Cell Lines and Growth Conditions

Enteroendocrine STC-1 (CRL-3254) cell line was obtained from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Corning) containing 4.5 g/L glucose, 2 mM l-glutamine, 10% (v/v) fetal bovine serum (Life Technologies), penicillin (100 U/mL), and streptomycin (100 μg/mL, Gibco). Cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. Cells were serially passaged using 0.25% Trypsin–EDTA (Gibco). Cells were seeded onto two 8-well glass slides (Ibidi) at a density of 1 × 10⁵ viable cells/well and incubated for 48 h. Then, the growth medium was replaced with fresh medium supplemented with either sodium nitrate, sodium nitrite, or vehicle. Cells were incubated for another 24 h before fixation.

Fixation was performed using 10% formalin (Fisher Scientific, 22-170-402) for 20 min at RT. Cells were then permeabilized for another 20 min at RT using 0.1% Triton X-100 (Bio-Rad, 1610407) in phosphate-buffered saline (PBS), pH 7.4. After discarding the permeabilization solution, wells were washed with PBS, twice. Samples were then blocked for 1 h at room temperature in PBS containing 5% Normal Goat Serum (Thermo Scientific, 50197Z) and 0.2% bovine serum albumin (BSA; Fisher Scientific, 501613336) followed by washing three times using PBS. Subsequently, the glass slide chamber was incubated at 4 °C overnight with anti-α-syn aggregate primary antibody (Abcam, ab209538; 1:250 dilution), isotype control (Abcam, ab172730; 1:566 dilution), or vehicle. The chambers were then washed with PBS followed by an incubation with anti-goat Alexa Fluor-488 secondary antibody (Abcam, ab150077; 1:500 dilution) for 1 h at room temperature in the dark. After washing three times, ∼10 drops of VECTASHEILD PLUS Antifade Mounting Medium with DAPI (Vector Laboratories, H-1900) were added to each well. All demarcation and staining steps were performed at RT, but the chamber slides were stored at 4 °C in the dark.

Images were acquired under identical conditions using a Zeiss Elyra 7 super-resolution microscope with a 63× oil immersion lens. Images were collected using 405 and 488 nm laser lines for excitation; emission filters used were BP 420-480 (DAPI) and BP 495-550 (AlexaFluor488). Sections were imaged in the absence of primary antibodies, and images were captured at the same gain as images with the primary antibody. No endogenous tissue fluorescence was observed in the absence of primary antibodies. Z-stack images were obtained and processed using SIM² scaled to raw image. Quantification of the fluorescence signal for each sample was determined by obtaining the mean intensity of the maximum intensity projection for each image given by Zen Black 3.0 software. The number of cells in each image was counted, and mean intensity per cell was calculated. For each biological sample, approximately 30–40 cells were evaluated. The background fluorescence signal was accounted for each replicate by subtracting the mean intensity per cell for the respective untreated sample. Results are expressed as arbitrary units (a.u.) of mean intensity per cell.