Avanti j 20xpi

The produced melanin was purified in a two-step process by acidic precipitation. A 5 M HCl solution was added to 400 mL of supernatant to reach pH 1.5, according to previously reported protocols [14 (link),21 (link)]. The samples were kept at 4 °C overnight and then centrifuged at 4 °C and 5000 rpm for 20 min (Avanti J-20XPI, Beckman Coulter, Brea, CA, USA). The precipitated melanin was then washed three times with MilliQ water, recovered each time by centrifugation as described above, and then dried at room temperature. A portion of the precipitated dried melanin (about 15.0%) was then further washed with a 5 M HCl solution under stirring conditions and at room temperature for 5 h, using a slightly modified protocol [12 (link),20 (link)]. This wash was performed to remove the eventual presence of proteins and nucleic acids bonded to the pigment. The sample was then centrifuged at 4 °C and 4500 rpm for 20 min (Avanti J-20XPI, Beckman Coulter, Brea, CA, USA), and the residual precipitated melanin was washed again three times with MilliQ water, dried as described above, and further used for characterization.

Streptomyces nashvillensis DSM 40314 was purchased from DSMZ (Braunschweig, Germany) and initially grown in a 1-L shake flask containing 200 mL of GYA medium (glucose (20.0 g/L), yeast extract (20.0 g/L), (NH₄)₂SO₄ (2.0 g/L), NaH₂PO₄·H₂O (5.8 g/L), Na₂HPO₄ (8.2 g/L) at pH 7.0), at 28 °C and under constant agitation of 250 rpm in a rotary air shaker (ISF-1-W, Kühner, Birsfelden (Basel), Switzerland) [25 (link)]. After 48 h of growth, the culture was centrifuged at 4 °C and 5000 rpm (Avanti J-20XPI, Beckman Coulter, Brea, CA, USA) and then resuspended in fresh GYA medium with 20% (v/v) glycerol to prepare cell stock solutions, according to a previously reported procedure [25 (link)]. The stocks were then stored at −80 °C. Bacterial growth was carried out in GEM III N medium at different pH conditions (6.0 or 7.0) (glucose (12.0 g/L), yeast extract (6.0 g/L), malt extract (30.0 g/L), with a buffer phosphate made of NaH₂PO₄·H₂O (11.9 g/L), Na₂HPO₄ (1.9 g/L) for pH 6.0 or NaH₂PO₄·H₂O (5.8 g/L), and Na₂HPO₄ (8.2 g/L) for pH 7.0) [25 (link)]. The media, devoid of glucose and Na₂HPO_4, was sterilized by autoclaving at 120 °C for 20 min (ALFA-junior, PBI International, Milano, Italy). Solutions of glucose and Na₂HPO₄, sterilized by filtration with 0.22 µm membranes (Merck Millipore, Guyancourt, France), were added later to the autoclaved media.

Postmortem SVZ human brain tissue samples were thawed on ice and transferred into a 1.5 mL Eppendorf tube. Tissue was homogenized in nuclear extraction buffer (0.32 M Sucrose, 10 mM Tris–HCl pH8.0, 5 mM CaCl₂, 3 mM MgCl₂, 1 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100) using a disposable pellet pestle (∼30 strokes).
Nuclei were isolated by using a discontinuous sucrose gradient. The homogenized tissue solution was transferred into a 50 mL centrifuge tube (3115-0050, Thermo Fisher Scientific) and a sucrose solution (1.8 M sucrose, 10 mM Tris–HCl pH8.0, 3 mM MgCl₂ and 1 mM DTT) was added at the bottom to form the gradient. Samples were centrifuged at 75600 × g at 10°C, for 1 h in a Beckman Coulter centrifuge Avanti J-20 XPI with a fixed angle JA-25.50 rotor. Nuclei were stained with a mouse anti-neuronal nuclei (NeuN) Alexa Fluor 488 conjugated monoclonal antibody (Alexa488NeuN antibody) (MAB377X, Millipore) and DAPI (D1306, Thermo Fisher Scientific).