Fig. 4: SP remodels bone homeostasis via nuclear factor erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (KEAP1) signaling pathways in vitro.

A WB analysis of SOD2, HO-1, NRF2, GPX1, GPX4, and KEAP1 in macrophages stimulated with varying doses of SP. B Semi-quantification of grayscale value in SOD2, HO-1, NRF2, and KEAP1 in panel A. C WB analysis of SOD2, HO-1, NRF2, GPX1, GPX4, and KEAP1 in BMMs stimulated with M-CSF, RANKL, and SP for 24 h. D Semi-quantification of grayscale value in SOD2, HO-1, NRF2, and KEAP1 in panel C. E Illustration of the protein degradation assay of KEAP1 (Created in BioRender. Yang, X. (2025) https://BioRender.com/nriylby). F WB analysis of HO-1, NRF2, KEAP1, and GPX4 in macrophages stimulated with varying doses of SP, cycloheximide (CHX, 50 nM), and MG132 (10 μM) for 8 h. G Semi-quantification of grayscale value in NRF2 and KEAP1 in panel F. H Ubiquitylation analysis of KEAP1 in 293T cells treated with MG132 (10 μM) and varying doses of SP using HA-KEAP1 and Myc-ubiquitin plasmids with HA-tagged beads. Three independent experiments were repeated. I WB analysis of HO-1, NRF2, KEAP1, and GPX4 in macrophages stimulated with varying doses of SP, CHX (50 nM), and chloroquine (Chlq, 25 nM) for 8 h. J Semi-quantification of greyscale value in NRF2 and KEAP1 in panel I. K Immunofluorescence analysis of KEAP1 and ubiquitin in macrophages stimulated with varying doses of SP for 24 h. Scale bar = 20 μm. Zoomed-in scale bar = 5 μm. L Quantification of the mean fluorescence intensity of KEAP1 and the merged color, as shown in panel K. M Immunofluorescence analysis of KEAP1 and LC3 in macrophages stimulated with varying doses of SP for 24 h. Scale bar = 20 μm. Zoomed-in scale bar = 5 μm. N Quantification of the mean fluorescence intensity of KEAP1, LC3, and the merged color, as shown in panel M. All data presented as mean (SD) are from three replicates. Statistical analysis was performed using the one-way ANOVA followed by Tukey’s post-hoc test. The exact p-values were labeled.