Fig. 2

HM increased COX-2 protein and gene expression levels in the gastric AGS cells while COX-1 was not affected. A Representative Western blot showing the stimulatory effect of both LPS and HM on COX-2 protein expression in the untreated AGS cells and the cells treated after 72 h incubation. The bar graph shows the relative expression levels of COX-2, calculated as a ratio of the expression to GAPDH. B Bar graph showing the relative expression level of COX-2 mRNA determined by RT-qPCR analysis in the AGS cells after 24, 48 and 72 h incubation with 10 µg/mL of LPS or HM, compared with the untreated cells, calculated as a ratio of the expression to GAPDH mRNA. ^*p < 0.05 and ^**p < 0.01 compared with the control, from three independent experiments. C Representative photomicrographs of the immune-cyto-fluorescence staining of COX-2 expression targeted by monoclonal anti-COX-2 conjugated to FITC, revealing the COX-2 nuclear, cytoplasmic, plasma membrane, and intercellular junctions localizations in the untreated AGS cells and cells exposed to 10 µg/mL of LPS or HM after 24, 48 and 72 h incubation. A higher magnification of an example of the AGS cells depicted in the insert including the arrows pointing to the different COX-2 cellular localizations as “Cy” standing for cytoplasmic, “P” for plasma membrane-bound form, “N” for nuclear, and “I” for intercellular junctions. Scale bar = 5 µm. D Bar graph showing the arbitrary quantification of the COX-2 protein expression levels analyzed using ImageJ software (from 6 random fields) at the cellular compartments after 72 h incubation, compared to the COX-2 expression level measured in the untreated cells, the control. E Representative gel electrophoresis showing the modulatory effect of both LPS and HM on COX-1 gene expression in the untreated AGS cells and the cells treated after 72 h incubation. The bar graph shows the relative gene expression levels of COX-1, calculated as a ratio of the expression to GAPDH mRNA. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001 compared with the control, from three independent experiments