Novex zymogram system

We measured the profiles of 52 cytokines (Table 2) that were secreted into the conditioned media using fluorescent beads-based multiplex assay (Bio-Plex, Bio-Rad, Hercules, CA, USA), using human cytokine 27-plex (#M500KCAF0Y, Bio-Rad), cytokine 21-plex (#MF0005KMII, Bio-Rad), VCAM-1 (#171B6022M, Bio-Rad), ICAM-1 (#171B6009M, Bio-Rad), and human TGF-β 3-plex (#171W4001M, Bio-Rad) assays according to the manufacturer’s protocol. Cytokine secretion was normalized for the wet weight of tissue as measured by the end of culture. The effect of pyridone 6 or IL-6 on cytokine secretion was estimated by calculating the ratio of cytokine secretion in the presence and absence of pyridone 6 or IL-6 (POST) to the basal cytokine secretion (PRE) (Fig. 1). Immunoblotting was performed for phosphorylated STAT3 (Tyr705, #9145, Cell Signaling Technology, Danvers, MA, USA), total STAT3 (#4904, Cell Signaling Technology), and GAPDH (#MAB374, Millipore), which served as the internal reference. Gelatin zymography was performed using the Novex zymogram system (#EC6175BOX, Thermo Fisher Scientific) according to the manufacturer’s instruction.