Mounting medium with dapi

To determine miPB-IPC’s multipotency and their neuronal cell differentiation (Figure S1), miPB-IPC were treated with 100 ng/mL neuronal growth factor (NGF, R & D Systems) + human neuronal stem cell growth medium (iXCells Biotechnologies, San Diego, CA, USA) for 3–5 days, in 24-well tissue culture-treated plates, at 37 °C in 5% CO₂. The differentiated cells were characterized by immunocytochemistry [29 (link)] with mouse anti-human tyrosine hydroxylase monoclonal Ab (mAb, Clone LNC1, Catalogue # MAB 318, at 1:100 dilution) and rabbit anti-Synapsin I polyclonal Ab (Catalogue # AB1543, at 1:100 dilution) (EMD Millipore, Temecula, CA, USA). The FITC-conjugated AffiniPure donkey anti-mouse 2nd Ab and Cy3-conjugated AffiniPure donkey anti-rabbit 2nd Ab were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Isotype-matched IgG served as negative control for immunostaining. After covering with Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA), cells were photographed with Nikon A1R confocal microscope on Nikon Eclipse Ti2 inverted base.

Evans blue, methamphetamine (METH) and MLA were purchased from Sigma-Aldrich (St. Louis, MO). Dynabeads M-450 Tosylactivated was purchased from Invitrogen (Carlsbad, CA). Ulex europaeus I (UEA I) lectin and mounting medium with DAPI were purchased from Vector (Buringame, CA). The Gp41 ectodomain peptide (gp41-I90) was prepared as described previously [9 (link), 11 (link)]. A senescence β-Galactosidase Staining Kit (Cat#9860) was bought from Cell Signaling Technology. A Fluoro-Jade B (FJB) staining kit was obtained from Histo-Chemo Inc. All primary antibodies (Ab) were purchased from the commercial sources: a rabbit anti-α7 nAChR Ab from Genescript (Piscataway, NJ); Anti-NF-κB/p65 mAb from Cell Signal Technology (Cell Signal Technology, #3033); an antibody against dimethyl-histone H3 (Lys9) from Millipore (Millipore, cat #.07–212), an anti-mouse CD146 Ab FITC-conjugated and a mouse anti-neuron (NeuN) Ab from eBiosciences (San Diego, CA); a mouse anti-CD44 Ab (sc-7297); a rabbit anti-CD54 Ab (ICAM-1, 250593) from Abbiotec (San Diego, CA); a rabbit anti-β-actin (sc-7210) and an anti-GFAP Ab from Santa Cruz Biotechnology (Santa Cruz, CA); an anti-mouse CD146 Ab FITC-conjugated from Biolegend (San Diego, CA), and a rabbit anti-S100B Ab from BD Biosciences.

Sperm and oocytes were retrieved as described above. Sperm were capacitated in HTF medium (ARK Resource) at 37°C for 1 h. Sperm were collected 2 h after insemination with cumulus oocyte complex, smeared on a slide glass, and air-dried. Control sperm were collected an additional 2 h after incubation with HTF medium. Sperm were fixed with ethanol at -20°C for 10 min and incubated with 10% FBS at RT for 30 min, incubated with anti-IZUMO antibody (1:200, 73–045, B-bridge) at RT for 1 h, and then incubated with Goat anti-Rat IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate (Thermo Fisher, A-11006). VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, H-1200) was used for nuclear counterstaining. We counted sperm stained only in the acrosomal cap as acrosome unreacted, and sperm stained in the equatorial or whole head as acrosome reacted [25 (link)]. We counted at least 100 sperm.

MDCK cells were seeded at 5 × 10⁴ cells per coverslip one day before transfection of a plasmid encoding the indicated gene using Lipofectamine LTX according to the manufacturer’s instructions. Nucleozin was added on the next day at a concentration of 1 μM. Coverslips were harvested at 8 hours post-drug addition and fixed using 3.7% formaldehyde in PBS for 10 min. Cells were then permeabilized using 0.1% Triton X-100 for 4 min and blocked with 10% FBS in PBS for 30 min before incubation with mouse primary antibody against NP (Millipore) in 1% FBS in PBS for 1 h at 37 °C. Coverslips were then incubated with rabbit secondary antibody against mouse (AF488; Invitrogen) in 1% FBS in PBS for 1 h at 37 °C. Coverslips were placed “face down” onto a drop of Mounting Medium with Dapi (Vectashield) on a microscope slide before sealing of the coverslip with nail polish. Fluorescent images were obtained using a Carl Zeiss LSM710 upright laser scanning confocal microscope with a Plan Apochromat 63 × 1.4 oil immersion objective (The University of Hong Kong Li Ka Shing Faculty of Medicine Faculty Core Facility).

Cells were seeded onto coverslips and treated with selenite for 24 h. After fixing with 5% paraformaldehyde for 10 min, the cell membranes were permeabilized with 0.2% Triton for 30 min. After blocked with 1% BSA for 1 h to avoid nonspecific binding, cells were stained with primary antibodies at 4 °C overnight and with Cy3- or FITC-conjugated secondary antibodies for 1 h at room temperature. The slides were preserved using Mounting Medium with DAPI (Vectashield, Burlingame, CA, USA). Images were acquired using an Olympus laser scanning confocal FV1000 microscope (Olympus, Tokyo, Japan) and analyzed with Olympus Fluoview software.

Immunofluorescence analysis was done as we previously described [50 (link), 51 (link)]. Cells were fixed in 100% methanol for 15 min at room temperature. For staining, samples were permeabilized for 15 min in freshly prepared PBS containing 0.25% Triton-X100, then blocked for 1h in 5% donkey serum, 0.1% fish gelatin, 0.2% Tween-20 and PBS. Samples were then incubated in 37°C water bath for 1 h with 1 mg/ml of primary antibody diluted in blocking solution. Samples were transferred to a 1:500 dilution of Goat anti-mouse IgG Rhodamine (Thermo Fisher Scientific, Waltham, MA) or Goat anti-rabbit IgG Fluorescein in blocking solution and incubated for in 37°C water bath for 1 h. Cells were mounted on a glass slide with mounting medium with DAPI (Vectashield, Burlingame, CA) and visualized with an inverted light microscope (Olympus IX81 and CellSens Dimension software, Center Valley, PA).