Fig 1: Galectin-3, prohibitin 1, and prohibitin 2 are abundantly expressed in human cholangiocyte models. (A) Immunohistochemical staining of galectin-3 and prohibitin 1 in control human liver sections with sequential cytokeratin 19 staining as a positive control for bile ducts (annotated by arrows) (scale bar, 20 µm). (B) Heatmaps depicting expression levels of galectin-3 and prohibitins 1 and 2 in human cholangiocyte models (positive for EPCAM and KRT19 and negative for hepatocyte markers ALB and ASGR1). The left shows log2-transformed expression values of four different derivations of human cholangiocyte organoids (ERCP, BCO, ECO, and ICO). The right shows log2-transformed expression values of human H69 cholangiocytes. (C) LGALS3 (26 kDa) and PHB1 (30 kDa) expression and knockdown quantification in H69 sham transduced and respective shRNA knockdown cell lines. LGALS3 KD is normalized by GAPDH (37 kDa) (9 cell samples from n = 3 independent experiments). PHB1 KD is normalized by beta-actin (42 kDa) (9 cell samples from n = 3 independent experiments). Data are represented as means with standard deviations. (D) Relative mRNA expression of LGALS3, PHB1, and PHB2 in H69 sham transduced and respective shRNA knockdown cell lines (9–12 cell samples from n = 3–4 independent experiments, 1 outlier value excluded from the sham and PHB1 dataset). Data are represented as the starting concentration (N0) of target genes over the geomean of the reference genes 36B4 (RPLP0) and HPRT N0 values normalized to sham. Levels of significance: (C) LGALS3 KD **** p < 0.0001 and PHB1 KD **** p < 0.0001, unpaired t-tests. (D) LGALS3 KD **** p < 0.0001, PHB1 KD **** p < 0.0001, PHB2 KD **** p < 0.0001, unpaired t-tests. ALB, albumin; A.U, arbitrary units; ASGR1, asialoglycoprotein receptor 1; BCO, bile-collected organoid (from resected gallbladders and PTC drainage); ECO, extrahepatic cholangiocyte organoid; EPCAM, epithelial cell adhesion molecule; ERCP, ERCP-derived cholangiocyte organoid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICO, intrahepatic cholangiocyte organoid; IHC, immunohistochemistry; kDa, kilodalton; KD, knockdown; KRT19, cytokeratin 19; LGALS3, galectin-3; N0, starting concentration; PHB1, prohibitin 1; PHB2, prohibitin 2; WT, wild type.
Fig 2: Single lentiviral shRNA knockdown of PHB1 or PHB2 or double PHB1/2 knockdown demonstrates a clear damaging effect against toxic bile acids in human cholangiocytes. 22,23-3H-GCDC permeation assay in (A) sham, PHB1 KD, PHB2 KD, and combined PHB1/2 KD cholangiocytes (representative experiments of n = 3). Relative quantification of 22,23-3H-GCDC permeation per time point in (B) sham, PHB1 KD, PHB2 KD, and combined PHB1/2 KD cholangiocytes (8–9 samples from n = 3 independent experiments). GCDC-induced Caspase-3/7 activity in (C) sham, PHB1 KD, PHB2 KD, and combined PHB1/2 KD cholangiocytes (9–12 samples from n = 3–4 independent experiments). Data are represented as means with standard deviations. Levels of significance: (B) in order of sham to PHB1 KD, to PHB2 KD, to PHB1/2 KD: 1 min: ns, p = 0.3731, ns, p = 0.9966, ns, p = 0.5143. 4 min: ns, p = 0.7795, ns, p = 0.9155, ** p = 0.0060. 16 min: ns, p = 0.1287, ns, p = 0.9999, ns, p = 0.4846. 64 min: ns, p = 0.7828, ns, p = 0.9940, ns, p = 0.9902, one-way ANOVA. (C) In order of sham to PHB1 KD, to PHB2 KD, to PHB1/2 KD: *** p = 0.0005, ns, p = 0.4392, ns, p = 0.9985, one-way ANOVA. Ctrl, control; DPMI, disintegrations per minute; GCDC, glycochenodeoxycholic acid; KD, knockdown; ns, not significant; PHB1, prohibitin 1; PHB2, prohibitin 2; WT, wild type.
Fig 3: Pharmacological inhibition of prohibitins and recombinant protein substitution of prohibitin 1 do not demonstrate a clear damaging or protective effect against toxic bile acids in human cholangiocytes. 22,23-3H-GCDC permeation assay in (A) WT and rocaglamide-treated cholangiocytes; (B) WT, recombinant low-dose prohibitin 1 0.25 µg/ml, and recombinant high-dose 0.5 µg/ml-treated cholangiocytes (representative experiments of n = 3). Relative quantification of 22,23-3H-GCDC permeation per time point in (C) WT and rocaglamide-treated cholangiocytes, and (D) WT, recombinant low-dose prohibitin 1 0.25 µg/ml and recombinant high-dose 0.5 µg/ml-treated cholangiocytes (8–9 samples from n = 3 independent experiments). GCDC-induced Caspase-3/7 activity in (E) WT and rocaglamide-treated cholangiocytes, and (F) WT, recombinant low-dose prohibitin 1 0.25 µg/ml and recombinant high-dose 0.5 µg/ml-treated cholangiocytes (9–12 samples from n = 3–4 independent experiments). Data are represented as means with standard deviations. Levels of significance: (C) 1 min: ns, not significant p = 0.0634. 4 min: * p = 0.0167. 16 min: **** p < 0.0001. 64 min: ** p = 0.0041, unpaired t-tests. (D) In order of sham to 0.25 µg/ml, to 0.5 µg/ml: 1 min: ns, p = 0.7354, ns, p = 0.8690. 4 min: ns, p = 0.0737, ns, p = 0.9288. 16 min: ns, p = 0.4439, ns, p = 0.1545. 64 min: ns, p = 0.0791, ns, p = 0.9104, one-way ANOVA. (E) ns, p = 0.5108, unpaired t-test. (F) In order of sham to 0.25 µg/ml, to 0.5 µg/ml: ns, p > 0.9999, ns, p = 0.8369, one-way ANOVA. Ctrl, control; DPMI, disintegrations per minute; GCDC, glycochenodeoxycholic acid; KD, knockdown; ns, not significant; PHB1, prohibitin 1; WT, wild type.
Supplier Page from Fine Biotech Co., Ltd. for Recombinant Human PHB1