Phosphorylated β catenin

AML cells (MV4-11 and MOLM-13) were drugged for 24 h, followed by protein extraction using RIPA buffer (Cell Signaling Technology) supplemented with PMSF. Cell pellets were incubated in RIPA buffer with constant agitation for 30 min, followed by sonication. Protein lysate was quantified using Pierce™ BCA Protein Assay Kit (ThermoFischer Scientific). 30 µg of protein was loaded into a Mini-PROTEAN TGX Stain-Free Precast Gel (Bio-Rad) and ran at 120 V for 1–2 h. Blots were developed using a ChemiDoc Chemiluminescence Imaging System (Bio-Rad) or by the Odyssey CLX Fluorescence Imaging System (Li-COR). Antibodies used in the presented studies purchased from Cell Signaling Technology included total β-catenin (#8480S), phosphorylated β-catenin (#9561S), non-phosphorylated β-catenin (cat#8814S), and β-actin (#3700S). Antibodies purchased from Abcam include LRP6 (ab134146) and phosphorlated-LRP6 (ab76417).

Antibodies against CD90, CD133, EpCAM, SOX2, OCT4, β-catenin, phosphorylated β-catenin, β-actin, and Tubulin were all purchased from Cell Signaling Technology (Danvers, MA, USA), antibody against CRBP-1 was purchased from Santa Cruz biotechnology (Dallas, TX, USA), antibody against WIF1 was purchased from BD Biosciences (San Jose, CA, USA), antibodies of c-Myc, and CyclinD1 were purchased from Proteintech Group (Wuhan, China), and antibodies of RARα used for Chromatin immunoprecipitation assay was purchased from Abcam (Shanghai, China). MG-132 and retinoic acid (RA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sorafenib tosylate (Nexavar) was purchased from Bayer (Shanghai, China). Human retinoic acid ELISA kit was purchased from CUSABIO Technology LLC (Wuhan, China).

Total protein was obtained from cells after lysed in RIPA buffer. The Western blot was performed as described in our previous work.32 The information of primary antibodies was HA (Cat No. 3724, Cell Signaling Technology), SFRP2 (Cat No. 06‐004, millipore), KDM2A (Cat No. ab31739, abcam), BCOR (Cat No. 12107‐1‐AP, proteintech), phosphorylated β‐catenin (Cat No.2009, Cell Signaling Technology), GAPDH (Cat No. C1312, Applygen) and β‐actin (Cat No. C1313, Applygen).

