Fig. 3: SOX9 up-regulates the protein expression of COL10A1 and its transcriptional activity by directly binding to the COL10A1 promoter.

a, b SOX9 and COL10A1 expression were detected using western blot analysis by transfecting GC cell lines with SOX9 siRNA and SOX9-sense plasmids. c, d Visualization of SOX9 (red) and COL10A1 (green) staining in SOX9-silenced and COL10A1-silenced MKN45 cells and in SOX9-overexpressing and COL10A1-overexpressing BGC823 cells by immunofluorescence microscopy. e The transcription factor-binding site (TFBS) of SOX9 in the COL10A1 gene promoter was used to design biotin-labeled, unlabeled, and mutant probes for EMSA. f EMSA using nuclear extracts from SGC7901 and AGS cells is shown. Shifted bands representing that endogenous SOX9 bound to the biotin-labeled probe were detected (lane 2). When a 100-fold excess of unlabeled probe was added to bind to endogenous SOX9 and compete with the biotin-labeled probe, the bands displayed a lower intensity (lane 3). However, when a 100-fold excess of mutant probe that could not bind to endogenous SOX9 was added, the intensity of the bands recovered (lane 4). With the addition of SOX9 antibody, a significantly lower intensity of the band was evident, which further indicated that the SOX9 protein could bind to the TFBS in the biotin-labeled probe (lane 5). g Schematic diagram depicting the positions of the primers used for the ChIP assay. h, i ChIP analysis was performed using a negative control immunoglobulin G (IgG) or anti-SOX9 antibody in SGC7901 and AGS cells. PC positive control (anti-RNA polymerase II antibodies). **P < 0.01. j Schematic diagram depicting the sequences in the wild type and mutant COL10A1 promoter. WT wild type, MT mutant type. MKN28 cells were harvested for analysis of luciferase activity and SOX9 protein stimulated COL10A1 activity. Scale bars represent 60 μm in (c, d)