Fig. 4
Necroptosis of primary osteocytes induced by tumor necrosis factor-α (TNF-α) stimulation. a Isolation of Topaz-positive cells with fluorescence-activated cell sorting (FACS). b Morphology of Topaz-positive cells under a fluorescence microscope. Scale bar = 50 μm. c Immunofluorescence staining for phosphorylated (p)-RIP3 (red) and DAPI staining (blue) in primary osteocytes treated with TNF-α, SM-164, and zVAD (TSZ) for the indicated time. White arrows indicate p-RIP3-positive osteocytes. Scale bar = 100 μm. d Immunofluorescence staining for p-RIP3 (red) and DAPI staining (blue) in primary osteocytes treated with TSZ, Nec-1, GSK872, and NSA for 6 h. White arrows indicate p-RIP3-positive osteocytes. Scale bar = 100 μm. e, f Percentage of p-RIP3-positive osteocytes (n = 3). g Immunofluorescence staining for p-MLKL (red) and DAPI staining (blue) in primary osteocytes treated with TSZ for the indicated time. White arrows indicate p-MLKL-positive osteocytes. Scale bar = 100 μm. h Immunofluorescence staining for p-MLKL (red) and DAPI staining (blue) in primary osteocytes treated with TSZ, Nec-1, GSK872, and NSA for 6 h. White arrows indicate p-MLKL-positive osteocytes. Scale bar = 100 μm. i, j Percentage of p-MLKL-positive osteocytes (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.
