Pdx1 effects on cell viability and Pdx1 internalization.

<p>(A) hBTSCs and HepG2 were exposed to different concentrations of Pdx1 (0.1 μM or 0.5 μM). Incubation with 0.1 μM Pdx1 determined the preservation of cell viability. When cells were treated with 0.5 μM Pdx1, cell viability significantly decreased after 48 hours in both hBTSCs and HepG2 as observed by Trypan blue exclusion assay (data are means ± SD of 6 experiments; **p< 0.01; ***p< 0.001). (B) After 24 hours of treatment with 0.1 μM Pdx1 recombinant protein, Western Blot analyses showed the presence of Pdx1 recombinant protein (His-Tagged protein) in treated hBTSCs and HepG2. The densitometry histograms showed an equal amount of His-Tagged protein in hBTSCs and HepG2 cells treated with 0.1 μM Pdx1 (data are means ± SD of 3 experiments). (C) Immunofluorescence analysis showed the internalization and nuclear translocation of His-Tagged recombinant protein (red nuclei) in treated cells. Nuclei are displayed in blue (DAPI). Original Magnification = 20X left image or 40X right images.</p

Human biliary tree stem cell (hBTSC) differentiation towards pancreatic fate induced <i>in vitro</i> by Pdx1 peptide.

<p>(A) In HepG2 cells the insulin mRNA level was not affected by 14 days of 0.1 μM Pdx1 administration, while it increased significantly (average 6 folds) in hBTSCs cultured for 14 days in Kubota’s Medium (KM) containing 0.1 μM Pdx1in comparison with control medium (KM) (data are means ± SD of 6 experiments; ***p< 0.001). The effect of Pdx1 administration on hBTSCs was similar to treatment with a Hormonally Defined Medium for Pancreatic islet cell differentiation (HDM-P). PANC-1 cells and pancreatic islets (islets) were used as positive controls. (B, C) In hBTSCs cultured for 14 days in KM containing 0.1 μM Pdx1, the glucagon and somatostatin mRNA levels increased 6- (data are means ± SD of 6 experiments; ***p< 0.001) and 2-folds (data are means ± SD of 6 experiments; **p< 0.01), respectively, in comparison with KM. (D) In hBTSCs cultured for 14 days in KM containing 0.1 μM Pdx1, MafA (4.5-folds) (data are means ± SD of 6 experiments; **p< 0.01) or Pdx1 (2-folds) (data are means ± SD of 6 experiments; **p< 0.01) gene expression increased in comparison with KM, while EpCAM mRNA decreased (average 4-folds) (data are means ± SD of 6 experiments; *p< 0.05).</p

Pdx1 effects on cell viability and Pdx1 internalization.

<p>(A) hBTSCs and HepG2 were exposed to different concentrations of Pdx1 (0.1 μM or 0.5 μM). Incubation with 0.1 μM Pdx1 determined the preservation of cell viability. When cells were treated with 0.5 μM Pdx1, cell viability significantly decreased after 48 hours in both hBTSCs and HepG2 as observed by Trypan blue exclusion assay (data are means ± SD of 6 experiments; **p< 0.01; ***p< 0.001). (B) After 24 hours of treatment with 0.1 μM Pdx1 recombinant protein, Western Blot analyses showed the presence of Pdx1 recombinant protein (His-Tagged protein) in treated hBTSCs and HepG2. The densitometry histograms showed an equal amount of His-Tagged protein in hBTSCs and HepG2 cells treated with 0.1 μM Pdx1 (data are means ± SD of 3 experiments). (C) Immunofluorescence analysis showed the internalization and nuclear translocation of His-Tagged recombinant protein (red nuclei) in treated cells. Nuclei are displayed in blue (DAPI). Original Magnification = 20X left image or 40X right images.</p

Pdx1 induced hBTSC-derived β-pancreatic islets are functioning active.

