Fig. 3

Different treatments affect Pg EV-induced osteoclastogenesis. a Schematic diagram of the different physical and biological treatments applied to EVs. The vesicular structure of EVs is disrupted by ultrasound, and the LPS, serine proteins, lipoproteins, and serine proteases in the EVs are removed or degraded by pretreatment with Pm B, Pro K, lipase, and PMSF, respectively. b EVs or RANKL were pretreated with Pm B (0.1 μg/μL), Pro K (0.1 μg/μL), lipase (0.1 μg/μL) and PMSF (5 mM) for 1 h at RT and then incubated at 4 °C. RAW264.7 cells were subsequently treated with Pm B-, Pro K-, lipase-, and PMSF-pretreated EVs or RANKL for 6 days, after which TRAP staining was performed. The red dashed line represents TRAP-positive osteoclasts. c TRAP-positive multinuclear cells with more than 3 nuclei were counted. ns P > 0.05, ^***P < 0.001