Pha l

LX-2 cells, an immortalized hepatic stellate cell line^{47 (link)}, were cultured in Dulbecco’s modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS) which was kindly provided by Dr. Yi-Tsau Huang (National Yang-Ming University, Taipei, Taiwan). Antibodies including anti-p-Erk1/2, anti-p-Akt, anti-Erk1/2, anti-Akt, anti-p-smad2, anti-p-smad3, anti-smad2, and anti-smad3 were from Cell Signaling. Anti-neuropilin (NRP)-1 and α-smooth muscle actin (α-SMA) antibodies were from GeneTex and Millipore. The polyclonal anti-Gal-1 antibody was purified from Gal-1-immunized rabbit serum. Recombinant PDGF and TGF-β were from R&D Systems. Inhibitors of N- and O-glycosylation [swainsonine (SW) and benzyl-N-acetyl-α-galactosaminide (BαG)] were from Calbiochem. Sorafenib and SIS3 were from MedChemexpress (MCE). Biotinylated lectins including L-PHA, LEL, SNA, PNA and DyLight® 488 streptavidin were from Vector Labs. Thiodigalactoside (TDG) was from Carbosynth.

FFPE muscle tissue sections (3 μm) from IIM patients (n=22) and healthy controls (n=11) were analysed by lectin histochemistry. For glycan profile determination, sections were deparaffinised, rehydrated and endogenous peroxidase activity blocked (3% H₂O₂). Sections were then incubated with the biotinylated Phaseolus Vulgaris Leucoagglutinin (L-PHA) lectin, which recognises β1,6GlcNAc-branched N-glycans, or with the biotinylated Galanthus Nivalis agglutinin (GNA) lectin, which is used for the detection of terminal α-1,3 mannose residues. The avidin-biotin-peroxidase complex was detected using the Vectastain ABC kit and the colour was developed using 3,3'-diaminobenzidine (DAB, Thermo Scientific, DE, USA). L-PHA, GNA and the Vectastain ABC kit were obtained from Vector Laboratories, Burlingame, CA, USA. Finally, all sections were counterstained with haematoxylin, dehydrated and preserved in appropriate embedding medium (Entellan® new, Merck Millipore).

Cells were washed with FACS buffer (PBS containing 0.1% (w/v) sodium azide and 2% BSA) and stained with fluorescent conjugated Phaseolus vulgaris leukoagglutinin lectin (L-PHA, 2 μg/ml), anti-CD4 (RPA-T4), anti-CD69 (FN50), anti-CD25 (BC96), anti-CTLA-4 (14D3) and anti-IL-17A (eBio64Dec17) for human, and anti-CD4 (RM4-5), anti-CD25 (PC61.5), anti-IL-17A (eBio17B7) and anti-FoxP3 (FJK-16s) for mouse (antibodies from eBioscience, L-PHA from Vector Labs). Proliferation was assessed by staining cells with 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE: ThermoFisher Scientific). After staining, cells were washed twice with FACS buffer and analyzed for CFSE dilution by the BD Accuri, BD FACSCanto II, or Attune Acoustic Focusing Cytometry. Data analysis was performed using FlowJo software.

For flow cytometry, cells were harvested and incubated with anti-sialyl-Tn (TKH2 antibody [75 (link)]), anti-galectin-3 (M3/38, ATCC TIB166), biotinylated lectins Erythrina cristagalli (ECA), Phaseolus vulgaris (L-PHA), Sambucus nigra (SNA) and Maackia amurensis (MAL) (from Vector Laboratories) or biotinylated-Arachis hypogaea (PNA) (Sigma-Aldrich). Subsequently cells were washed with PBS, and primary antibodies were detected with anti-mouse-Alexa488 or anti-rat-Alexa488 antibodies, or streptavidin-Alexa488 (all from Invitrogen) for 45 min. Alternatively, cells were incubated with DyLigth488 labelled-hrGal-3. For the cleaved caspase3/7 assay, cells were treated for 48h in the presence or absence of cisplatin (12,5μM). Caspase-3/-7 evaluation was performed accordingly to manufacturer's instructions (Vybrant FAM Caspase-3 and -7 Assay kit, Life Technologies). Analyses were made using the flow cytometer CyAn™ ADP Analyzer from Beckman Coulter. Data were subsequently evaluated with FlowJo vX 0.7 software. For detailed protocol see supplemental experimental procedures.

Experiments involving neural pathway-tracing were as follows: (Exp. 2) Retrograde tracing from the dpLHA following unilateral pressure injection of either Fluorogold (FG, Fluorochrome LLC, Denver, CO; 2% in 0.9% NaCl), or cholera toxin B subunit conjugated to (red fluorophore) Alexa Fluor 594 (CTB-AF594, 0.25% in 0.9% NaCl, Life Technologies, Carlsbad, CA; Cat. No. C-22842). Pressure injections were performed using a microinfusion pump as described above; injection volumes were 100 nl (Exp. 4). Anterograde tracing from the vHP field CA1 following unilateral iontophoretic injection of Phaseolus vulgaris-leucoagglutinin (PHAL, Vector Labs, Burlingame, CA; 2.5% in 0.1 M sodium phosphate-buffered saline, pH 7.4) was performed using a precision current source (digital midgard precision current source, Stoelting) as described previously (Hahn and Swanson, 2010 (link)). PHAL immunoreactive axons in the LHA confirmed the exclusively ipsilateral nature of vHP to LHA projections (Figure 6—figure supplement 1; representative vHP PHAL injection site, Figure 6).