Journal: Nature Communications
Article Title: Co-delivery of sorafenib and an FSP1 inhibitor triggers dual ferroptosis in tumor cells and immunosuppressive macrophages for enhanced immunotherapy in mouse models of hepatocellular carcinoma
doi: 10.1038/s41467-025-65056-9
Figure Lengend Snippet: a Schematic illustration of the induction of M2 TAMs, the 1-6/TAM spheroids formulation, the transwell co-incubation, and their respective treatments. b Western blot analysis of GPX4 and FSP1 expression in M2 TAMs after different treatments. The experiment was repeated three times independently with similar results. Uncropped blots in Source Data. c Quantification of grayscale intensity of GPX4 and FSP1 in ( b ) ( n = 3 independent experiments). Protein expression levels in PBS group were normalized to 1. d Live/dead staining of M2 TAMs after different treatments (Scale bar = 200 μm). Viable cells were stained with calcein-AM (green), and dead cells were stained with PI (red). The experiment was repeated three times independently with similar results. e Quantification of the percentage of viable and dead M2 TAMs in ( d ) ( n = 3 independent experiments). f Flow cytometric analysis of Annexin V-FITC/propidium iodide (PI)-stained M2 TAMs (gated on F4/80 + CD206 + macrophages) after different treatments. g Quantification of the percentage of apoptotic cells in ( f ) ( n = 3 independent replicates). The experiment was repeated twice independently with similar results. h The cell proliferation of M2 TAMs after different treatments measured by CCK8 ( n = 3 independent experiments). The concentrations of SF and vF contained in nanoparticles were both 3 μM. i Fluorescence images of C11 BODIPY 581/591 -stained M2 TAMs after different treatments (Scale bar = 100 μm). The experiment was repeated three times independently with similar results. j Fluorescence images of live/dead stained 1-6/TAM spheroids after different treatments (Scale bar = 500 μm). Viable cells were stained with calcein-AM (green), and dead cells were stained with PI (red). Z-stack scanning of the spheroids was performed with slices distanced by 14.36 μm. The experiment was repeated three times independently with similar results. k Flow cytometric analysis of anti-CRT-stained Hepa1-6 cells after different treatments. l Quantification of the percentage of CRT positive cells in ( k ) ( n = 3 independent replicates). The experiment was repeated twice independently with similar results. m The ATP assay of Hepa1-6 cells after different treatments ( n = 3 independent experiments). n The HMGB1 assay of Hepa1-6 cells after different treatments ( n = 3 independent experiments). o Flow cytometric analysis of anti-CD80/CD86-stained BMDCs (gated on CD11c + DCs) after different treatments. p Quantification of the percentage of mature DCs in ( o ) ( n = 3 independent replicates). The experiment was repeated twice independently with similar results. q The assay of TNF, IL-6, and IL-12 in BMDCs after different treatments ( n = 3 independent experiments). Unless specified otherwise, error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test ( c , g , h , l – n , p , q ) and P -values were indicated. Source data are provided as a Source Data file. The elements in Fig. 5a were created by Adobe Illustrator.
Article Snippet: PerCP/Cyanine5.5 anti-mouse F4/80 (1:200, #E-AB-F0995J), APC anti-mouse CD206 (1:200, #E-AB-F1135E), PE/Cyanine7 anti-mouse CD86 (1:200, #E-AB-F0994H), Fluor Red 780 anti-mouse CD80 (1:200, #E-AB-F0992S), APC anti-mouse CD11c (1:200, #E-AB-F0991E), Fluor Violet 450 anti-mouse CD3 (1:200, #E-AB-F1013Q), Fluor Red 780 anti-mouse CD4 (1:200, #E-AB-F1097S), PerCP/Cyanine5.5 anti-mouse CD8 (1:200, #E-AB-F1104J), FITC anti-human/mouse CD44 (1:200, #E-AB-F1100C), PE anti-mouse Foxp3 (1:200, #E-AB-F1238D), FITC anti-mouse MHC II (1:200, #E-AB-F0990C), APC anti-mouse CD62L (1:200, #E-AB-F1011E), APC anti-mouse PD-L1 (1:200, #E-AB-F1132E), PerCP anti-human CD45 (1:200, #E-AB-F1137F), Fluor647 anti-human CD68 (1:200, #E-AB-F1299M), FITC anti-human CD206 (1:200, #E-AB-F1161C), PE anti-human CD80 (1:200, #E-AB-F1232D), APC anti-human HLA-DR (1:200, #E-AB-F1111E), PE anti-human CD11c (1:200, #E-AB-F1118D), APC anti-human CD3 (1:200, #E-AB-F1001E), and FITC anti-human CD8 (1:200, #E-AB-F1110C) were purchased from Elabscience (Wuhan, China).
Techniques: Formulation, Incubation, Western Blot, Expressing, Staining, Fluorescence, ATP Assay
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