Fig 2: Molecular alterations underlying GDFs induced histopathological alterations of murine hypopharyngeal mucosa (HM), in vivo, linked to p-NF-?B activation, increased cell proliferation, cell-cell adhesion changes, and STAT3 activationImmunofluorescence (IF) staining and automated quantitative analysis (AQUA) for (a) p-NF-?B (p65 S536) (green) and ?Np63 (red); (b) Ki67 (green) and E-cadherin (red); (c) CK14 (green) and ?-catenin (red); and (d) p-STAT3 (Tyr705) (green) (from left to right): Normal untreated-HM, acid alone treated-HM, and hyperplastic/dysplastic neutral-bile, acidic-bile, DCA, and CDCA treated-HM derived from C57Bl6J mice. DAPI (blue) was used for nuclei staining. (DyLight®488 for green and DyLight®549 for red). The boxplots, represent the means ranks. The upper line indicates the highest value, the lower line the lowest value and the middle line the mean of AQUA normalized quantities of each variable. Statistically significant difference between AQUA-score means is indicated for GDFs vs. acid (left) and GDFs or acid treated vs. normal-untreated (right) (*p < 0.01; **p < 0.001;***p < 0.0001; ONE-WAY ANOVA, Kruskal-Wallis and Dunn's multiple comparison tests; Graph Pad Prism 6.0).

Fig 3: Murine hypopharyngeal mucosa (HM) exposed to glucose or saline present normal histological features and immunofluorescence (IF) staining patterns similar to normal-untreated HMA. IF staining for (a) p-NF-?B (p65 S536) (green) and ?Np63 (red); (b) Ki67 (green) and E-Cadherin (red); (c) CK14 (green) and ?-catenin (red); and (d) p-STAT3 (Tyr705) (green). DAPI (blue) was used for nuclei staining. (DyLight®488 for green and DyLight®549 for red). B. Graph presents linear correlation between AQUA means of glucose or saline-treated HM and normal untreated-HM (Paired-t test, GraphPad Prism 6.0).

Fig 4: In vivo GDFs-induced NF-?B activation in murine hypopharyngeal mucosa (HM)A. Immunohistochemical analysis (IHC) for p-NF-?B (p65 S536) (from left to right): control (normal) untreated-HM, cytoplasmic staining; acid alone treated-HM, nuclear or cytoplasmic staining mainly of cells of basal layer and weak cytoplasmic staining of suprabasal layers; hyperplastic/dysplastic neutral-bile, acidic-bile, DCA and CDCA treated-HM, intense nuclear and cytoplasmic staining of cells of basal and suprabasal layers; glucose and saline treated-HM, cytoplasmic staining sporadically of a few basal/parabasal cells. B. Image analysis algorithm(s) (red, nuclear positive staining of p-NF-?B; orange, intense positive cytoplasmic staining of p-NF-?B; yellow, weak cytoplasmic staining of p-NF-?B; blue, negative p-NF-?B staining).

Fig 5: Analysis of influence of cell media acidification (96 h) and 10 µM mambalgin-2 (72 h) on expression and activity of molecules mediating progression in mel P cells: CD44 (a), CD133 (b), Frizzled 4 (c), connexin 43 (d), SNAI (pSer246) (e), NF?B p65 (pSer536) (f), and STAT3 (pTyr705) (g). Analysis was performed by flow cytometry. The representative cell distribution histograms after staining by antibodies and expression levels are shown at the left and right figure panels, respectively. Data presented as normalized MFI ± SEM (n = 4). * (p < 0.05) and ** (p < 0.01) indicate significant difference between the data groups by One-Way ANOVA followed by Tukey’s hoc test. Cells stained only by secondary antibodies (Abs) were used as a negative control.