For immunohistochemistry and immunofluorescence, sections were incubated overnight at 4°C with primary antibodies against cytokeratin (CK)7 (mouse monoclonal, code: M7018, dilution: 1:100, Dako, Glostrup, Denmark), EpCAM (Dako, mouse monoclonal, code: M3525, dilution: 1:100), CD68 (Dako, mouse monoclonal, code: M0876, dilution: 1:100), CD206 (monoclonal mouse, code: MAB25341; dilution: 1:25, R&D Systems, Minneapolis, USA), Arginase-1 (polyclonal mouse, code: ab118884; dilution: 1:150, Abcam, Cambridge, United Kingdom), Caspase-3 (polyclonal rabbit, code: #9662; dilution: 1:200, Cell Signaling Technology, Danvers, USA), CD163 (monoclonal mouse, code: ncl-CD163; dilution: 1:200, Novocastra, Milan, Italy), S100A9 (polyclonal rabbit, code: ab92507; dilution: 1:200, Abcam, Cambridge, United Kingdom), Wnt3a (polyclonal rabbit, code: #09–162; dilution: 1:200, Merck Millipore, Darmstadt, Germany), SOX9 (polyclonal rabbit, code: AB5809; dilution: 1:200, Millipore, Darmstadt, Germany), and phosphorylated (p) β-catenin (Cell Signaling Technology, rabbit polyclonal, code: #4176, dilution 1:100). For immunohistochemistry, samples were than incubated for 20 minutes at room temperature with secondary biotinylated antibody and, successively, with streptavidin-Horse radish peroxidase (LSAB+, Dako, code K0690). Diaminobenzidine (Dako, code K3468) was used as the substrate and the sections were counterstained with hematoxylin or Sirius Red [25 (link)].
For immunofluorescence, non-specific protein binding was blocked with 5% normal goat serum. Sections were incubated with primary antibodies, and subsequently incubated with labeled isotype-specific secondary antibodies (anti-mouse AlexaFluor-488 and anti-rabbit Alexafluor-594, Invitrogen Ltd, Paisley, UK) for 1 hour; nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI) [17 (link)].
To perform double immunostaining with two mouse or rabbit primary antibodies (CD68/CD206), we followed a 3-step protocol [11 (link), 26 (link)]: sections were incubated with anti-CD68 (or anti-SOX9), an anti-mouse (or anti-rabbit) secondary fluorescent antibody (alexafluor-488) was applied, and the second primary antibody was pre-labeled with a fluorophore using APEX-594 labeling Kit (Invitrogen) and applied to the section. All antibodies were diluted (1:50) and incubated for 1 hour. Slides were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). For all immunoreactions, adequate negative controls were also preformed.
Sections were examined with a Leica Microsystems DM 4500 B Microscopy (Weltzlar, Germany) equipped with a Jenoptik Prog Res C10 Plus Videocam (Jena, Germany) and with an Olympus Fluoview FV1000 confocal microscope equipped with FV10-ASW version 4.1 software. Only biopsies containing at least 5 portal spaces were considered [27 (link)].
The extension of ductular reaction (DR) was evaluated by CK7 immunoreactivity. CK7 stained slides were scanned by a digital scanner (Aperio Scanscope CS System, Aperio Digital Patology, Leika Biosystems, Milan, Italy) and processed by ImageScope [11 (link), 17 (link)]. The area occupied by CK7+ cells was quantified by an image analysis algorithm. The extension of DR was expressed as the percentage of the parenchymal area occupied by reactive ductules [28 (link)]. Cholangiocytes lining the interlobular bile ducts were excluded from the counts.
To assess the commitment of progenitor cells toward a hepatocyte fate, the presence of EpCAM+ hepatocytes has been investigated by immunohistochemistry. EpCAM+ hepatocytes have been shown to represent the progeny of stem/progenitor cells within bile ductules [29 (link), 30 (link)]. The presence of EpCAM+ hepatocytes was scored as: 0 = no positive cells, 1 (level 1) = single occasional, and 2 (level 2) = clusters of EpCAM+ hepatocyte [29 (link), 31 (link)].
The extension of portal fibrosis was evaluated on Sirius Red stains and the area occupied by Sirius Red-positive fibers in the entire section was quantified as previously [32 (link)].
The number of macrophages with an anti-inflammatory phenotype was calculated as the number of CD206+ cells per High Powered Field (HPF) [4 (link), 33 (link)]. In DHA treated patients, Arginase1 and CD163 have been further used as markers of anti-inflammatory macrophages. The presence of macrophages with an inflammatory phenotype was calculated as the number of S100A9+ cells per High Powered Field (HPF) [4 (link), 33 (link), 34 (link)].
Wnt3a expression by CD68+ macrophages was evaluated in serial sections and by double immunofluorescence as the number of positive macrophages per HPF. pβ-catenin expression by CK7+ HPCs was evaluated in serial sections and by double immunofluorescence; the average number of positive cells was divided by the average number of HPCs and data were expressed as a percentage of positive cells [11 (link)]. Given the differences in term of DR extension among examined biopsies, pβ-Catenin-positivity was further calculated as the ratio between the extension of positive DR (by ImageScope, Aperio) and the total DR extension.
For confocal microscopy imaging, fluorochrome unmixing was performed by acquisition of automated-sequential collection of multi-channel images, in order to reduce spectral crosstalk between channels. The average number of cells that displayed a colocalization of Caspase-3/CD68 or Arginase1/CD163 was assessed by counting 5 fields acquired using 20x.