Figure 3
From: Cortisol modulates calcium release-activated calcium channel gating in fish hepatocytes
Cortisol stimulates Ca2+ entry via CRAC channel. (a) Results are shown as % of cortisol-treated fura2 intensity, which is represented by the horizontal line at 100%. Representative fluorescence images taken at 1 min either with or without the addition of cortisol are shown above (ai–ci), while the corresponding quantification is shown below as a bar graph (a–c). Bar graph showing fura2 intensity ([Ca2+]i) changes in hepatocytes at 60 s relative to cortisol treatment with either control, thapsigargin or Tg + Cortisol. (b) Bar graph showing fura2 intensity ([Ca2+]i) changes in hepatocytes at 60 s relative to cortisol treatment with either control, Cpd5J-4 or Cpd5j-4 + Cortisol. (c) Bar graph showing ([Ca2+]i) changes in hepatocytes at 60 s relative to cortisol treatment with either control, Cortisol exposure to cells treated with cortisol antibody (1:100) or Cortisol exposure to cells treated with ORAI1 antibody (1:250) (d) ORAI-1expression (~ 51 Kda) was detected in the crude liver membrane (CLM), plasma membrane purified (PLMP) and liver homogenate (LH) of trout using western blotting. See Figure S4 for the image of the blot with the molecular marker. (e) Representative immunofluorescence localization of ORAI1 in control and cortisol-treated (5 min) hepatocytes. The top panel shows increase ORAI1 expression on the surface of cortisol treated cell relative to the control (diffused distribution). The middle panel shows DAPI staining (nucleus) and the lower panel shows a merged image. (f) Colocalization of ORAI-1 with Caveolin-1 in control cells and cells treated with cortisol for 5 min. Colocalization was confirmed by IMAGE J- colocalization analyzer tool. Green indicates ORAI1 expression, while the red shows Caveolin-1 expression. The merged image showing yellow colour represents colocalization, and it is higher in the cortisol hepatocytes compared to the control. All bars represent mean ± SEM (n = 5–6 independent fish); Bars with different letters are significantly different (one-way ANOVA; p < 0.05). Image scale bar represents an approximate cell diameter of 25 μm (field of view at 100x has 650 × 520 pixel with 7.7 pixel/μm).
