Control vector

Mice at 4–6 weeks of age were killed with CO₂ and DRGs were quickly collected in cold PBS after removing axons and meningeal tissue and digested for 30 min at 37 °C in trypsin, collagenase and DNAse. Neurons were plated on poly-L-lysine coated coverslip and cultured in Neurobasal medium plus B27 and N2 supplement (Gibco) without adding growth factors or mitotic inhibitor. At 18–20 h after plating, cultured DRG neurons were transfected using the calcium phosphate method with the following plasmids: ORF of mouse TRPA1 C- terminally myc-tagged (a kind gift from Manuela Schmidt, Max Planck Institute für Experimentelle Medizin, Göttingen), or a control vector (Origene Inc.).
At 24 h after transfection, the cells were treated with Sema4C (see above) for 15 min, washed and fixed in 4% PFA for 20 min at room temperature. Immunofluorescence analysis was performed using an anti-myc antibody (mouse (9E10) 1:1000, sc-40 Santa Cruz) anti-β-tubulin III (rabbit 1: 1000; T2200, Sigma) and secondary antibodies conjugated with Alexa488 or Alexa 594 (Molecular Probes, Invitrogen) using standard protocols. The relative levels of TRPA1 expression at the cell membrane vs. cytoplasm were analyzed using the line profile tool in confocal microscopy (Leica TCS AOBS), or Fiji software.

HEK293T, H1299, HCT116, HeLa, and PC9 were purchased from ATCC (USA). All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI-1640 medium with 10% fetal bovine serum (FBS) and 100 μg/ml penicillin/streptomycin. HCT116 shN and HCT116 shR cells were constructed previously. H1299 shN, H1299 shR, PC9 shN, and PC9 shR cells were generated by stable integration of a retroviral shRNA vector specific for REGγ or a control vector from OriGene (Rockville, MD). H1299 shN & H1299 shR cells stably expressing NIP30 WT, NIP30 4A, or NIP30 4D were generated in our laboratories. 293T-REGγ knockout cells were generated using TALENs. 293T & 293T KO cells with stable expression of NIP30 (221–235) WT or NIP30 (221–235) 4A or NIP30 (221–235) 4D were also generated for this study. The 293-REGγ inducible cell lines were previously described^{1 (link)}.

Transfection was conducted using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions. The control vector, HK2 overexpression vector, control shRNA, or HK2 shRNA was obtained from OriGene and Santa Cruz Biotechnology, respectively. Control siRNA, HK2 siRNA, control mimic, miR-145 mimic, miR-148a mimic, miR-497 mimic, control inhibitor, miR-145 inhibitor, miR-148a inhibitor, or miR-497 inhibitor, was purchased from Invitrogen. When cells achieved 70% confluence, the above plasmid, shRNA, siRNA, or miRNA mimic (or inhibitor) was transfected into CC cells for 48 h. Finally, stable HK2-overexpressing or HK2 knockdown CC cell lines were established by selecting cells using G418 (Sigma-Aldrich) or Puromycin (Sigma-Aldrich) for 4 weeks.