Uchl1

Reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Antibodies for mTOR, Rictor, UCHL1, phospho-NF-kB p65, phospho-Akt^Ser473 were purchased from Cell Signaling Technologies (Danvers, MA), β-actin and NF-kB from Invitrogen (Carlsbad, CA), goat anti-mouse IgG (Horseradish Peroxidase) from Jackson ImmunoResearch Laboratories. The anti-human goat NAMPT pAb was custom-generated as previously described^{10 (link),11 (link)}. The eNAMPT-neutralizing humanized mAb was provided by Aqualung Therapeutics (Tucson, AZ) as previously described^{11 (link)}. See Supplemental Materials and Methods for more details.

5 nm AuNPs
functionalized with tannate, citrate, and PVP, all purchased from
NanoComposix (San Diego, CA, USA), were used for this study. Phosphate-buffered
saline (PBS), F-12 nutrimix media, penicillin/streptomycin, 0.25%
trypsin-EDTA, fetal bovine serum (FBS), and RPMI 1640 were purchased
from Thermo Fisher Scientific (Paisley, UK). CytoTox 96 nonradioactive
cytotoxicity and GSH/GSSG-Glo luminescence assay kits were acquired
from Promega (Southampton, UK). Menadione, staurosporine, dimethyl
sulfoxide (DMSO), tris-base, glycine, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide), dichlorofluorescin diacetate (DCFDA), and sodium chloride
were purchased from Sigma-Aldrich (Dorset, UK). Bovine serum albumin
(BSA), acrylamide/bis-acrylamide, and ponceau were bought from Alfa
Aesar (Lancashire, UK). The Alexa fluor 488 Annexin V/PI cell death
reagent kit was purchased from Thermo Fisher Scientific (Paisley,
UK). The PVDF membrane and ECL were purchased from GE HealthCare (Buckinghamshire,
UK). USP7, USP8, USP10, UCHL-1, AKT, p-AKT, mTOR, GSK-3β, β-catenin,
p-β-catenin, GAPDH, caspase-3, caspase-9, and PARP primary antibodies
were purchased from Cell Signaling Technology (CST) (London, UK).

We lysed cells and sEV with 2% SDS, 2 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The lysates were centrifuged and the supernatants were separated by SDS-polyacrylamide gel electrophoresis and blocked with BSA. After blotting onto a nitrocellulose membrane (Bio-Rad Laboratories, UK), the membrane was incubated with TSG101 (Abcam, cat. #: ab83, 1:1,000), CD63 (Abcam, cat. #: ab193349, 1:1,000), CD9 (Abcam, cat. #: ab223052, 1:1,000), Alix (Abcam, cat. #: ab76608, 1:1,000), Runx2 (Cell Signaling, cat. #: 12556, 1:1,000), FN1 (Proteintech, cat. #: 15613-1-AP, 1:1,000), FOXO4 (Proteintech, cat. #: A21535-1-AP, 1:1000), and CBL (ABclonal, cat. #: A7881, 1:1000), β-actin (Cell Signaling Technology, cat. #: 4970S, 1:4000), GAPDH (Cell Signaling Technology, cat. #: 2118, 1:5000), UCHL1 (Cell Signaling Technology, cat#13179, 1:1000), MAP2 (Cell Signaling Technology, cat#: 4542 S, 1:1000). Secondary antibodies for western blot anti-rabbit IgG, (cat# 7074, dilution 1:2000) from Cell Signaling Technology (MA, USA), anti-mouse IgG, (cat# A9044, dilution 1:2000) from Sigma Aldrich (MO, USA). The membrane was then analyzed using specific antibodies and visualized by an enhanced chemiluminescence^{50 (link)} kit (Amersham Biosciences, Piscataway, NJ, USA).

UCH-L1^+/+, UCH-L1^+/− and UCH-L1^−/− mice testis tissues were collected at post-natal days 0, 7, 17, 30, 60, 90 and 120, fixed in Bouin’s solution or 4% paraformaldehyde (PFA) and embedded in paraffin. Serial paraffin-embedded sections were prepared and stained with hematoxylin and eosin (H&E) for histology examination. Immunohistochemistry was performed on PFA-fixed embedded sections following deparaffination, rehydration and heat-induced antigen retrieval steps. The sections were incubated with primary antibodies against PLZF (HPA001499, Sigma,), LIN28A (8641, Cell Signalling Technologies, Danvers, MA, USA), UCH-L1 (13179, Cell Signalling Technologies) and SOX9 (sc-17341, Santa Cruz Biotehnologies, Dallas, TX, USA,), followed by an incubation with species-specific secondary Alexa Fluor 488, 555 or 647 antibodies. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (VECTH1200, BioLynx, Brockville, ON, Canada). At least 50 cross-sections were processed per sample from 3 tissue sections 50 µm apart. For comparison purposes, PFA-fixed archival testis sections from other mammalian species—adult human, adult buck, adult cat, adult rat, adult sheep, juvenile porcine, juvenile goat, juvenile horse and juvenile monkey—were also processed and analyzed.

AuNPs of sizes 5, 10, and 80 nm coated with citrate were used in this study. They were all purchased from NanoComposix (San Diego, CA, USA). Phosphate-buffered saline (PBS), F12 nutrimix media, penicillin/streptomycin, 0.25% trypsin-EDTA, fetal bovine serum (FBS), and RPMI 1640 were purchased from Thermo Fisher Scientific (Paisley, UK) and the CytoTox 96^® non-radioactive cytotoxicity assay was from Promega (Southampton, UK). Chlorpromazine hydrochloride, wortmannin, nystatin, simvastatin, methyl-β-cyclodextrin (MβCD), menadione, staurosporine, dimethyl sulfoxide (DMSO), tris-base, glycine, and sodium chloride were purchased from Sigma Aldrich (Dorset, UK). Bovine serum albumin (BSA), acrylamide/bis-acrylamide, 4% paraformaldehyde, and ponceau were bought from Alfa Aesar (Lancashire, UK). Alexa fluor 488^® Annexin V/PI cell death and CellRox deep red reagent kits were purchased from Thermo Fisher Scientific (Paisley, UK). The PVDF membrane and ECL ™ were bought from GE HealthCare (Buckinghamshire, UK). USP7, USP8, USP10, UCHL-1, caspase-3, caspase-9, and PARP primary antibodies were purchased from Cell Signaling Technology (CST) (London, UK).

Cells were grown on glass coverslips and then fixed with 4% paraformaldehyde. After a PBS wash, the cells were permeabilized using 0.1% Triton X-100, incubated in a blocking solution (PBS with 3% bovine serum albumin), and further incubated overnight at 4 °C with rhodamine phalloidin (Cytoskeleton, Denver, Colorado, USA) and the primary antibody to UCHL1 (Cell Signaling Technology). The fluorescent conjugated secondary antibody was Alexa Fluor 488 (Invitrogen, Carlsbad, California, USA), and DAPI (Sigma Aldrich, St Louis, MO) was used as a nuclear counterstain for 10 min. The coverslips were finally mounted onto slides with fluorescent mounting medium and immediately observed via confocal microscopy.