siRNA-mediated knock down of NHLH1

HEK293 cells were seeded at 100,000 cells/well in a 6-well plate the day before transfection. Cells were transfected using Lipofectamine and 50 nM of siRNA designed to target human NHLH1 or negative control siRNA (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 hours, cells were harvested and total RNA extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesized using the RevertAid H- First Strand cDNA Synthesis Kit (Thermo Fischer Scientific, Waltham, MA, USA) using random hexamer primers and qPCR was performed with TaqMan assays for NHLH1, TMEM232, SLC25A4, WDR36, TSLP, CAMK4 and GAPDH on a StepOne Plus Real-Time PCR System (Life Technologies). Gene expression was normalized to GAPDH and expressed relative to cells transfected with negative control siRNA according to the ΔΔCt-method [61].