Ultraviolet gel imaging system

Total RNA was extracted from cultured VSMCs and BMMs using Trizol reagent (Invitrogen Life Technologies) following the manufacturer's instructions. RevertAid First strand cDNA Synthesis kit (Thermo Fisher Scientific) was used to synthesize cDNA from RNA and PCR was performed in a T100^TM-Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR products were analyzed by electrophoresis in a 2% agarose gel and imaged using an ultraviolet gel imaging system (Bio-Rad). The qPCR analysis was performed in optical 96-well plates using SYBR Green PCR master mix (Roche, Penzberg, Germany) and the Light Cycler 480 Real-time PCR Detection System (Roche) according to the manufacturer's instructions. Gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal control. The relative expression of the target genes was calculated by the standard curve method using the target Ct values and the Ct value for GAPDH.

Total RNA was extracted from cultured hMSCs and various breast cancer cells using Trizol reagent (Invitrogen Life Technologies) following the manufacturer’s instructions. RevertAid First strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to synthesize cDNA from RNA and subsequent PCR was performed in a T100^TM-Thermal Cycler (Bio-Rad, Hercules, CA, USA). The resulting PCR products were analyzed by electrophoresis in a 2% agarose gel and imaged using an ultraviolet gel imaging system (Bio-Rad). qPCR analysis was performed in optical 96-well plates using SYBR Green PCR master mix (Roche, Penzberg, Germany) and the Light Cycler 480 Real-time PCR Detection System (Roche), in accordance with the manufacturer’s instructions. Gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal control. The relative expression of the target genes was calculated by the standard curve method using the target Ct values and the Ct value for GAPDH.

Total RNA was extracted from cultured breast cancer cells using Trizol reagent (Invitrogen Life Technologies) following the manufacturer’s instructions. A RevertAid First strand cDNA Synthesis kit (Thermo Fisher Scientific) was used to synthesize cDNA from RNA and subsequent PCR was performed in a T100™-Thermal Cycler (Bio-Rad, Hercules, CA). The resulting PCR products were analyzed by electrophoresis in a 2% agarose gel and imaged using an ultraviolet gel imaging system (Bio-Rad). qPCR analysis was performed in optical 96-well plates using SYBR Green PCR master mix (Roche, Penzberg, Germany) and the Light Cycler 480 Real-time PCR Detection System (Roche), in accordance with the manufacturer’s instructions. The primers used are described in Supporting Information Table S1. Gene expression was normalized to that of a GAPDH internal control. The relative expression of the target genes was calculated by the standard curve method using the target Ct values and the Ct value for GAPDH.