RNA extraction and RT-PCR

Following the manufacturer's instructions, total RNA was extracted from cells using the TRizol reagent (Invitrogen, USA). The Prime Script RT Master Kit (Vazyme, China) was then used to generate cDNA from RNA. The Vazyme HI Script II One Step qRT-PCR SYBR Green Master Mix (Vazyme, #Q711, China) was used to amplify the reverse transcribed cDNA and assess the gene expression using the CFX96 real-time fluorescence quantitative PCR equipment system (Bio-rad, USA). Briefly, 1000 ng of RNA extracted were added to 4 μl of 4× gDNA into 14 μl of DEPC water, incubated for 2 minutes at 42°C then added 4 μl of 5× RT Super Mix and incubated at 50°C for 15 minutes and 85°C for 2 minutes. The qPCR reaction conditions was as follows: initial denaturation at 95°C for 2 minutes, followed by 40 cycles with 3 step PCR of 95°C for 10 seconds; 60°C for 30 seconds and 72°C for 30 seconds. Then the Melt cuve step (95°C for 15 seconds; 60°C for 1 minute and 95°C for 15 seconds). The relative expression of genes in each group of cells was calculated by the 2^-ΔΔCt method. The relative RNA expression level was normalized to GAPDH. The Table S2 displays the primer sequences that were used in this study.