Figure 3
Gene and protein expression patterns of HDMSCs and ASMSCs after LPS stimulation. (a) High-throughput qRT-PCR was performed to determine the gene expression of important molecules related to the LPS-induced autophagy process in ASMSCs (n=30) and HDMSCs (n=30). H1-H3 and A1-A3 represent three samples randomly selected from the 30 healthy donors and the 30 AS patients, respectively. The heat maps show the ΔΔCT values. Each column represents one sample, and each row represents one gene. The results indicated that ASMSCs (A) had lower expression of ATG5, ATG7, ATG12 and ATG16L1 than HDMSCs (H) after LPS stimulation but higher expression of TRAF4 both before and after LPS stimulation. (b, c) Standard qRT-PCR was performed to confirm the results of high-throughput qRT-PCR. The standard qRT-PCR results were consistent with those of high-throughput qRT-PCR (n=30). (d) The protein level of TRAF4 was evaluated in ASMSCs (n=30) and HDMSCs (n=30) before and after LPS stimulation. The TRAF4 protein expression levels were consistent with the TRAF4 mRNA expression levels: TRAF4 protein expression was much higher in ASMSCs than in HDMSCs both before and after the LPS stimulation. The figures in d are typical examples of MSCs from one healthy donor and one AS subject. The values in b–d are presented as the means±s.d.; *P<0.05. The high-throughput and standard qRT-PCR data were first normalized to GAPDH expression and then normalized to the values of H1 before LPS stimulation, and the relative expression levels of each gene were analyzed using the 2−ΔΔCt method. AS, ankylosing spondylitis; ASMSCs, ankylosing spondylitis mesenchymal stem cells; HDMSCs, healthy donors mesenchymal stem cells; LPS, lipopolysaccharide; MSCs, mesenchymal stem cells; qRT-PCR, quantitative real-time PCR.
