Quantstudio version 1

Harvested cells were washed with Dulbecco’s phosphate-buffered saline (dPBS), centrifuged at 125 × g for 5 min and then the flash-frozen pellets were stored at −80 °C. Extracted RNA (500 ng–2 µg) were used as templates to make cDNA libraries using a High Capacity RNA-to-cDNA kit (Applied Biosystems). TaqMan gene expression assays were designed using GAPDH (Hs03929097, VIC) as an endogenous control and CDH1 (Hs01023895_m1, FAM), GSTP1 (Hs00943350_g1, FAM), HSPA1A (Hs00359163_s1, FAM), MGMT (Hs01037698_m1, FAM), MLH1 (Hs00179866_m1, FAM), PKP3 (Hs00170887_m1, FAM), RHOXF2 (Hs00261259_m1, FAM), SOX17 (Hs00751752_s1, FAM), SRPX2 (Hs00997580_m1, FAM), or THBS2 (Hs01568063_m1, FAM) as targets (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed in 10 µL reactions by using TaqMan Universal Master Mix II with UNG and 2 μL of diluted cDNA from each sample (Applied Biosystems). Gene expression levels were calculated by “delta delta Ct” and normalized to control samples using ViiA7 version 1.2.2 or QuantStudio version 1.3 software (Applied Biosystems by Life technologies).

Total RNA was extracted using the NucleoSpin RNA kit (Macherey-Nagel, catalog 740955.250). The cDNA was synthesized by reverse transcription (RT)-PCR from 1 μg of purified RNA with M-MLV reverse transcriptase (Thermo Fisher Scientific, catalog 28025-013) and random primers (Thermo Fisher Scientific, catalog 58875). qPCR reactions were performed in a Viia 7 Real-Time PCR System (Thermo Fisher Scientific) using PerfeCTa SYBR Green SuperMix reagent (Quantabio, catalog 95056-500) and gene-specific primers (Supplemental Table 4). Data were analyzed using QuantStudio version 1.3 (Thermo Fisher Scientific). Relative expression was calculated using the 2^–ΔΔCt method. The RPLP0 gene was used as housekeeping gene.