Fig 2: The charts show the dCt, the fold change of gene expression (relative to the housekeeping gene GAPDH) in the large intestine. (Panel A) shows CDH1, (Panel B) shows CDH2, (Panel C) VCL and (Panel D) OCLN. LI—large intestine. * p (two-tailed) ≤ 0.05.

Fig 3: PCR array analysis and validation of genes differentially expressed between shNT and shCSNK1D cells(A) Scatter plot of gene expression analysis by RT2 Profiler PCR Array in the shNT MDA-MB-231 cells compared to shCSNK1D cells. The central line indicates unchanged gene expression. (B) Taqman validation of genes identified in the RT Profiler array. The histogram represents fold change of genes up-or down-regulated in shCSNK1D compared to shNT control cells. (C) Western blot analysis of the expression of occludin (OCLN), junction adhesion molecule A (F11R) and claudin-1 (CLDN-1) in control (shNT) and inactivated cell lines (shCSNK1D). (D) Detection of OCLN and F11R by immunofluorescence in shCSNK1D compared to control shNT cells. Nuclei were stained with Dapi. Arrows indicate the increased expression of tight junction proteins.

Fig 4: UAS03 is a potent anti-inflammatory structural analogue of UroA and induces tight junction proteins. a Chemical structures of UroA, UAS03. UroA/UAS03 stability was examined in the presence of gastric pH 2.0 and digestive enzymes. UroA and UAS03 (0.2 mg/ml) were incubated with digestive enzymes (esterases and proteases, 100 U/ml) for 12 h at 37 °C and compound levels were quantified. b BMDMs were stimulated with LPS (50 ng/ml) without or with UroA (blue line)/UAS03 (purple line) (0.1, 1, 10, 25, and 50 µM) for 6 h. IL-6 and TNF-a levels in supernatants were measured. c C57BL/6 mice (n = 3–4) were pretreated with UroA (20 mg/kg) and UAS03 (20 mg/kg). After 4 h, LPS (2 mg/kg) was injected intraperitoneally. Post 4 h of LPS administration, serum levels of IL-6 and TNF-a was measured. d–f HT29 or Caco2 cells were treated with vehicle (DMSO-0.01%) or UroA/UAS03 (50 µM) for 24 h. d The fold changes in mRNA levels of claudin 4 (Cldn4), occludin (Ocln), and Zona occludens 1 (ZO1) in HT29 cells were determined by RT PCR method. e UroA/UAS03 induced protein expression of Cldn4, Ocln, and ZO1 in HT29 cells were determined by immunoblots and quantified by Image J software. f Caco2 or HT29 cells were grown on coverslip bottom FluroDish and treated with Vehicle, UroA/UAS03 for 24 h. The cells were stained with anti-Cldn4 followed by secondary antibody tagged with Alexa-488. Nucleus was stained using DAPI. The confocal images were captured. The green intensity (n = 15–20 cell membrane regions) was measured. Scale bars for Caco2 and HT29 cells indicate 50 and 25 µm respectively. g Monolayer HT29 or Caco2 cells on transmembranes were treated with vehicle or UroA/UAS03 (50 µM) for 24 h followed by treatment with LPS (50 ng/ml) for 2 h. FITC-dextran was added to these cells (top of the membrane) and incubated for 2 h and FITC-dextran levels in bottom chamber well was measured. Results are representative of three independent experiments with triplicates for each concentration. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t-test between Veh, UroA, or UAS03. Error bars, ±SEM. Source Data are provided as a Source Data File