Viafect transfection reagent

C2C12 cells were seeded in GM at 13,000 cells/well in 24-well plates. Twenty-four hours after seeding, cells were transfected using ViaFect Transfection Reagent (Promega) according to the manufacturer’s instructions using 1000 ng DNA/well and 8 μL of ViaFect per well. Four hours after transfection, media was replaced with fresh GM containing 0 mM or 1 mM theophylline. Forty-eight hours after transfection, when cells had reached 90–100% confluency, GM was replaced with fresh DM containing 0 mM or 1 mM theophylline. DM was replaced every 24 h until the indicated timepoint.
At the indicated timepoint, total RNA was extracted using the RNeasy Plus kit with QIAshredder (Qiagen) for homogenization following manufacturer’s instructions. RT-qPCR analysis was performed in triplicate using Luna Universal One-Step RT-qPCR Kit (New England BioLabs) and a QuantStudio 3 (Applied Biosystems). Thirty nanograms of total RNA was added to each 20 μL reaction in a 96-well plate. mRNA levels were normalized to beta-actin and plotted as the fold change from the expression level of the indicated switch control (generally sTRSVctrl or sTRSV).
The following primer sequences used for RT-qPCR were obtained from the Protein and Nucleic Acid Facility at Stanford University.
ACTB1:
HRAS:
JAK1:
MYOG:
MyHC (Myh 1):

Glioma cell lines H4, U-118 and U-87 were obtained from the American Type Culture Collection (ATCC). The purchased cell lines were immediately frozen after recultivation of the original aliquot and all the experiments were performed on cultures sourced from these secondary aliquots within a maximum of 12 passages. Cells were cultured in Dulbecco's modified eagle medium (DMEM) high glucose (HyClone) supplemented with 10% FBS (ATCC) and 1% penicillin–streptomycin (HyClone). Cells were transfected with a pJ509 plasmid containing either CSMD1 cDNA optimized for expression in human cells or mock pJ509-GFP plasmid using ViaFect transfection reagent (Promega) followed by antibiotic selection (0.5 µg/ml puromycin) as described [10 (link)]. Mycoplasma contamination was routinely checked in cells by Eurofins Genomics. CSMD1-overexpressing cells were coded as CSMD1, mock pJ509-GFP transfected cells were coded as Ctrl and untransfected cells were coded as (Wild type) WT throughout the manuscript.
TNF was purchased from Immunotools (Cat no: 11343015), resuspended in sterile 0.1% BSA in dH₂O and used for subsequent experiments.

661W, COS-7, and HEK-293 cells were grown in DMEM supplemented with 10% fetal calf serum (FCS). hiPSCs were obtained and cultured as previously described (15 (link)). Briefly, 4.5 × 10⁵ human iPS cells were expanded in COAT-1 (Cellartis def-cs 500, TaKaRa) coated six-well plates in Basal medium (Cellartis def-cs 500, TaKaRa). The culture supernatant was collected for ELISA after 48 h. ViaFect transfection reagent (Promega) and opti-MEM were used to transfect the plasmids in accordance with the supplier's protocol. The amounts of plasmids and oligonucleotides, cell numbers, and plates used were as follows: 2 ng of plasmids and 12 ul transfection reagent into 4 × 10⁵ cells using 6-well plate. Nuclear protein was extracted using the CelLytic NuCLEAR Extraction Kit (Sigma) following the manufacture's protocol. The protein concentration was determined with the BCA protein assay kit (Thermo Scientific).

Isomeric purity of (Z)-4-undecenal (Z4-11Al) was 98.6%, according to gas chromatography coupled to mass spectrometry (6890 GC and 5975 MS, Agilent Technologies, Santa Clara, CA, USA). Isomeric purity of (Z)-4-nonenal (Z4-9Al) and (Z)-6-undecenal (Z6-11Al) were 97.4 and 96%, repectively. Chemical purity of these synthetic aldehydes was >99.9%. Ethanol (redistilled; Merck, Darmstadt, Germany) was used as solvent.
For the OR screening assays, the following chemicals were used: Dulbecco’s MEM medium (#F0435), FBS superior (#S0615), L-glutamine (#K0282), penicillin (10000 U/ml)/streptomycin (10000 μg/mL) (#A2212), trypsin/EDTA solution (#L2143) (Biochrom, Berlin, Germany), CaCl2∗2H2O (#22322.295), D-glucose (#101174Y), dimethyl sulfoxide (DMSO) (#83673.230), HEPES (#441476L), potassium chloride (#26764.230), and sodium hydroxide (#28244.295) (VWR Chemicals BDH Prolabo, Leuven, Belgium), sodium chloride (#1064041000, Merck, Darmstadt, Germany), ViaFect™ Transfection Reagent (#E4981, Promega, Walldorf, Germany), D-luciferin (beetle) monosodium salt (#E464X, Promega, Walldorf, Germany), Pluronic® PE 10500 (#500053867, BASF, Ludwigshafen, Germany), (R)-(−)-carvone (#W224908, Sigma-Aldrich, Steinheim, Germany).

Plasmids (2 μg) containing WT METTL23-FLAG, splicing 1–FLAG (skip exon 2), and splicing 2–FLAG (skip exons 2 and 3) were transfected into subconfluent HEK293T, COS-7 (ATCC), and 661W cells (62 (link)) in a 6-well plate using ViaFect transfection reagent (Promega), respectively. Forty-eight hours later, the cells were harvested by RIPA buffer with protease and phosphatase inhibitors (Roche), PMSF, and aprotinin. Equal amounts of samples were subjected to an Any kD Mini-PROTEAN TGX Gel and transferred onto a PVDF membrane using the Transblot Turbo system (Bio-Rad). Blots were probed with a Can Get Signal and PVDF Blocking Reagent Set (TOYOBO). Every blot was also probed with an anti-actin antibody to ensure equal protein loading. Protein detection was achieved with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) using the Bio-Rad system (ChemiDoc XRS+).