Formalin-fixed, paraffin-embedded 5 μm intestinal slices from Lean Control, (Chow+PP2Ainh), NAFLD, (NAFLD+PP2Ainh), and (NAFLD+PP2Ainh+p47phox KO) groups were used for immunofluorescence imaging. (A)(i-iv) Immunofluorescence images depicting Smad2/3 (red) and Smad4 (green) co-localization events in the small intestine, counterstained with DAPI (blue) of Lean Control, (Chow+PP2Ainh), NAFLD, (NAFLD+PP2Ainh), and (NAFLD+PP2Ainh+p47phox KO) mice groups. Images were taken at 20X magnification, (B)(i-v) Immunofluorescence images depicting the proximity ligation (red) of Smad2/3 and Smad4 proteins in the small intestine, counterstained with DAPI (blue) of Lean Control, (Chow+PP2Ainh), NAFLD, (NAFLD+PP2Ainh), and (NAFLD+PP2Ainh+p47phox KO) groups of mice. Images were taken at 20X magnification. (C) Morphometric analysis of Smad2/3-Smad4 co-localization events. Y-axis shows % positive immunoreactive area (% ROI) (n=3, analysis from three separate microscopic fields) (*p < 0.05, #p< 0.01). (D) Morphometric analysis of Smad2/3 and Smad4 proximity ligation. Y-axis shows % positive immunoreactive area (%ROI) (n=3, analysis from three separate microscopic fields) (*p < 0.05, 0.05<**p < 0.01). Significance was tested by performing unpaired t-test between the groups (*p< 0.05, 0.05<**p < 0.01, #p< 0.01), followed by Bonferroni Dunn Post hoc corrections. Results were expressed as mean ± SEM.