Protein analysis kit

The MDA-MB-231 cells were seeded in 6-well plates at a concentration of 6.0 × 10⁴ cells/well and treated with SchA (0, 40 and 80 µM) for 24 h. RIPA lysis kit (Beyotime) and protein analysis kit (Takara, Kyoto, Japan) were used for cell lysis and total protein quantification analysis, respectively. The total protein was then separated on 10% SDS-PAGE gel electrophoresis and afterward transferred onto a nitrocellulose membrane by electroblotting (Millipore, Billerica, MA, USA). After blocking the membrane using 5% BSA for 2 h at room temperature, it was incubated overnight at 4°C with primary antibodies (p-EGFR, PIK3R1, Cleaved-caspase 3, AKT1, p-AKT1 and MMP9), in a dilution of 1:1000, (Cell Signaling Technology, Danvers, MA, USA). They were then incubated with secondary antibodies at room temperature for 1 h. Later, the membrane was washed in three tris buffered saline + Tween (TBST) cycles for 10 minutes. The proteins were then visualized by adding ECL (Thermo) and subsequently scanned and imaged using a FluorChem FC 3 system (ProteinSimple, USA). Finally, image J software was used to analyze the results.

Total RNA was isolated from cells using TRIzol reagent (Sigma-Aldrich). The preparation of cDNA and qRT-PCR of miR-497 was performed using MicroRNA First-Strand Synthesis and miRNA Quantitation kits (Takara, Dalian, China). Expression of Cdc25A was detected using a CellAmp Direct RNA Prep kit for qPCR and a Protein Analysis kit (Takara, Dalian, China) according to the manufacturer’s instructions. The reaction was as follows: 10 min at 95°C, then 40 cycles of 1 min at 95°C, 2 min at 63°C, and 1 min at 72°C, and then a final annealing step at 72°C for 10 min. All primers were synthesized by Takara as follows: Cdc25A: 5′-GTGAAGGCGCTATTTGGCG-3′ (forward) and 5′-GGTCCATAGTGACGGTCAGGT-3′ (reverse); GAPDH: 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-ACCACAGTCCATGCCATCAC-3′ (reverse). U6 was used as the internal control to normalize the relative expression of miR-497, and Ct values of GAPDH were used to normalize the relative expression of Cdc25A. All PCRs were performed in triplicate. Relative expression levels were presented using the 2^-ΔΔCT method.

TRIzol reagent (Sigma-Aldrich) was used to isolate the total RNA from cultured cells. The cDNA of miR-202 was prepared using MicroRNA First-Strand Synthesis and miRNA Quantitation kits (Takara, Dalian, P.R. China) according to the manufacturer’s instructions. The CellAmp Direct RNA Prep Kit for quantitative polymerase chain reaction (qPCR) and a protein analysis kit (Takara) were used to test the expression of HSF2 and Hsp70. Ct values of U6 and GAPDH were used as the internal control to normalize the relative expression of miR-202 as well as HSF2 and Hsp70. All PCRs were performed in triplicate. Relative expression levels were determined using the 2^−ΔΔCt method.