Sc 281692

Murine SPZ collected from caput and cauda epididymis were dried on slides as above reported and fixed in 4% paraformaldehyde (sc-281692; Santa Cruz Biotechnology, Heidelberg, Germany) for 20 min at RT and then permeabilized with 0.2% Triton X-100 (X100; Sigma-Aldrich, Milano, Italy). Blocking was carried out with 10% of donkey serum (ab7475; Abcam, Cambridge, UK) for 30 min at RT and then cells were separately incubated with different primary antibodies [IZUMO1 (ab211623) from Abcam Cambridge, UK; PNA (L21409) from Invitrogen, Milano, Italy; TSSK6 (sc-514076) from Santa Cruz Biotechnology, Heidelberg, Germany] overnight at 4°C. Following three washes in DPBS (1X), a fluorescein isothiocyanate (FITC) conjugated was used as secondary antibody (711-095-152; Jackson ImmunoResearch, Cambridge, UK) for 1 h at 37°C. Nuclei were labeled with DAPI (D9542; Sigma-Aldrich, Milano, Italy), while F-actin was labeled with phalloidin (21834; Thermo Fisher Scientific, USA). All samples were analyzed under an optical microscope (Leica DM 5000 B + CTR 5000) with a UV lamp. Densitometric analysis of immunofluorescence was performed with ImageJ Software (version 1.53 g) and adjusted relatively to DAPI fluorescence intensity.

Embryoid body (EB) formation was performed by plating single cells in AggreWell800 (Stem Cell Technologies, 34811, Vancouver, Canada) in medium containing Knockout DMEM F-12 (Gibco,12660-012, Grand Island, NY, USA), 20% Knockout Serum (Gibco, 10828-028, Grand Island, NY, USA), non-essential amino acids-1x (Gibco, 11140-050) and Glutamax-1x (Gibco, 35050-061). Medium was changed 48 hours later and thereafter till day 7 in an every-other-day mode. On day 7, EB spheres were collected and plated onto plates coated with 0.1% Gelatin (Millipore, ES-006-B) and medium containing Dulbecco’s modified Eagle’s medium (DMEM) (Gibco 11965-092), 20% fetal bovine serum (FBS) (Gibco, SH30071), non-essential amino acids-1x (Gibco, 11140-050) and Glutamax-1x (Gibco, 35050-061). Medium was changed every other day for 7 days. On day 7 post plating, EBs were fixed with 4% paraformaldehyde (Santa Cruz, SC-281692, Dallas, TX, USA), and stained for detection of cells of the three germ layers with antibodies for the following antigens: SOX17 (R&D Systems, AF1924, Minneapolis, MN, USA) for endoderm, PAX6 (BioLegend, PRB-278P, San Diego, CA, USA) for ectoderm and SMA (Millipore, CBL171) for mesoderm.

Sperm cells collected from caput/cauda epididymis of CTRL (n = 4) and HFD (n = 4) mice, dried on slides as reported above, were used for immunofluorescence analysis. In detail, sperm cells were fixed with a fix solution consisting of 4% paraformaldehyde (sc-281692; Santa Cruz Biotechnology, Heidelberg, Germany) for 20 min at RT. Then, a permeabilization step was carried out by using 0.1% Triton X-100 (X100; Sigma-Aldrich, Milano, Italy). Blocking was conducted with 10% of donkey serum (ab7475; Abcam, Cambridge, UK) for 30 min at RT, and then sperm cells were incubated with anti-FUS antibody (PA5-52610; Invitrogen, Milano, Italy) overnight at 4 °C. A negative control consisting of primary antibody omission and an isotype control by using the same isotype (IgG) at the same concentration of FUS primary antibody (rabbit IgG, polyclonal isotype control (ab37415) from Abcam, Cambridge, UK) were carried out (Figure S6). Slides were washed 3 times in Dulbecco’s PBS (DPBS, 1X) until the addition of Texas Red conjugated antibody (Jackson ImmunoResearch, Cambridge, UK) for 1 h at 37 °C. Nuclei visualization was performed, incubating the slides with DAPI solution (D9542; Sigma-Aldrich, Milano, Italy). Immunofluorescence analysis was conducted under an optical microscope (Leica DM 5000 B + CTR 5000) with a UV lamp.

Cells were seeded on Chamber slide (C6932, Sigma-Aldrich) with the seeding density of 30 × 10³ per well. The cells were allowed to attach to the wells overnight before transfecting with siRNA. After transfection with the siRNA for 72 h, slides were incubated with 4% paraformaldehyde (sc-281692, Santa Cruz, Heidelberg, Germany) 20 min at room temperature; PBS 5 min 3 times, 0.1% Triton X-100 (11332481001, Sigma-Aldrich, Saint Louis, MO, USA) 10 min at room temperature, PBS 5 min 3 times, 3% BSA block 1 h at 37 °C. After that, the slides were incubated with anti-MUC2 antibody (ab11197, Abcam, Cambridge, UK, 1:500) for 3 h at 37 °C, PBS 5 min 3 times, and with anti-mouse IgG (ab150113, Abcam, Cambridge, UK, 1:1000) for 1 h at 37 °C, PBS 5 min 3 times. The nucleus was dyed with 1 ug/mL DAPI (62248, Thermo Scientific) for 2 min at room temperature, PBS 2 min 3 times. The slides were mounted with Limonene Mounting Medium (ab104141, Abcam, Cambridge, UK,) and covered by coverslips (7695031, Th. Geyer, Renningen, Germany). Confocal microscopy was performed with Leica SP8, and images were saved as TIFF file formats. For each group in every experiment, we randomly selected three images with similar numbers of cells and uniform background.

MRC-5, A549, and AGS cells were treated with the pH probe, CS-1. The working solution was prepared to a concentration of 20 μM in phosphate-buffered saline (PBS) and incubated at 37 °C for 30 min. Cells were washed with PBS and fixed with 4% paraformaldehyde (sc-281692; Santa Cruz Biotechnology, USA) for 10 min before fluorescent imaging. For the CS-1 and lysosome co-staining study, the cells were incubated with CS-1 (20 μM) and LysoTracker® (100 nM, LysoTracker® Red DND-99 (L7528), Molecular Probes, Eugene, OR, USA) at 37 °C for 30 min. For the CS-1 and mitochondria co-staining study, the cells were incubated with a solution consisting of CS-1 (20 μM) and MitoTracker® (250 nM, MitoTracker® Red CMXRos (L7512), Molecular Probes) at 37 °C for 30 min. To achieve membrane staining, cells were incubated with a solution of CS-1 (20 μM) and CellMaskTM Deep Red Plasma Membrane stain (250 nM, C10046, Molecular Probes) at 37 °C for 30 min prior to imaging. For the CS-1 and ER-Tracker® (E34250; Molecular Probes) co-stain studies, a solution of CS-1 (20 μM) and ER-Tracker® in cell culture medium was used with the test cell culture held at 37 °C for 30 min.