Fig 4. CHO-K1, COS7, and HEK293 cells express PAR1 and PAR2 receptors.

A. CHO-K1, COS7, and HEK293 cells naturally express high levels of PAR1 and PAR2 mRNA but express little or no PAR3 and PAR4 mRNA. qPCR analysis was used to quantify the mRNA expression. Specific primers for each of PAR1, PAR2, PAR3, and PAR4, were used to quantify the respective mRNA expression using cDNA made from each cell line as the templates. β-actin primers were used to quantify β-actin mRNA expression as the internal controls. The relative mRNA expression of PAR1, PAR2, PAR3, and PAR4 were first normalized using β-actin expression, and then normalized using the PAR1 expression level in CHO-K1 cells, which is arbitrarily set as 100%. The relative expressions of other genes are represented as percentage of PAR1 mRNA level in CHO-K1 cells. The results shown are mean ± sd (n = 3). Statistical analysis (One-Way ANOVA) shows that compared with the mRNA expression of PAR4, which is undetectable in these cells, CHO cells express high levels of mRNAs for PAR1 (** p = 0.0037), PAR2 (* p = 0.023), and PAR3 (* p = 0.035); COS7 and HEK293 cells express high level of mRNAs for PAR1 (** p = 0.0029, * p = 0.032, respectively) and PAR2 (** p = 0.0013, ** p = 0.0027, respectively) without expressing detectable PAR3 and PAR4 mRNAs. B, C, D. CHO-K1, COS7, and HEK293 cells naturally express PAR1 and PAR2 receptors and respond to thrombin (PAR1 ligand) and trypsin (PAR2 ligand) stimulations. FLIPR assays were used measure receptor activation as indicated by intracellular Ca²⁺ mobilization. Relative fluorescent units (RFU) are the readout for fluorescent intensities for Ca²⁺ mobilization signals. Various concentration of thrombin or trypsin were used as the ligands to activation the receptors. The assays were performed in triplicates at each data point and mean ± sd are shown. E. Sequencing analysis of the genomic DNA from par1 & par2 knock out HEK293 cells. The results show that a 270 bp deletion in par1 gene and a 347 bp deletion in par2 gene have been achieved. The deletions removed the coding regions from TM2 to TM3 for both PAR1 and PAR2 proteins. The vertical lines indicate the deletion sites. F. Characterization of par1 & par2 knock-out HEK293 cells. FLIPR assays were used to characterize receptor activation as indicated. Wild type HEK293 cells were used as the positive control. The assays were performed in triplicates at each data point and mean ± sd are shown.