Fig. 3.

Regulatory relationship of Reg IV and SOX9 in MKN-45 cells. a Reg IV overexpression vectors (PEGFP/Reg IV) and control empty vectors (PEGFP) were added to the cells. Cells were harvested after 48 h, total RNA were extracted and converted to cDNA. Real-time PCR was performed to examine the mRNA level of Reg IV and SOX9 in PEGFP/Reg IV and PEGFP treated cells. Relative expression values of mRNA levels were normalized by GAPDH mRNA expression; (b) after transfection with PEGFP/Reg IV and PEGFP, the cells and medium were collected. Western blot analysis was done using anti-Reg IV antibody and SOX9 antibody, and bands were visualized (left) and the gray intensity was analyzed (right). β-actin expression level was used as an internal control; (c) compared with control scramble siRNA (siR-NC), the mRNA levels of three Reg IV siRNAs (siR-R1, siR-R2, siR-R3) were examined by real-time PCR and siR-R3 showed higher silencing efficiency. d the mRNA level of SOX9 was examined in siR-R3-treated cells and the negative control (siR-NC) by real-time PCR; (e) after transfection with siR-NC and siR-R3, western blot analysis was done using anti-Reg IV antibody and SOX9 antibody, and bands were visualized (left) and the gray intensity was analyzed (right); (f) compared with control scramble siRNA (siR-NC), the mRNA levels of threeSOX9 siRNAs (siR-S1, siR-S2, siR-S3) were examined by real-time PCR and siR-S1 showed higher silencing efficiency; (g) the mRNA level of Reg IV was examined in siR-S1-treated cells and the negative control (siR-NC) by real-time PCR; (h) After transfection with siR-NC and siR-S1, western blot analysis was done, and bands were visualized (left) and the gray intensity was analyzed (right). The results are shown as Mean ± SEM, n = 3. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, N.S. = not significant