siRNA-Mediated Knockdown of ESM1 in Human ACC

The siRNA treatment was conducted as previously described (42 (link)). The human SW13 cell, one of the human ACC cell lines, was transfected with 50 nmol of ESM1 siRNA (siESM1) or negative control siRNA (siNC) in a special medium (CM0451, Procell, Wuhan, China) for 48 h. SW13 cells were then lysed by TRIzol reagent (Invitrogen, Waltham, MA, USA) for total RNA isolation. The cDNA was obtained by oligo-dT primers and reverse transcriptase kit (Invitrogen, USA). Quantitative real-time PCR (qRT-PCR) was performed by SYBR Green PCR Master Mix (Qiagen, Hilden, Germany) and specific primers in an ABI Prism 7500 analyzer (Applied Biosystems, Waltham, MA, USA). GAPDH was an endogenous reference gene. Three replicates were set for all reactions. The 2^−ΔΔCt method was applied to calculate the relative expression of ESM1 in ACC samples. The related primers are listed in Supplementary Table S4.