10 cm cell culture dishes

Vero cells (ATCC, CCL-81), PK15 cells (ATCC, CCL-33), Panc-1 (ATCC, CRL-1469) and primary rat embryonic fibroblasts (REFs) were all maintained in Dulbecco’s Modified Eagle’s Media (DMEM, Cytiva) supplemented with 10% FBS (Omega Scientific) and 1% penicillin-streptomycin (Hyclone), and incubated in a 5% CO₂ incubator at 37°C.
REFs were collected from E16–17 Sprague-Dawley rat embryos (Charles River Laboratories), as follows: Animal work was performed in accordance with all applicable regulations and guidelines, and with the approval of the Institutional Animal Care and Use Committee (IACUC) at Arizona State University (protocol #20–1799R). The animal care and use program at Arizona State University has an assurance on file with the Office of Laboratory Animal Welfare (OLAW), is registered with the USDA, and is accredited by AAALAC International. Briefly, embryos were decapitated and internal organs removed. Remaining skin and connective tissue was trypsinized (Trypsin-EDTA, Gibco) at 37°C, pipetted vigorously in complete DMEM, and supernatants were plated onto 10cm cell culture dishes (Celltreat). REFs were passaged no more than 4 times before use in experiments [76 ].

All cell cultures were maintained at 37°C and 5% CO₂. HeLa cells were plated in 10-cm cell culture dishes (CellTreat) at a density of 1.5 x 10⁵ cells/mL in DMEM + 10% FBS (Mediatech, Gibco). Knockdown of the mtFASII pathway was achieved using Qiagen Flexitube siRNAs specific for the gene for ACP (NDUFAB1) as described in Parl et al., 2013[25 (link)]. Control cells were transfected with Allstars negative control siRNA (Qiagen). Additionally, as a negative control cells were transfected with siRNAs specific for METTL9, a nuclear gene encoding a mitochondrial protein, HeLa cells were transfected with siRNA using HiPerfect Transfection Reagent (Qiagen). In order to maintain the knockdown, cells were re-transfected after 48 h: cells were trypsinized, resuspended in double their original volume of DMEM + 10% FBS, then plated in a 15-cm dish for an additional 48 h. At 96 h, cells were harvested by trypsinization and centrifugation. Knockdown efficiency was measured after 96 h using real time quantitative RT-PCR. Cell viability was unaltered in the siRNA treated cells as determined by cell count and trypan blue uptake.

Vero cells (ATCC, CCL-81), PK15 cells (ATCC, CCL-33), Panc-1 (ATCC, CRL-1469), and primary REFs were all maintained in Dulbecco’s Modified Eagle’s Media (DMEM, Cytiva) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 1% penicillin-streptomycin (Hyclone) and incubated in a 5% CO₂ incubator at 37°C. MRC5 cells (ATCC, CCL-171) were maintained in Eagle’s Minimal Essential Media (Cytiva) supplemented with 1% FBS and 1% penicillin-streptomycin, as stated above.
REFs were collected from E16-17 Sprague-Dawley rat embryos (Charles River Laboratories) as follows: briefly, embryos were decapitated and internal organs were removed. The remaining skin and connective tissue were trypsinized (Trypsin-EDTA, Gibco) at 37°C, pipetted vigorously in complete DMEM, and supernatants were plated onto 10 cm cell culture dishes (Celltreat). REFs were passaged no more than four times before use in experiments (78 (link)).