Nb100 449

Cells lysates were prepared using lysis buffer (150 mM NaCl, 1% tritonX-100, 0.5 % sodium deoxycholate, 0.1% SDS, 50 mM tris pH 8) containing protease inhibitor and phosSTOP phosphatase inhibitor cocktail (Roche). Protein concentration was measured using BCA protein assay (Pierce) for western blot analysis. Primary antibodies were purchased from Santa Cruz for transferrin (sc-374441), TfR1 (sc-32272), DRD2 (sc-5303), DRD3 (sc-136170), DRD4 (sc-136169), DRD5 (sc-376088), TH (sc-25269), PAH (sc-271258), and DDC (sc-293287); from Cell Signaling Technology for SOX2 (#3728), CD44 (#3570), pSrc (#6943), Src (#2108), pSTAT3 (#9131), STAT3 (#9139), pERK (#9101 S), ERK (#9102 S), and PARP (#9542 S); from Novus Biologicals for HIF1α (NB100-449) and DRD1 (NBP2-66807); from Abcam for 4-HNE (ab46545), β-actin (ab6276), and HFE (ab133369). Secondary antibodies include HRP conjugated anti-mouse IgG (ab6728) from Abcam and anti-rabbit IgG (G-21234) from Innovative Research. Antibody concentration was 1:1000 for DRD1, DRD2, DRD3, DRD4, DRD5, TH, PAH, and DDC; 1:2000 for transferrin, TfR1, SOX2, CD44, pSrc, Src, pSTAT3, STAT3, pERK, ERK, PARP, HIF1α, and HFE; 1:3000 for 4-HNE; 1:5000 for β-actin and secondary antibodies.

PBMCs (250,000/coverslip) were added in 100μL PBS on poly-L-lysine pre-coated coverslips (BioCoat™) and left to adhere 30 min at 37°C. After attachment coverslips were washed in PBS and fixed 10 min at RT using 4% PFA. Cells were blocked using 3% BSA and antibody detection of CD4 (R&D systems, #AF-379-NA) and Alexa 647 conjugated CD8 (Abcam, #ab196193) performed prior permeabilization 10 min using 0.2% Triton x-100 and subsequent primary antibody incubation targeting HIF-1α (Novus Biologicals, #NB-100-449) and HIF-1β (Abcam, #ab2771). Secondary antibodies, Alexa Fluor 488 (Invitrogen, #A32731), Alexa Fluor 568 (Invitrogen, #A-11004), and Alexa Fluor 647 (Invitrogen, #A-21447) were incubated 1h at RT. Cells were counterstained using DAPI and mounted with Prolong Gold Antifade reagent (Thermofisher). Images were acquired using Nikon single point scanning confocal microscope with 60x/1.4 oil objective. All samples were analysed in two technical replicates and detection threshold was set using secondary antibody controls. Image analysis was performed in Imaris (Bitplane) using detection of Nucleus, Cell and Vesicles detection according to pipeline in Figures S4A and S4B. Graphical representation was performed in Imaris and statistical analysis was performed using Mann-Whitney U-test in Prism 8.4.3 (GraphPad Software) (significance level, p<0.05).