Total RNA Extraction and qPCR Analysis

Total RNA in MDA-MB-231 cells stably expressing FOXM1 shRNA and NC shRNA, MDA-MB-231 cells, MDA-MB-468, cells and MDA-MB-231 xenografts was extracted with TRIzol reagent (Vazyme, Nanjing, Jiangsu, China) according to the manufacturer’s instructions [31 (link)]. cDNA was synthesized using a HiScript II one-step RT-PCR kit (P612-01, Vazyme, Nanjing, China) with 1.0 μg of total RNA in a 20 μl reaction system. The resulting cDNA was diluted 1:2 in nuclease-free water, and 1.0 μl was used per Q-PCR in triplicate. Q-PCR was carried out using ChamQ SYBR Q-PCR master mix (Q311-02, Vazyme, Nanjing, China) on a QuantStudio 3 real-time PCR-detection system (Life Tech, New York, USA) including a nontemplate negative control. GAPDH was used to normalize the level of mRNA expression. The sequences of the primers are listed in Supplementary Table 2.