Mpk1096

For electroporation, cells were dissociated to singlets as described above. For testing of electroporation conditions, cells were resuspended at a concentration of 20,000 cells/μL in Neon Buffer R, and pMax-GFP was added at 100 ng/μL of buffer. After electroporation with a 10 μL Neon tip (ThermoFisher MPK1096), cells were seeded onto a single well of a collagen-coated 24-well plate with MM + Y and allowed to recover overnight at 37°C. Media were replaced after 24 h with MM, and cells were analyzed for transfection and survival after 72 h. After optimization, all other transfections were performed using the Neon Electroporator 100 μL tips (ThermoFisher Scientific MPK10096) with preset #5 (1,700 V, 1 pulse, 20 ms), electroporating 6,000–12,000 cells/μL and plating onto a single collagen-coated well of a six-well plate. Media was changed after 24 h with MM. For transfections requiring selection, media were changed with MM + puromycin (2 μg/mL, Sigma P8833) after 72 h and every other day thereafter for 7 days. To account for experimental variability in single plasmid integration rates, the transfection efficiency for each plasmid was normalized to the efficiency of a control transfection using a constitutively expressing EGFP plasmid (Figure 4B).

Mice ACCs were prepared as described previously.^[26] Briefly, the adrenal glands were isolated from anesthetized animals (25% ethyl carbamate), then the cortex was removed, cut into pieces, and digested by papain enzyme for 40 min at 37 °C under 5% CO₂. The pieces were then triturated gently through a 200‐µL pipette tip. After centrifugation, the cells were plated on coverslips pre‐coated with 0.1% poly‐L‐lysine, incubated at 37 °C under 5% CO₂, and used within 24–96 h. Chromaffin cells were transfected for genetic manipulations using a 10‐µL Neon electroporation system (Invitrogen, MPK1096) according to the manufacturer instruction.

A frozen stock of HDFs was thawed and cultured for four days, and then 1 × 10⁵ cells were collected by trypsinization. With SeV, HDFs were transduced with the CytoTune-iPS 2.0 (c-MYC) or CytoTune-iPS 2.0L (MYCL) Sendai Reprogramming Kit (DV-0304, DV-0305, ID Pharma). With EpiP, HDFs were electroporated with 1.2 μg of plasmid mixtures with the Neon Transfection System (MPK1096 and MPK10096, Invitrogen). The plasmid mixtures included pCXLE-SOX2, -KLF4, -OCT3/4-shp53, -LIN28A, and pCXWB-EBNA1 with wild-type or mutant pCXLE-c-MYC or -MYCL^{17 (link)}. The mixing ratio of SOX2, KLF4, OCT3/4-shp53, LIN28A, EBNA1, and c-MYC/MYCL was 1:1:2:1:0.5:2. After that, the cells were plated in a 6-well plate and cultured in StemFit AK03N without bFGF with iMatrix-511 at 0.25 μg/cm² in SeV or 0.125 μg/cm² in EpiP. The culture medium was changed the next day and every three days after that. The colonies were counted 21 days after plating.