Cyclers

RT-qPCR was performed following the method of Cha et al. [61 (link)]. Total RNA was extracted from cells using RNAiso Plus (Takara Bio Inc., Kusatsusi, Japan), and cDNA was prepared using the PrimeScript™ cDNA synthesis kit (Takara Bio Inc.) according to the manufacturer’s instructions. cDNA samples were analyzed using the SYBR^® Premix Ex Taq™, ROX plus (Takara Bio Inc.) on Bio-Rad cyclers (Bio-Rad, Hercules, CA, USA). Gene expression was normalized to that of the endogenous housekeeping control gene cyclophilin, which was not influenced by ST-I4C or MGO. Relative expression was calculated for each gene using the ΔΔCT (where CT is the threshold cycle) method. Statistical analysis of the PCR data was based on triplicate samples. The primer sequences are presented in Table 1.

Cells were harvested on the 4, 9, and 14 days of differentiation. Total ribonucleic acid (RNA) was extracted from the cells using RNAiso Plus (Takara Bio, Kusatsu, Japan), according to the manufacturer’s protocol. Complementary deoxyribonucleic acid (cDNA) was synthesized from 2 μg of total RNA using the PrimeScript™ cDNA synthesis kit (Takara Bio), according to the manufacturer’s protocol. The samples of cDNA were then analyzed using the SYBR^® Premix Ex Taq™ and ROX plus (Takara Bio) on Bio-Rad cyclers (Bio-Rad, Hercules, CA, USA). Gene expression was normalized to the endogenous housekeeping control gene, cyclophilin. Relative expression was calculated for each gene using the ΔΔCT (where CT is the threshold cycle) method. The primer sequences used are listed in Table 1 [43 (link)].

Total RNA was prepared by using TRIzol reagent (Sigma), and cDNA was synthesized using the PrimeScript™ cDNA synthesis kit (Takara Bio). The cDNA samples were analyzed using the SYBR^® Premix Ex Taq™ (Takara Bio) on Bio-Rad cyclers (Bio-Rad, Hercules, CA, USA). Relative gene expression was normalized to the GAPDH, and calculated using the 2^−ΔΔCT method. The KIF2A primer sequences were forward, 5′-GCCGAATACATCAAGCAAT-3′ and reverse, 5′-CTCTCCAGGTCAATCTCTT-3′. GAPDH primer sequences were 5′-AATCCCATCACCATCTTCCAG-3′ and 5′-TCATGAGTCCTTCCACGATACC-3′.