Lectin pna

All reagents unless otherwise noted were from Sigma. FluoZin™-3, AM (FZ3; zinc probe) from ThermoFisher (F24195) was reconstituted with DMSO to a stock solution of 500 μM. Lectin PNA (A. hypogea/peanut agglutinin) conjugated to Alexa Fluor™ 647 (PNA-AF647) was from Invitrogen™ (L32460). Fluo-4 NW (calcium probe) from ThermoFisher (F36206) was reconstituted using kit provided assay buffer. Hoechst 33342 (H33342) from Calbiochem (382065) was reconstituted with H₂O to a stock solution of 18 mM. PI from Acros Organics (AC440300010) was reconstituted with H₂O to a stock solution of 1 mg mL⁻¹. Proteasomal inhibitors were from Enzo Life Sciences: MG132 (BML-PI102) was reconstituted with DMSO to a stock solution of 20 mM; epoxomicin (Epox, BML-PI127) was reconstituted to a stock solution of 20 mM (using MG132 stock); and clasto-lactacystin β-Lactone (CLBL, BML-PI108) was reconstituted with DMSO to a stock solution of 5 mM. Zn-chelator TPEN from Tocris (16858-02-9) was resuspended with 1:100 EtOH:H₂O to a stock solution of 1 mM. Bovine serum albumin was from Sigma (A4503). Anti-phosphotyrosine antibody, clone 4G10^® was from EMD Millipore (05-321).

Sperm capacitation and acrosome reaction were measured using Arachis hypogaea (peanut) agglutinin (Lectin PNA; Molecular Probes, Eugene, USA) as previously described [47 (link)]. Sperm were incubated in G-IVF + 10% HSA for 1 h in 6% CO₂ and 5% O₂ at 37 °C, washed in PBS, and incubated in Lectin PNA Alexa 594 antibody (1:100) in PBS for 45 min. Samples were stained with Hoechst to identify sperm nuclei. A minimum of 200 sperm were counted per sample and were classified as capacitated, non-capacitated, or acrosome reacted.

Testes were fixed with 4% paraformaldehyde (PFA) diluted with PBS and then were gradient dehydrated with 5, 10, 12.5, 15, and 20% sucrose in PBS. After embedded in 50% Tissue-Tek O.C.T. compound (Sakura Finetek, 4583) in 20% sucrose on liquid nitrogen, the 5μm cryosections were spread onto Superfrost Plus slides and stored in −80 °C. After antigen retrieval by 0.01 mM Citrate or Tris-EDTA (PH = 9.0) and washing three times in PBS (10 min/wash), sections were incubated with primary antibodies (Supplementary Table 4) in a humidified chamber at 4 °C overnight. The slides were washed three times with PBS and then incubated with a secondary antibody (Supplementary Table 4) at RT for 1 h. After the primary SC was cultured, the sections were permeabilized with 0.2% Triton X-100 at RT for 5 min and washed with PBS. Then the sections were incubated with primary and secondary antibodies, mounted with DAPI (H1200, Vector laboratories), and photographed. For F-ACTIN staining, cryosections were rinsed with PBS and permeabilized by 10% Triton X-100 at RT for 30 min. Then the sections were incubated with phalloidin (Molecular Probes) and Lectin PNA (Molecular Probes) at RT for 1 h. After washing three times with PBS (10 min/wash), the slides were counterstained with DAPI and photographed with a fluorescence microscope (Olympus, Japan).

Polyclonal antibodies against VCP were developed in our laboratory as described previously36 (link). Anti-CHOP antibody was purchased from Santa Cruz Biotechnology (CA, USA); anti-M-opsin and anti-actin antibodies were purchased from Millipore (MA, USA); anti-cleaved caspase-3, anti-phospho-mTOR (Ser 2481), anti-phospho-mTOR (Ser 2448), and anti-mTOR antibodies were purchased from Cell Signaling (MA, USA). Lectin PNA was purchased from Molecular Probes (MA, USA).

Immunofluorescence and confocal microscopy were used to detect the expression and localization of OR51E2 in human spermatozoa and HeLa cells. In HeLa cells (kindly provided by Dr. Fabiola Moretti), we overexpressed FlagRho-OR51E2 (kindly provided by Dr. Jennifer Pluznick) to verify the specificity of the α-OR51E2 antibody. Human sperm cells, purified as described above, were treated with PA 50 mM or Ringer’s solution (CTR) for 10 min at room temperature. Then, the sperm cells were washed and smeared on polylysine slides, and then allowed to air-dry. Then, HeLa and sperm cells were fixed in 3.7% formaldehyde for 15 min at room temperature (RT) and subsequently treated with 5% BSA to block nonspecific binding. Then, both cellular models were treated with polyclonal anti-OR51E2 (1:200) overnight (Aviva System Biology, San Diego, CA, USA) antibody, washed three times by PBS, and incubated for 45 min with goat Alexa Fluo-488 anti-rabbit IgG (A11034 Molecular Probes, Thermo Fisher, Waltham, MA, USA). For the HeLa cells, DNA was stained with Prolong Gold DAPI (P36935 Molecular Probes), while in sperm cells, acrosomal compartment was counterstained with lectin PNA (L32459 Molecular Probes) following the manufacturer’s instructions.

Sulforaphane (Cas No: 4478-93-7), Annexin V FITC assay kit and JC-1 were purchased from Cayman Chemical Company (Cayman Chemical, Michigan, Ann Arbor, USA). Lectin PNA and H2DFCDA were purchased from Thermo Fisher. All other chemicals used in the study were purchased from Sigma (Sigma, Aldrich Chemical Company, Burlington, Massachusetts, USA).