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Lentiviruses were generated by transfecting the pLKO.1 vectors described in the Method Details, each containing a different shRNA sequence directed against its cognate target (Parp1 or Nmnat1), into 293T cells, together with the expression vectors for the VSV-G envelope protein (pCMV-VSV-G, Addgene plasmid no. 8454), the expression vector for GAG-Pol-Rev (psPAX2, Addgene plasmid no. 12260), and a vector to aid with translation initiation (pAdVAntage, Promega) using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, L3000015) according to the manufacturer’s instructions. The viruses were collected in the culture medium, concentrated by using a Lenti-X concentrator (Clontech, 631231), and used to infect 3T3-L1 cells. The infected cells were selected with 2 μg/mL puromycin (Sigma, P9620), expanded, and frozen in aliquots for future use.

Mouse preadipocytes (derived from inguinal white adipocyte tissues, gift from Dr. Shingo Kajimura) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and penicillin/streptomycin. To differentiate into adipocytes, preadipocytes were cultured to ~95% confluence before a differentiation cocktail was added to the following concentrations: 5 μg/mL of insulin (Sigma, #I0516), 1 nM T3 (Sigma, #T2877), 125 mM indomethacin (Sigma, #I-7378), 5 μM dexamethasone (Sigma, #D1756), and 0.5 mM IBMX (Sigma, #I5879). After 2 days, the cells were switched to DMEM supplemented with 10% FBS, 5 μg/mL of insulin, and 1 nM T3. After another 2 days, fresh media of the same composition were supplied. Differentiated adipocytes were usually analyzed 6 days after the addition of the differentiation cocktail.
To generate cell lines expressing the GFP-GLUT4-HA reporter, lentiviruses were produced by transfecting 293T cells with a mixture of plasmids including GFP-GLUT4-HA,^{74 (link)} pAdVAntage (Promega, #E1711), pCMV-VSVG, and psPax2. Lentiviral particles were collected 40 h after transfection and every 24 h thereafter for a total of four collections. Lentiviruses were pooled and concentrated by centrifugation in a Beckman SW28 rotor at 25 000 rpm for 1.5 h. The viral pellets were resuspended in PBS and were used to transduce preadipocytes.

We generated stable HeLa CCL2 cells expressing YFP-CASP4(C258S) (31 (link)) by lentiviral transduction. Lentivirus was produced in HEK293T by transient transfection with the lentiviral backbone pMX-CMV-YFP CASP4(C258S) and the three packaging plasmids pVSV-G (#138479, Addgene), pAdvantage (E1711, Promega), and pGag-Pol (#14887, Addgene). DNA, mixed with FuGENE 6 Transfection Reagent (Promega) in Opti-MEM (Gibco), was incubated at RT for 15 min, then added dropwise into HEK293T cells. After 48h, the supernatant containing the lentivirus was collected, filtered using a 0.45 μm membrane filter, and supplemented with polybrene (Sigma) at 8 μg/mL. Lentivirus was then transduced into HeLa CCL2 cells by centrifugation of the HEK293T supernatant for 2 h at 1000 × g at 30°C. Two days later, HeLa CCL2 containing YFP-CASP4(C258S) were selected and maintained with 2 μg/mL of puromycin.