RNA Isolation and Gene Expression Analysis

Cultured cells were washed once with ice-cold PBS and collected with TRIzol reagent (Thermo Fisher Scientific, USA). Total RNA of cells was isolated with chloroform. The quantity and concentration of RNA were evaluated with a NanoDrop system (Thermo Fisher Scientific, USA). A total of 1 μg RNA was reverse transcribed into cDNA with PrimeScript™ RT Master Mix (TaKaRa, Japan) [16 (link)]. The mRNA levels of the genes were quantified with a real-time PCR kit (Tiangen, China) on a Bio-Rad CFX96 real-time PCR system according to the manufacturer’s instructions. GAPDH was used as the internal control. The sequences of specific primers used in this study are listed below: LIMD2-F: 5′-CAGGAAGACCCTACCAAATATC-3ʹ LIMD2-R: 5ʹ- CCCAACAGGGCTGATTTAC-3ʹ GAPDH-F: 5ʹ-GTATGACAACAGCCTCAAGAT-3ʹ GAPDH-R: 5ʹ-GTCCTTCCACGATACCAAAG-3ʹ. Three biological replicates were performed.