Nano instrument

Total RNA was obtained from the TM cells cultured in 10 ng/ml CRYAB with a Total RNA kit (Omega, USA) and was reverse transcribed into cDNA (1 μg of isolated total RNA) using a qPCR RT Kit (TOBOYO, Japan) according to the manufacturer's instructions. The RNA concentrations were quantified with a Nano instrument (NanoDrop Technologies, Wilmington, DE, USA). Rt-qPCR was carried out using a SYBR Green PCR Master Mix (TOYOBO, Japan). The system of each PCR reaction contained 4 μl of cDNA, 0.8 μl of forward and reverse primers (PCR primers were listed), and 10 μl of SYBR Green PCR Master Mix, with a final volume of 20 μl. All the PCR amplification reactions were performed on a MiniOpticon qPCR (Bio-Rad, USA). The specific primers were the following: zo-1 (forward primer (F), 5′-ACCAGTAAGTCGTCCTGATCC-3′ and reverse primer (R), 5′-TCGGCCAA ATCTTCTCACTCC-3′); claudin-1 (F, 5′-CGAGAGCTACAC GTTCACGG-3′ and R, 5′-GGGTGTCGAGG GAAAAA TAGG-3′); cadherin-E (F, 5′-AAAGG CCCATTTCCTAAAAACCT-3′ and R, 5′-TGCGTTCTCTATCCAGAGGCT-3′); cadherin-N (F, 5′-AGCCAACCTTAAC TGAGGAGT-3′ and R, 5′-GGCAAGTT GATTGGAG GGATG-3′); and vimentin (F, 5′-GACGCCATCAACACCGAGTT-3′ and R, 5′-CTTTGTCGTTGGTTAG CTGGT-3′). The mRNA expression was normalized to the endogenous reference gene GAPDH (F, 5′-TGGAGAAAAT CTGGCACCAC-3′ and R, 5′-ACCACCCTGTTGCTGTAGCCA A-3′) and was analyzed using the 2^−ΔΔCt comparative threshold method.