Effect of siRNA knockdown of TPM1 or TPM4 in ASM on proliferation and cell size

Mouse ASM cultures were grown in 6-well plates using SMC media with smooth muscle growth supplement (Cell Biologics). ASM cultures were transfected with 20 nM TPM1 siRNA, TPM4 siRNA, or control siRNA by using transfection reagents siTran1.0 (Origene) and Opti-MEM (Thermo Fisher Scientific) according to the manufacturers’ instructions. The transfected ASM cells were used 24 hours after the transfection in all experiments including quantitation of ASM proliferation, ASM cell size, and expression of TPM1 and TPM4 mRNA. Detection of expression of TPM1 and TPM4 mRNA by RT-qPCR was performed with TaqMan PCR Master Mix and mouse TPM1 or TPM4 primers (Life Technologies, Thermo Fisher Scientific). The relative amounts of transcripts were normalized to those of housekeeping gene (GAPDH) mRNA and compared between control siRNA–transfected samples and TPM1 siRNA– or TPM4 siRNA–transfected samples by the ΔΔ cycle threshold method as previously described in this laboratory (2, 8).