Fig 2: EST12 induces classical M1 activation relying on RACK1-JNK signaling pathway. (A-D) BMDMS isolated from WT, RACK1 KO C57BL/6 mice, respectively, were stimulated with EST12 protein (2 µM) for the indicated time, the expression of iNOS and Arg1 was determined by Western blot (A); NO in the supernatant was determined as described above (B); the secreted IL-6 (C) and TNF-a (D) levels were determined by ELISA. (E-H) RAW264.7 cells were pretreated with JNK inhibitor (10 µM), and then stimulated with EST12 protein (2 µM) for the indicated time, the expression of iNOS and Arg1 was determined by Western blot (E); NO in the supernatant was determined as described above (F); the expression of IL-6 (G) and TNF-a (H) were determined by ELISA. Two-way ANOVA with Tukey’s multiple comparison test was used for (B-D) and (F-H). The data are expressed as the mean ± SEM of three independent experiments. p > 0.05, not significant (ns), ***p < 0.001.

Fig 3: CeNP scavenged ROS and attenuated senescence of synoviocytes in vivo. (a) DHE staining and mean fluorescence intensity of ROS in the synovium (n = 5). (b–e) Immunohistochemical staining and quantitative results of P16, P21, iNOS and COX2 in the synovium (n = 5). ** p < 0.01 vs. sham; # p < 0.05 vs. ACLT; ## p < 0.01 vs. ACLT.

Fig 4: CeNP attenuated senescence and inhibited SASP in multiple passaged synoviocytes. (a) Representative fluorescence imaging of intracellular ROS and mean fluorescence intensity in NC, MP, and MP with CeNP treatment synoviocytes (n = 3). (b) SA-β-Gal staining and quantification of SA-β-Gal positive rate in NC, MP, and MP with CeNP treatment synoviocytes (n = 3). (c–e) RT-qPCR analysis of P16, P21, iNOS, COX2, MMP3, ADAMTS5, IL-6 and TNFα in NC, MP, and MP with CeNP treatment synoviocytes (n = 3). (f–h) Western blot results of iNOS, COX2, ADAMTS5 and MMP3 in NC, MP, and MP with CeNP treatment synoviocytes (n = 3). (i,j) Elisa assays of IL-6 and TNFα in NC, MP, and MP with CeNP treatment synoviocytes (n = 3). Data represent the mean ± SEM from three independent experiments, ** p < 0.01 vs. NC; ## p < 0.01 vs. MP.

Fig 5: CeNP attenuated H2O2-elicited senescence and inhibited SASP in synoviocytes. (a) Representative fluorescence imaging of intracellular ROS and mean fluorescence intensity in NC, H2O2 and H2O2 + CeNP group (n = 3). (b) SA-ß-Gal staining and quantification of SA-ß-Gal positive rate in NC, H2O2 and H2O2 + CeNP groups (n = 3). (c–e) RT-qPCR analysis of P16, P21, iNOS, COX2, MMP3, ADAMTS5, IL-6 and TNFa in NC, H2O2 and H2O2 + CeNP groups (n = 3). (f–h) Western blot results of iNOS, COX2, ADAMTS5 and MMP3 in NC, H2O2 and H2O2 + CeNP groups (n = 3). (i,j) Elisa assays of IL-6 and TNFa in NC, H2O2 and H2O2 + CeNP groups (n = 3). Data represent the mean ± SEM from three independent experiments, ** p < 0.01 vs. NC; # p < 0.05 vs. H2O2 group; ## p < 0.01 vs. H2O2 group.