<p>(A) Morphologically (Phase Contrast: Ph-C; Original Magnification = 10X), after 14 days in basal condition, hBTSCs formed large colonies composed of small, densely packed, and uniform EpCAM-positive cells (Immunofluorescence for EpCAM; Original Magnification = 20X). After 14 days in cultures containing 0.1 μM Pdx1, several islet-like structures were present (Phase Contrast: Ph-C; Original Magnification = 10X); these structures were mostly composed of insulin-positive cells (Immunofluorescence for Insulin; Original Magnification = 10X); and some glucagon-positive cells were located at the periphery of these islet-like structures (double immunofluorescence for Insulin and Glucagon; Original Magnification = 20X). (B) Functionally, hBTSCs in Kubota’s Medium (KM) or in KM plus 0.1 μM Pdx1 were exposed to low (5.5 mM) or high (28 mM) glucose concentrations to stimulate C-peptide secretion and the response was compared with human pancreatic islets (islets). Human C-peptide secretion in hBTSCs in KM was not affected by high glucose stimulation (data are means ± SD of 6 experiments). In hBTSCs cultured for 14 days in KM containing 0.1 μM Pdx1, C-peptide secretion was detected at low glucose levels and further increased after exposure to high glucose concentration (data are means ± SD of 6 experiments; *p< 0.05). Human pancreatic islets (islets) were used as a positive control of the high glucose challenge (data are means ± SD of 6 experiments; *p< 0.05).</p

Immunofluorescence for vimentin in cell cultures treated with the TGF-ΒR1 inhibitor LY2157299.

<p>Expression of vimentin was unchanged by LY2157299. However, IF for vimentin (red) highlighted evident morphological changes in mucin- and mixed-IHCCA cell cultures treated for 72 hrs with the TGF-ΒR1 inhibitor LY2157299 (50 μM). LY2157299 induced the loss of typical mesenchymal features, instead favoring the epithelial phenotype with formation of cell clusters (white arrows). No morphologic change was induced by LY2157299 in CK19-mucin cell cultures. Cell clusters with undefined edges, a typical feature of the epithelial phenotype, were observed in mucin- and mixed- IHCCA cells treated with LY2157299 (white arrows) and in CK19-mucin (yellow arrows, similar in treated and untreated cells). Mean +/- SD of N = 3–5 independent experiments.</p

Apoptosis (Annexin V-FITC/PI by FACS) of primary cell cultures exposed to TGF-ΒR1 inhibitor (LY2157299) and CK2 inhibitor (CX4945), and RT-PCR analysis of snail-1 and vimentin in primary cell cultures exposed to CX4945.

<p><b>A:</b>After 72 hrs of treatment, CX4945 10 μM induced apoptosis in both mucin- and mixed-IHCCA primary cell cultures ** = p<0.01 vs control. <b>B:</b>When CX4945 was combined with MK2206, an enhanced number of apoptotic cells was seen with respect to CX4945 or MK2206 alone. * = p<0.05 vs control and MK+CX; & = p<0.05 vs MK and CX alone. <b>C:</b> CX4945 30 μM did not induce apoptosis in CK19 mucin primary cells. <b>D:</b> CX4945 (10μM) treatment for 72h induced vimentin expression in all three primary cell cultures, whereas snail-1 expression was induced only in CK19-mucin, being down-regulated in mixed-IHCCA and unaffected in mucin-IHCCA. mRNA expression was normalized to control cells exposed to CX4945 carrier and considered equal to 1. * = p<0.05 vs control. Mean +/- SD of N = 3–5 independent experiments.</p

RTK human phosphoarrays in mucin-IHCCA cells resistant to CX4945 treatment.

<p>After 72h of CX4945 (10 μM) treatment, surviving cells showed contemporary deactivation of both EGFR and HGFR signalling. The black lines indicate the spots positive for EGFR and HGFR activation.</p

Clonogenic assays.

<p>The number of colonies (A and B) and the absorbance of the extracted dye (crystal violet) measured at 595 nm (C) indicate the clonogenic potential of CCA cell cultures. We tested the clonogenic capacity of mucin-IHCCA cells after sequential treatment with CX4945 (72 hrs) and MK2206 (48 hrs) versus their combination. The sequential treatment of CX4945 followed by MK2206 showed the highest inhibitory effect on mucin-IHCCA cell growth. We calculated the numbers of colonies and the absorbance of the extracted dye at 595 nm. & = p<0.05 vs non pre-incubated conditions. $ = p< 0.05 vs others columns. Mean +/- SD of N = 3–5 independent experiments.</p