Supplementary Figure S2 from Reprogramming of TLR–Ferroptosis Signaling and Immunometabolic Pathways Overcomes Myeloid Suppression to Improve Checkpoint Blockade in Prostate Cancer

PSMAi-C' dots trigger proinflammatory responses in prostate cancer and immune cells. (a) Gene expression levels, as fold changes over controls, using qRT-PCR and (b) normalized cytokine and chemokine expression levels by Proteome Profiler in Myc-CaP cells and supernatants, respectively, following incubation with 15 uM of PSMAi-C’ dots over 72 h. (c) IFN-a/b reporter B16 cell luminescence (OD620) after incubation with supernatants from particle-treated and untreated Myc-CaP cells. Percentage (%) of surface-expressed (d) IFNGR1, (e) MHC-I, (f) PD-L1, and (g) CD73 on vehicle- and particle-treated Myc-CaP cells by flow cytometry at 72-hour post-exposure. (h) Flow cytometry-based analyses of the %M1 and %M2 markers in vehicle- and particle-treated bone marrow-derived macrophages (BMDMs) over 72 h. (i) IFN-a/b reporter B16 cell luminescence (OD620) after incubation of BMDMs with supernatants from (h). Percentage of CD8+ T cell-specific (j) IFN-g and (k) TNF-a secretion from splenocytes by flow cytometry following a 72-hour incubation with or without PSMAi-C’ dots. (L) T cell cytotoxicity (expressed as luciferase (luc+) activity) following co-culture of mouse T cells with luc + Myc-CaP cells, with and without PSMAi-C’ dots for 48 h. All samples were run in triplicate. Numerical data are presented as mean ± s.e.m. 1-way ANOVA with Kruskal-Wallis test was performed in (a). Unpaired t-tests were performed in (d–g) and (i–l). 2-way ANOVA with Šídák's multiple comparisons test was performed in (c) and (h). *p < 0.05, **p < 0.01, ****p < 0.001.