Anti foxa2

Cells were seeded on matrigel-coated glass coverslips and they were fixed for 20 min in ice in 4% paraformaldehyde (PFA, Sigma), solution in phosphate-buffered saline (PBS, Euroclone). Then, cells were permeabilized for 30 min in a blocking solution, containing 0.5% Triton X-100 (Sigma-Aldrich) and 10% donkey serum (Sigma-Aldrich), and incubated overnight at 4 °C with the primary antibodies in a blocking solution. Then, cells were washed with PBS and incubated for 1 h at room temperature with Hoechst and with secondary antibodies. The following antibodies were used: anti-OCT4 (1:100, Abcam), anti-NANOG (1:100, Abcam), anti-FOXA2 (1:300, Abcam), anti-NESTIN (1:300 Millipore), anti-TH (1:200, Immunological Sciences), anti-MAP2 (1:500, Immunological Sciences), anti-TOMM20 (1:300, Novus), anti-α-Synuclein (clone LB509, 1:100, Thermo), anti-GFP (1:500, Thermo), anti-α-Synuclein (phosphoS129, 1:300, Abcam), anti-TAU (1:500, Millipore), anti-LAMP1 (1:500, Abcam), anti-Synapsin-1 (1:500, Synaptic Systems), anti-SMI311 (1:500, BioLegend), anti GM130 (1:300, BD), anti-GRIM19 (1:300, Abcam). All the secondary antibodies used for the immunofluorescence staining are Alexa Fluor^TM.

MCF-7 cells plated on coverslips were washed with PBS, fixed in methanol, and blocked with 3% BSA in 1× PBS plus 0.02% Triton for 1 h at room temperature. Samples were then probed overnight with anti-FOXA2 (1:200; Abcam ab60721, UK) and anti-FOXP2 (1:200; Cell Signaling Technology #5337, USA). After three washes with PBS, cells were incubated at room temperature with FITC-labeled anti-rabbit IgG (1:1,000; Beyotime A0562, China) or Texas Red-labeled anti-mouse IgG (1:1,000; Invitrogen PA1-28626, USA) secondary antibodies for 1 h. Cells were then washed three times with PBS/Triton and mounted on slides with Fluoroshield with DAPI (Beyotime C1002, China). The samples were examined with a TE2000 fluorescence microscope (Nikon Instruments Inc., Japan) (200×).

NSCs (6.5 × 10⁴ cells/cm²) were grown on poly-ornithine/laminin-coated coverslips and differentiated into dopaminergic progenitors. Cells were fixed in paraformaldehyde (PFA, 4% v/v in PBS) for 20 min and permeabilized with Triton X-100 (0.1% v/v) in PBS for 5 min at room temperature. Cells were then incubated with the following primary antibodies diluted 1:200 in BSA (1% w/v in PBS): anti aS (Abcam), anti-Pax6 (Sigma-Aldrich), anti-Nestin (Abcam), anti-Lmx1A (Abcam), and anti-FOXA2 (Abcam), with a secondary fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G antibody (DBA; dilution: 1:500) or a secondary Alexa Fluor 568-conjugated goat anti-rabbit (ThermoFisher Scientific; dilution: 1:500). The cells were observed with a confocal microscope (Leica, Buffalo Grove, IL, USA).