Fugene reagent

Cells at the logarithmic growth phase were plated into 96-well microtiter plates 24 h before transfection, in order to reach 60-80% of confluency. Plasmid DNA (containing SEPT-5 siRNA or miR-185 siRNA) was transiently transfected using FuGENE® reagent (Promega, Madison, WI, USA), according to the manufacturer's protocol. Briefly, 0.1 μl of the FuGENE® 6 Transfection Reagent was mixed into 10 μl of serum-free medium (Opti-MEM® I reduced serum medium) and was incubated for 5 minutes at room temperature. Then, 10 μl of the FuGENE® 6/Opti-MEM® I mixture was added to each well of cells to be transfected and was incubated at 37°C for 48 hours. There was no need to remove serum or change culture conditions or remove the transfection complex. Cells were collected and used in further experiments, 48 hours after transfection.

The DNA transfection followed a Promega FuGENE protocol by the manufacturer as described in Refs. 45 (link), 59 (link), and 60 (link). The HEK293 cells were transfected after reaching 30–50% confluence in a LabTeck 4-well cover glass chamber with the mixtures of bGCAP-GFP or hRD3-GFP coding plasmids with mOrange-RetGC1 coding plasmid, at ∼1/100 molar ratio (58 (link), 59 (link), 60 (link)), using 3 μl of Promega FuGENE reagent per 1 μg of DNA. Confocal images were taken after 24–30 h of incubation in 5% CO₂ at 37 °C, using an Olympus FV1000 Spectral instrument with the respective 543- and 488-nm excitation for the red and the green fluorochromes in sequential mode. The images were processed, and the PCC values in whole-cell images were determined using Olympus FluoView FV10-ASW software as previously described (43 (link), 44 (link), 45 (link), 58 (link), 59 (link), 60 (link)). No changes to the original images were made except for minor γ correction applied to whole image for more clear presentation in print. Quantitative analyses were performed using the original images without corrections.

All assays were performed with U2-OS cells (obtained from ATCC) grown in DMEM with 10% FBS. Cells were tested and confirmed free of mycoplasma contamination. Luciferase reporters carrying Nodal enhancers linked to a viral E1b promoter have been previously described^{49 (link)}. Generation of Y401X ZIC2 and deletion of ZIC2 binding sites within HBE was performed by site directed mutagenesis (NEB) using primers listed in Supplemental Table 5. Cells were transfected using Fugene reagent (Promega) with either HA-ZIC2 (Zic2), HA-ZIC3 (Zic3), Y401X ZIC2 (Zic2-iso) or an empty vector (pcDNA) along with a 5× excess of reporter plasmid. A Renilla luciferase reporter was co-transfected to control for transfection efficiency. Cells were harvested after 48–72 hrs and assayed using the Dual Luciferase Reporter assay system (Promega). Each data point represents the mean of three technical replicates performed during the same experiment, and each experiment was repeated three times, as shown on the graphs. Asterisks indicate results of a one tailed t-test of samples with unequal variance testing the null hypothesis either that transfection with ZIC2 does not result in a change in activation of a given reporter over transfection with pcDNA (Fig. 5b,d) or that there is no difference between the two indicated conditions (Fig. 5c). *p < 0.05, NS = not significant.

Cells were transfected with 0.5 μg/coverslip of LC3-GFP (microtubule associated protein 1 light chain 3) construct using Fugene reagent (Promega) as per manufacturer’s instructions. After 24 hours transfection, cells were incubated with LAAO (2 EC₅₀) for 1.5, 3 and 6 hours. Keratinocytes incubated with vehicle were used as negative control, while cells starved (incubated with Earle’s Balanced Salt Solution – EBSS- Sigma) for 30 min were used as positive control. Cells were fixed and stained with DAPI as described above.

The H9c2 WT cells were seeded on a 4-chamber glass bottom dish (Cellvis). After overnight incubation, the cells were transiently transfected with plasmid encoding ROMK2 fused with GFP. Transfection was performed using a Fugene reagent (Promega, Madison, WI, USA). Forty-eight hours after transfection, the mitochondria were additionally stained using 100 nM Mitored (Sigma, St. Louis, MO, USA). Confocal images of living cells were acquired using an Olympus FV 1200.

Stable HEK293T-packaged cells expressing Gluc or GlucB with sshBirA were generated by transduction with lentivirus expression plasmids (kindly provided by Dr Xandra O. Breakefield)16 (link). All plasmids were transfected with lentivirus packing vectors pCMVdelta8.2 and VSV-G using the FuGENE reagent (Promega, WI, USA). Pseudovirus-containing culture medium was collected after 72 h of transfection, and the viral titre was estimated. CT26 cells (2 × 10⁵) in a six-well plate received 10 μg ml⁻¹ of polybrene as well as an appropriate amount of viral stock in the medium. After selection by puromycin, the cells with the highest expression of EV-GlucB and sshBirA were sorted using a BD FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA). Details of other methods used in this study are described in the Supplementary Experimental Procedures, and the results were confirmed by confocal fluorescence microscopy (Nikon, Melville, NY, USA).

Validated pools of FBXW7α-, MCL1-, PLK1-siRNA and non-targeting siRNA as negative control were obtained from GE Dharmacon (ON-TARGETplus SMART pools L-004264, L-004501, L-003290, and D-001810). Transfections were carried out using DharmaFECT 2 reagent (GE Dharmacon) according to manufacturer's instructions. All siRNA pools were used at 50 nM. Cells were subjected to different treatments 24 h after silencing. Transient transfections of pCMVHA and pCMVHA-FBXW7 plasmids [28 (link)] were carried out using FuGENE reagent (Promega) according to manufacturer's instructions. Cells were subjected to different treatments 24 h after transfection.