Hanks balanced salt solution (hbss)

The uptake of AmB-MP at 3 × 10⁵ SCs /cm², cell concentration established on our previously published data [45 (link),46 (link)], was evaluated at four different concentrations—10 µg, 20 µg, 30 µg, and 40 µg/cm²—dispersed in HAM’S F-12 medium (Lonza, Verviers, Belgium) by exploiting the innate phagocytic capacity of SCs. Before loading, the exact amount of AmB-MP was weighed for all four concentrations, sterilized with UV light for 30 min, and suspended in 10 mL of HAM’S F-12 medium. Then, the four suspensions were sonicated and vortexed in the end to obtain a homogeneous dispersion by preventing the formation of lumps. Subsequently, the SCs were loaded and then incubated at 37 °C for 5 h [31 (link)]. Thereafter, the cell monolayer was detached by trypsin/ethylenediaminetetraacetic acid (EDTA) (Lonza, Verviers, Belgium) treatment at 37 °C for 8 min to promote the enzymatic reaction. After washing with 1 mL of HBSS (Sigma Aldrich, Milan, Italy), SCs viability was measured by an automated cell counter (INVITROGEN, Countess Automated Cell Counter, CA, USA) after trypan blue staining (Sigma–Aldrich, Milan, Italy) [47 (link)]. Finally, cell pellets were centrifuged at 1500× g for 6 min and stored at −80 °C.
Analysis was performed by extracting the drug from the pellets in methanol solution upon vortexing and sonication steps at room temperature (RT). After proper centrifugation (300× g, 5 min), the supernatants were recovered, properly diluted in methanol, and immediately submitted to HPLC analysis, as described above.
After determining the most suitable AmB-MP concentration, the uptake process was investigated over time at 5, 24, 72 h, and 7 days.

Mouse embryos were harvested following Cortical Primary Neuron Culture methods described above. Brains were dissected from embryos and stored in ice cold HBSS (Sigma Aldrich). Brains were cut using a razor blade down the midline evenly to separate hemibrains and each hemibrain was transferred into a 1.5 mL microcentrifuge tube containing neurobasal complete media used in cortical Primary Cortical Neuron Culture Maintenance described above. One hemibrain was subjected to 42 °C heat shock for 1 h while the complementary hemibrain was maintained at 37 °C for 1 h as a control. Samples were briefly centrifuged at 2000 g for 2 min to pellet hemibrain, then supernatant was carefully removed. The hemibrain pellets were immediately lysed in 100 μL of 1% SDS buffer (1% SDS in IP Lysis Buffer supplemented with 1X protease inhibitor, 1X phosphatase inhibitor, 100 mM N-Ethylmalamide), and homogenized with a pestle then boiled at 95 °C for 10 min. After boiling, 900 µL of ice-cold IP Lysis buffer was then added to the sample then samples were vortexed aggressively for 10 s and passed through a 18G insulin needle. Lysates were left on ice for 20 min with vortexing every 5 min for 10 s to ensure complete lysis. Next, the lysates were centrifuged at ~ 21000G for 20 min at 4 °C. Following the centrifugation, 1–3% of the supernatant was transferred into a microcentrifuge tube to be used as the Input sample. The remainder of the supernatants were equally divided (~ 450 uL each) into two separate 1.5 mL microcentrifuge tubes containing 10 μg of normal mouse IgG (Alpha Diagonstic 20008–250) or 10 ug of anti-SUMO2/3 clone [8A2] (Abcam ab8137) per sample. Samples were incubated overnight rotating at 4 °C. The next day, 50 μL of Protein G Dynabeads (Thermo Fisher Scientific) per sample were washed 3X in ice cold IP Lysis Buffer. The beads were resuspended in 50 μL IP Lysis Buffer per sample which was then added to each of the samples incubated with antibody. The samples were left to rotate at 4 °C for 45 min. The solution was added to a magnetic rack kept at 4 °C and the supernatant was collected for flowthrough to test immunodepletion or aspirated. The beads were washed 5X in ice cold IP Lysis buffer. Samples were eluted using 30uL (at 1 mg/mL) of synthetic SUMO2/3 peptide (Amino Acid Sequence: IRFRFDGQPI, synthesized through Genscript) per sample and incubating at 37 °C for 15 min at 900 rpm on a thermomixer. The input/flowthrough and immunoprecipitation samples were prepared with 4X Laemmli buffer containing 10% Beta-Mercaptoethanol and then boiled at 85 °C for 10 min. Samples were then analyzed by Western Blot using light chain specific secondary antibodies. Quantification of SUMOylation assays was performed through densitometry analysis using ImageLab (BioRad) to quantify the volume of the ~ 62 kDa anti-TDP-43 bands from the immunoprecipitation standardized to the loading control of the input. For quantification of changes in TDP-43 SUMOylation response, conditions were normalized to the 1 h sodium arsenite treatment.

HeLa cells were seeded into six-well plates, grown overnight to approximately 50% confluence, and transiently transfected with 1.5 μg/well of the pEYFPN1-SLC26A4 vector and 3 μl METAFECTENE PRO^® (Biontex, Munich, Germany), following the manufacturer’s instructions. This vector encodes SLC26A4 with the enhanced yellow fluorescent protein (EYFP) fused to its C-terminus (SLC26A4-EYFP). The medium was replaced 6–8 h after transfection, and cells were transferred on glass slides 56 h after transfection and processed 72 h after transfection.
Quantitative imaging was done as formerly described (Roesch et al. 2018 (link); Matulevicius et al. 2022 (link); de Moraes et al. 2016 (link)). Shortly, cells were fixed with 4% paraformaldehyde for 30 min, counterstained with 0.1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, thoroughly washed and imaged in Hank’s balanced salt solution (HBSS, Sigma-Aldrich). Imaging was performed with a Leica TCS SP5II AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with an HCX PL APO 63x/1.20 Lambda blue water immersion objective and controlled by the LAS AF SP5 software version 2.7.3.9723 (Leica Microsystems). Imaging parameters are given in the Additional file 2.

Pharmacological agents included the kainic receptor agonist kainic acid (Abcam and Tocris) and sodium salicylate (Sigma-Aldrich). A 10 mM kainic acid solution (volume of 4 µL) was applied at intact round window niche. The kainic acid solution was prepared in Hank’s balanced salt solution (HBSS, Sigma-Aldrich). No discernible effects of different solvents on the AVCN responses were observed (data not shown). sodium salicylate was dissolved in HBSS for local round window delivery at a concentration of 100 mM (volume of 4 µL) or dissolved in saline for IP injection at a dose of 200 mg/kg (volume of 0.3 mL).

Freshly excised testes from 2-week-old Holstein calves (Bos taurus) were obtained from Tockenham Corner Abattoir in Swindon, UK. The testes were immersed in basic medium and immediately transferred on ice to our laboratory, where they were processed as previously described [20 (link)]. The basic medium was prepared using sterile Hanks’ balanced salt solution (HBSS; Sigma-Aldrich, Missouri, USA) supplemented with 2% penicillin–streptomycin (Sigma-Aldrich), 0.1 M sucrose (Sigma-Aldrich), and 10 mg/ml bovine serum albumin (BSA; Thermo Fisher Scientific, Massachusetts, USA). Isolated ITTs were dissected into fragments approximately 3 × 3 × 3 mm³ in size. These fragments were then stratified into three experimental groups based on the cryopreservation technique utilized: controlled slow freezing (in which the rate of freezing was controlled), uncontrolled slow freezing (in which the rate of freezing was uncontrolled), and vitrification.