Fig. 1

CathL-induced Gal1 secretion in HOMECs is transcriptionally regulated via activation of NFκB. a Cells were seeded and starved overnight in media supplemented with 2% FCS. Cells were then treated ± CathL (50 ng/ml) and supernatants were collected after 4 min, 30 min, 2, 4, 8 and 24 h treatment. A commercially available ELISA kit was used to assess the levels of extracellular Gal1 using a SpectraMax plate reader. Results are mean ± SD and represented as fold change in secreted Gal1 vs control. n = 4–6. b Cells were treated ± CathL (50 ng/ml) and lysed after 6 or 24 h treatment. Real-time PCR was performed on extracted RNA using a Roche LightCycler 96 and the data were normalised to GAPDH and β2 M. c Gal1 secretion was assessed with or without CathL ± sulfasalazine (100 µmol/l) for 8 h and the supernates were analysed as above. d CathL induces activation of NFκB (p65) in HOMECs. Cells were incubated with sulfasalazine (sulf, 100 µmol/l) or media alone for 24 h and then treated ± CathL (50 ng/ml) or positive control TNF-α (160 pg/ml) and in the absence or presence of sulfasalazine (100 µmol/l). After 4 h treatment, the phosphorylated level of NFkB was assessed using a commercially available cell-based ELISA kit. Results are mean ± SD and are represented as fold change in phosho-NFkB relative to total NFkB (compared to control). e CathL induces an upregulation of LGALS1 gene expression via NFkB activation. After overnight incubation and pre-incubation, cells were treated with CathL ± sulfasalazine for 6 h and analysed for gene expression as above. Results are mean ± SD and are represented as fold change in LGALS1 gene expression (relative to control), n = 4. *p < 0.05, **p < 0.01 vs control. #p < 0.05, ##p < 0.01 vs CathL. n.s. denotes not significant