnor, PA) with 1% penicillin/streptomycin (Corning, Corning, NY), 1% MEM non-essential amino acids (Corning) and 1% MEM vitamins (Corning). All cell lines were genotyped to confirm their authenticity (IDEXX Bioresearch short tandem repeat marker profiling every 3 months), and were used between 2-12 passages from first thaw. All experiments were carried out in low glucose DMEM (VWR Life Science) supplemented as above. The pLX304 SPHK1-V5 lentiviral expression vector was purchased from DNASU (Tempe, AZ). The constitutively active HIF1α (P402A/P564A) and HIF2α (P405A/P531A) plasmids were a gift from William Kaelin (27 (link)) obtained through Addgene (Cambridge, MA). 5HRE-GFP reporter was a gift from Martin Brown and Thomas Foster obtained through Addgene (28 (link)). SPHK1 siRNA was obtained from GE Dharmacon (Lafayette, CO). Metformin, phenformin, CoCl₂, and thiazolyl blue tetrazolium bromide were purchased from Sigma Aldrich (St. Louis, MO). S1P (Huzzah S1P) was purchased from Avanti Polar Lipids (Alabaster, AL). Hypoxia was induced through 1% O₂/5% CO₂ using Hypoxia Incubator Chamber by STEMCELL Technologies (Vancouver, BC, Canada).