Metafectene

pCDH-CFP, pCDH-CFP-NRAS Q61R and pCDH-CFP-BRAF V600E lentiviral expression vectors were packaged into lentiviral particles using the psPAX2 packaging plasmid DNA (Addgene #12260) and the pMD2.G envelope plasmid DNA (Addgene #12259). Briefly, 2.5 × 10⁶ HEK293T (human embryonic kidney; CVCL_0063) cells were plated onto poly-L-lysine- (Sigma-Aldrich) coated 25 cm² flasks. Twenty-four hours later, cells at 70% confluence were co-transfected with 2.5 µg of each pCDH-vector, 1.5 µg psPAX2 and 1 µg pMD2.G, using METAFECTENE (Biontex, München, Germany) at a final ratio of 1 µg DNA: 4.5 µL METAFECTENE. The medium was removed 6 h later and replaced with 5 mL of fresh DMEM supplemented with 10% FBS. CFP fluorescence was monitored to confirm transfection efficiency. Culture supernatants were collected after 72 h and cell debris were pelleted by centrifugation at 300× g in an Eppendorf 5810 centrifuge. Lentiviral supernatants were then filtered through a 0.45 μM filter and stored for further usage in cell transduction. Briefly, PCCL3 or FRTL5 cells were grown in 6-well plates to 60% confluence and incubated with 1 mL lentiviral supernatants and 8 µg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). 6-well plates were spun for 30 min at 37 °C at 1200× g. 48 h after transduction, cells were treated accordingly to each experimental assay.

To suppress endogenous Inpp5e, mouse Inpp5e siRNA (Dharmacon, Cat# M-041108-00-0005) was transfected as follows. Inpp5e siRNA was diluted in 1x siRNA Buffer to attain a stock concentration of 20 µM. Transfections were performed with a final siRNA concentration of 50 nM. Cells for siRNA transfection were plated in plastic 6-well plates. The first transfection of siRNA was performed 3–6 h after cell plating using Metafectene (Biontex Laboratories, Munich, Germany) following the manufacturer´s instructions (1 µg siRNA per 2 µl Metafectene). Twenty to twenty-four hours after the first transfection, cells were re-plated on poly-L-lysine coated glass cover slips. Plasmid DNA and a second round of siRNA transfection was performed 24 h after the second plating using Metafectene (1 µg siRNA or DNA per 3 µl Metafectene). Twenty-four hours after transfection, formation of primary cilia was induced by starvation with Opti-MEM for 24 h. Cells were fixed on day 5. Non-targeting siRNA (Dharmacon, Cat# D-001206-13-20) was used as negative control.

SW620 cells were seeded at 0.5 × 10⁵ cells/ml in 96-well plates and incubated at 37°C until 50–60% confluent. Cells were then treated with 100 ng MISSION shRNA plasmid DNA (TRCN0000029305, Sigma) or empty vector control (SHC002, Sigma) with the transfection reagent metafectene (Biontex) in a 1:4 plasmid:metafectene (w/v) ratio for 6 hr at 37°C. The transfected cells were selected by treatment with 8 μg/ml puromycin over 72 hr, followed by 48 hr with 4 μg/ml puromycin. Cells were then released by trypsin, suspended in complete culture medium to highly-diluted cell suspension and plated in a 96-well plate. Wells containing a single cell were identified by microscope. Following further culture to allow cell proliferation and colony formation, galectin-3 expression was determined by galectin-3 immunoblotting and immunohistochemistry to allow separate selection of galectin-3 expressing (SW620^Gal3+) and knockdown (SW620^Gal3−) cells/colonies.

For BRET assays, HEK293 cells were transfected 4–6 h after seeding. Transfection was performed using 8 µL FuGene HD (Promega, Mannheim, Germany) and 1.4 µg DNA in Opti-MEM (Gibco, Waltham, MA, USA). The BRET partners were co-transfected using a NL/HT ratio of 1:5 (200 ng: 1 µg). In addition, 200 ng Carrier DNA (pGEM3Z) was added. For GloSensor™, HEK293 cells were transfected 24 h after seeding with 60 ng plasmid DNA and 0.6 µL Metafectene (Biontex, Munich, Germany), according to the manufacturer’s protocol. An additional 60 ng of pGloSensor™-22F cAMP Plasmid was added. For reporter gene assays, HEK293 cells were transfected 24 h after seeding with 45 ng plasmid DNA and 0.45 µL Metafectene (Biontex, Munich, Germany), according to the manufacturer’s protocol. A total of 45 ng of reporter DNA (pGL4.3(luc2P/NFAT/Hygro)) was additionally transfected per well in MEM without supplements.

Simian SV-40 transformed monkey kidney cells (COS-7 ATCC CRL-1651) were cultured in DMEM (Invitrogen) containing 10% FCS (Sigma) under standard conditions (95% humidified atmosphere, 37 °C, 5% CO₂). Cells were transiently transfected with pcDNA4/HisMax plasmid coding for mAtgl, mPnpla6, mPnpla7, mHsl, mMgl, or β-galactosidase (lacZ) using Metafectene^TM (Biontex GmbH, Munich, Germany) as described (7 (link), 43 (link), 44 (link)). Cells were harvested 48 h after transfection. For the preparation of cell lysates, cells were resuspended in sucrose solution (see above) and disrupted by sonication (SONOPLUS ultrasonic homogenizer HD 2070) on ice. Nuclei and unbroken cells were removed by centrifugation at 1,000 × g for 10 min at 4 °C. Protein concentrations were determined as described below.

rSlfn1 (NCBI Reference Sequence: NC_005109.3) cDNA expression constructs were chemically synthesized (Neuron Biotech, China). The rSlfn1 cDNA was first subcloned into a pGS-1 vector, and then 293 cells were infected with the Ad-Slfn1 recombinant adenovirus in a 12-well plate with MetafecteneTM (Biontex) according to the manufacturer's protocol. Finally, the recombinant Ad-Slfn1 was collected 8 days after transfection and amplified in the 293 cells, producing 2 mL of viral stock. The adenoviruses expressed GFP under a separate promoter, which allowed the infection to be verified. All PCR-amplified cloning and fragment junctions were confirmed by DNA sequencing (Sangon, Shanghai, China). An adenovirus encoding a green fluorescent protein (Ad-control) was used as a control. The Ad-Slfn1 and Ad-control viruses were used for functional assays (see below).

COS-7 cells were transiently transfected with the different CGI-58 clones and pcDNA4/HisMax coding for His-tagged ATGL (28 (link)) with Metafectene^TM (Biontex GmbH) as described earlier (29 (link)). The cells were disrupted by sonication and resuspended in lysis buffer (0.25 m sucrose, 1 mm dithiothreitol, 1 mm EDTA, 20 μg/ml of leupeptine, 2 μg/ml of antipain, 1 μg/ml of pepstatin, pH 7.0). Then, nuclei and unbroken cells were removed by centrifugation at 1000 × g at 4 °C for 5 min, and the supernatants were used for triglyceride hydrolase activity assays.