Supplementary Figure S1 from Reprogramming of TLR–Ferroptosis Signaling and Immunometabolic Pathways Overcomes Myeloid Suppression to Improve Checkpoint Blockade in Prostate Cancer

Tunable structural properties of ultrasmall PSMAi-PEG2-Cy5-C’ dots promote enhanced binding/uptake and potency in prostate cancer cell lines. (a) C’ dot-encapsulating deep red fluorescent dye Cy5 (1), surface functionalized with DFO chelator (2) and the PSMA targeting peptide, PSMAi (3). (b) Chemical structure of PSMAi-PEG2 peptide. (c) Cell binding of PSMAi-PEG-C’ dots and second generation PSMAi-PEG2-C’ dots in LNCaP cells. (d) FCS correlation curve and fit of PSMAi-PEG2-Cy5-C’ dots. (e) GPC elugram of PSMAi-PEG2-Cy5-C’ dots with Gaussian fit. (f) Competitive binding curves for various PSMAi-targeted C’ dot constructs versus PSMAi peptide in LNCaP cells. (g) Flow cytometry of concentration-dependent PSMAi-PEG2-Cy5-C’ dots particle uptake (4 h) in human and murine prostate cancer cell lines, expressed as mean fluorescence intensity (MFI). (h) Specific uptake of PSMAi-PEG2-Cy5-C’dots in LNCaP, Myc-CaP, and PC-3 cells without and with addition of anti-PSMA antibody by flow cytometry, expressed as MFI. (i) Differential PSMA expression in LNCaP, Myc-CaP, PC-3, and ID8 cells by western blot. (j) Confocal microscopy of PSMAi-PEG2-Cy5-C' dot uptake in LNCaP cells (Red: particle, Green: 70 kDa FITC-dextran, Blue: DAPI). All numerical data are presented as mean ± s.e.m. (n = 3); scale bar: 5 um. Unpaired t-tests were performed in (c) and (h). **p < 0.01, ***p < 0.005, ****p < 0.001, ns – not significant.