Normal donkey serum (nds)

For immunolabelling, cultured cells or histological sections were permeablized with saponin (0.05% in PBS) for 10 min and non-specific binding sites were blocked with 5% (v/v) normal donkey serum (GIBCO^®Invitrogen) in PBS for 1 h. Cultures or slices were incubated with mouse anti-MyHc (MF20, DSHB; 1:25) antibody and either rabbit anti-CXCL11 (Biorbyt, 1:100), goat anti-CXCR4 (ab1670, Abcam; 1:100), rabbit anti-CXCR7 (AP17961PU-N; Acris; 1:100), rabbit anti-CXCR3 (Bioss; bs-2209R; 1:100), or mouse monoclonal anti CXCR3 (clone No. 49801, R&D Systems; 1:100) antibodies overnight in a humid chamber at 4 °C. Primary antibodies were detected by incubating slices for 2 h at room temperature with appropriate Alexa 488-labelled and Alexa 555-labelled secondary antibodies (1:200; Invitrogen, Carlsbad, CA). Cell nuclei were counterstained with DAPI (AAT Bioquest, Sunnyvale, CA) and sections were mounted with Dako glycergel. Sections were examined under a Zeiss confocal laser scan microscope (LSM).

The iPSC-derived MNs were plated on day 14, on top of 1mm glass coverslips (Fisher scientific) coated with Matrigel (37°C, overnight) at a density of 80,000 cells/coverslip. Cells were washed once with PBS and fixed with 4% PFA in PBS for 20 min at RT. Following fixation cells were washed three times with PBS and permeabilized with PBS-Triton X-100 0.2% for 45 min at RT. To block the non-specific binding of primary antibody to unrelated epitopes, cells were treated with 10% Normal Donkey Serum (Jackson ImmunoResearch) in PBS-Triton X-100 0.1%, for 1h at RT and then incubated with primary antibody, diluted in 2% Normal Donkey Serum in PBS-Triton X-100 0.1%, overnight at 4°C. Next day, coverslips were washed three times with PBS and incubated with the fluorophore-conjugated secondary antibody (Alexa Fluor 488, or Alexa Fluor 647 or Alexa Fluor 555, all from Invitrogen) at a dilution 1:1,000 in 2% Normal Donkey Serum in PBS-Triton X-100 0.1%, for 2h at RT protected from light. Cells were washed once with PBS, incubated with DAPI 1:1,000 in PBS for 10 min at RT, washed again three times with PBS and coverslips were mounted on glass slides using Fluoromount-G (Southern Biotech).

For immunofluorescence assays, HepG2 and Bel-7402 cells were seeded in 12-well plates and treated with GA for 48 h. Then, cells were fixed with 4% paraformaldehyde for 15 min, 0.5%Triton X-100 was used for permeabilization, and 10% normal donkey serum (Invitrogen) was added and incubated for 1 h. Cells were incubated with primary antibody against β-catenin (Cat.NO. 8480S, 1:100; Cell Signaling Technology, United States) at 4°C for overnight and then probed with donkey anti-rabbit IgG-Alexa Fluor 594 (Absin, Beijing, China) in dark at 37°C for 1 h. Finally, the cells were washed with PBS and stained with DAPI (Beyotime). The images were captured by using a confocal microscope (Nikon, Tokyo, Japan). For immunohistofluorescence examination, the specimens were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Sections (5 µm) were used to analyze Ki-67 and β-catenin expression. After being counterstained with DAPI, the images were captured using a Zeiss Axiophot two microscope.

Cultured cells on coverslips were fixed with 4% PFA in PBS for 15 min, permeabilized by 0.1% Triton X-100 for 5 min, and blocked in 10% normal donkey serum (Invitrogen) in PBS for 1 h at room temperature^{54 (link),62 (link)}. Cells were then labeled with primary antibodies against p150^Glued (BD Biosciences, #610474, 1:200, recognizing p150^Glued but not p135+), tyrosine hydroxylase (TH, Pel-Freez, #P40101-150, 1:2000), microtubule-associated protein 2 (MAP2, Abcam, #92434, 1:1000), binding immunoglobulin protein (BiP, also referred to as GRP78, Abcam, #ab21685, 1:500), and α-tubulin (Abcam, #ab89984, 1:1000) overnight at 4 °C in a humidified chamber. After three washes with PBS, secondary antibodies conjugated to Alexa Fluor (Invitrogen, 1:1000) were applied and incubated for 1 h at room temperature in the dark. After extensive washes, coverslips were mounted on glass slides with prolonged diamond antifade reagent containing DAPI (Invitrogen), and fluorescence signals were detected using LSM 880 laser-scanning confocal microscope (Zeiss). The paired images in all the figures were collected at the same acquisition settings, uniformly processed, presented as either a single optic layer or maximum-intensity projection of confocal stacks, and analyzed with ImageJ (NIH).

1500 cells per well were seeded in 80 µl medium in 96-wells plates with clear flat bottoms for imaging (Greiner) and treated in fresh medium 24 h later. After incubation with indicated concentrations of drugs or vehicle (DMSO) for 72 h, cells were fixed with 4% paraformaldehyde (Electron Microscopy [USA]) for 20 min at room temperature, washed once with PBS and permeabilized with 0.05% Triton X-100 (Amresco) in PBS for 20 min. After washing, primary antibodies against GM130 (1:100, sc-16268 Santa-Cruz) diluted in 5% normal donkey serum (Jackson ImmunoResearch) were added for overnight incubation at 4° C. The next day cells were washed three times with PBS and incubated with appropriate secondary antibody (1:2000, Life Technologies) and Hoechst (1:2500, Life Technologies) diluted in 5% normal donkey serum for one hour. Finally, samples were washed five times with PBS. Pictures were acquired with an Olympus Biosystems IX81 inverted microscope at 20 × magnification using Olympus ScanR 2.5.0. acquisition software. For each replicate 9 fields per well were acquired.