Cell culture

HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2-null (CRL-2993), and Mfn1-null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA). GP2-293 cells were purchased from Takara Bio USA (#631458). All cells were maintained at 37°C with 5% CO₂ in a Forma Steri-Cycle i160 CO2 Incubator (NC1207547, Thermo Fisher Scientific). Cells were cultured in 10% FBS in DMEM with sodium bicarbonate. Cell lines were authenticated using STR profiling. Mycoplasma contamination was absent in monthly tests using Mycoplasma Detection PCR. To obtain MFN2 re-expression cell line, introduce the ER-mito tethering structure, or express CaMKII-WT, CaMKII-DN, MLCK-CA, or MRLC-GFP, the MSCV-puro vector (Takara Bio USA, #634401) were used. We cloned the gene of interest into the MSCV vector and cotransfected the vector plasmid along with the envelope plasmid pVSVG, at a ratio of 1:1, into GP2-293 cells using Lipofectamine 3000 (Invitrogen L3000015). Virus supernatant was collected at 48 hpt and 72 hpt and further concentrated with Lenti-X concentrator (Clontech 631232). MEF cells were transduced with concentrated retrovirus in a complete medium and then selected with 2 µg/ml puromycin (Gibco A1113803) starting the next day. The stable lines were generated after puromycin selection for 1 wk. To generate ROCK or MLCK knocking down lines in Mfn2-null MEF cells, pLKO.1 lentiviral constructs with shRNAs were obtained from Sigma-Aldrich (shROCK: TRCN0000022903, shMLCK: TRCN0000024037), and SHC 003 was used as a non-targeting control. The plasmid of lentiviral constructs together with pCMV-dR8.2 dvpr (Addgene #8455) and pCMV-VSV-G (Addgene #8454), at a ratio of 10:7.5:2.5, were cotransfected into HEK293T cells with Lipofectamine 3000 (Invitrogen L3000015) to produce lentivirus. Virus supernatant was collected at both 48 hpt and 72 hpt, and further concentrated with Lenti-X concentrator (Clontech 631232) before transduction. 2 µg/ml puromycin (Gibco A1113803) was added into the complete medium on the next day for selection.