Bradford assay

Cells were lysed at 4 °C for 15 min in ice cold lysis buffer [150mMNaCl, 50 mM Tris pH 7.5, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.5% sodium deoxycholate containing PhosSTOP phosphatase inhibitors and a complete protease inhibitor mixture (Roche Life Science)], and centrifuged at 20,000 g. Protein concentration was estimated in the supernatant by Bradford assay (Applichem). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Maine Manufacturing). For phospho-(Ser129)-αSyn detection, the membrane was heated at 65 °C overnight in PBS. Nonspecific binding sites were blocked in TBS/ 0.1% Tween 20/ 5% skimmed milk for 1 hour at 20 °C followed by overnight incubation with primary antibodies diluted in the same buffer. Incubation with appropriate HRP-conjugated secondary antibodies (Thermo) was for 2 hours at room temperature and protein bands were visualized using the Clarity Western ECL Substrate (BIO-RAD). Densitometric analysis was performed using ImageJ software (NIH). All blots were derived from the same experiment and were processed in parallel. Original unprocessed blots are shown in Supplementary Fig. 7.

An enzymatic activity assay was used to measure the levels of ALP activity, an early osteogenic marker produced from the pre-osteoblastic cells cultured on different materials. The ALP activity method is based on the ability of the enzyme to use para-nitrophenyl phosphate (pNPP) as a substrate and hydrolyze it into 4-nitrophenol (pNP) and a phosphate group [58 (link)]. Cells were cultured for 7, 14 and 21 days in complete medium, lysed in 200 μL lysis buffer (0.1% Triton X-100 in 50 mM Tris-HCl pH 10.5) after rinsing with PBS, and subjected to two freeze-thaw cycles. Then, 200 μL of a solution containing 2 mg/mL p-nitrophenyl phosphate (pNPP) (Sigma-Aldrich, Burlington, MA, USA) in 50 mM Tris-HCl and 2 mM MgCl2 at pH 10.5 were added to each sample and left at room temperature for 60 min. The reaction was stopped with the addition of 50 μL of 1 N NaOH. The absorbance of the reaction product p-nitrophenol (pNP) was measured at 405 nm in a spectrophotometer (Synergy HTX Multi-Mode Microplate Reader, BioTek, Winooski, VT, USA). The absorbance values were translated to pNP concentrations by means of a calibration curve, and these were normalized to cellular protein determined using the Bradford assay (Applichem, Darmstadt, Germany) in the cell lysates. Quadruplicates of three independent experiments were analyzed (n = 12).

ALP activity was used as an early marker of osteogenesis after days 14/1, 28/14, and 42/28 in mono- and co-cultures of hBM-MSCs, based on a previously described protocol [39 (link)]. Briefly, at the specific time points, cells were rinsed twice with PBS and lysed with 100 μL lysis buffer (0.1% Triton-X in dH₂O) and were subjected to two freeze–thaw cycles. An aliquot of 50 μL was mixed with 50 μL of ALP reaction solution (2 mg/mL p-nitrophenyl phosphate (pNPP, Sigma, Burlington, MA, USA) in 50 mM Tris-HCl and 2 mM MgCl₂ at pH 10.5). Afterwards, samples were left for 1 h at 37 °C and the absorbance of the reaction was measured at 405 nm in a spectrophotometer (Synergy HTX Multi-Mode Microplate Reader, BioTek, Winooski, VT, USA). The absorbance values were translated into pNP concentrations, with the use of a calibration curve constructed from known pNP values. Normalization was performed to total cellular protein, determined using the Bradford assay (Applichem, Darmstadt, Germany) in the cell lysates.