Fig 2: TRAF4 acts as an E3 ubiquitin ligase to ubiquitinate the K119 site of Smurf2 through the K48 ubiquitin chain and degrade Smurf2. After coexpression of HA-Ubiquitin, Myc-TRAF4 and Flag-Smurf2 into 293T cells for 36 h, cell lysates were harvested, and Flag-Smurf2 was immunoprecipitated with anti-Flag. a Western blotting demonstrated that Myc-TRAF4 significantly induced the ubiquitination of Flag-Smurf2. b, c Myc-TRAF4 significantly induced K48-linked ubiquitination of Flag-Smurf2 rather than K63-linked ubiquitination. d Deletion of either the RING domain or the TRAF domain from TRAF4 caused TRAF4 to lose the ability to induce the ubiquitination of Smurf2. e A catalytically inactive mutant of Smurf2 (cysteine 716 to alanine) did not affect the TRAF4-dependent ubiquitination and degradation of Smurf2. f Coomassie blue staining of immunoprecipitated Flag-Smurf2 coexpressed with Myc-TRAF4 or empty vector separated by SDS-PAGE. g Mutation of the 119 lysine residues to arginine (K119R) significantly reduced TRAF4-mediated ubiquitination of Smurf2, and TRAF4 lost the ability to degrade the expression of Smurf2 (K119R). The results shown in (e) and (g) are presented as the means ± SD. *p < 0.05 (n = 3 independent experiments)

Fig 3: Smurf2 functioned as an E3 ubiquitin ligase to ubiquitinate and degrade PTX3. After coexpression of HA‐UBB, Flag‐Smurf2, Flag‐C716A, and Myc‐PTX3 in 293T cells for 36 hours, the cell lysates were harvested. A, Western blots showing that Flag‐Smurf2 significantly induced the ubiquitination of Myc‐PTX3, whereas Flag‐ C716A did not exert a significant effect. B,C, Flag‐Smurf2 significantly induced both the K63‐linked and K48‐linked ubiquitination of Myc‐PTX3 (n = 3 independent experiments). D, Myc‐PTX3 was transiently overexpressed in mesenchymal stem cells (MSCs) in patients with ankylosing spondylitis (ASMSCs) to avoid interfering with the NF‐κB pathway (n = 10 different ASMSC lines per group), and then the cells were treated for 12 hours with the proteasome inhibitor MG132 (10 μM) or lysosome inhibitor chloroquine (50 μM). Western blots showed that MG132 reversed the degradation of Myc‐PTX3 mediated by Flag‐Smurf2, whereas chloroquine exerted less of an effect. E, Protein lysates were prepared at the indicated time points after treatment with 10 μM CHX, and PTX3 protein levels were evaluated using Western blotting. Compared to that in MSCs in patients with healthy donors (HDMSCs), the half‐life of PTX3 was shorter in ASMSCs. F, Quantitative analysis of the rate of PTX3 degradation in HDMSCs and ASMSCs (n = 10 different HDMSC or ASMSC lines per group, P < .01). G, Myc‐PTX3 was transfected into 293T cells along with Flag‐Smurf2 or empty vectors. At 36 hours after transfection, protein lysates were prepared at the indicated time points after treatment with 10 μM CHX. The rate of Myc‐PTX3 degradation was higher when coexpressed with Flag‐Smurf2. H, Quantitative analysis of the rate of PTX3 degradation with Flag‐Smurf2 and empty vectors (n = 3 independent experiments, P < .01). All data are presented as the means ± SD. *P < .05, **P < .01

Fig 4: Expression levels of SnoN and p-Smad2 in NRK-52E cells transfected with recombinant adenovirus. (A) Immunofluorescence analysis of the expression of smurf2 in NRK-52E cells infected with AD-smurf2. (B) RT-qPCR analysis was performed to detect the mRNA levels of smurf2, SnoN and Smad2, and western blot analysis was performed to detect the expression levels of smurf2, SnoN and p-Smad2 in NRK-52E cells infected with AD-smurf2. (C) Immunofluorescence analysis of the expression of smurf2 in NRK-52E cells treated with AD-shsmurf2. (D) RT-qPCR analysis was performed to detect the mRNA levels of smurf2, SnoN and Smad2, and western blot analysis was performed to detect the expression levels of smurf2, SnoN and p-Smad2 in NRK-52E cells infected with AD-shsmurf2. ***P<0.001, vs. control. SnoN, ski-related novel protein N; p-Smad2, phosphorylated small mothers against decapentaplegic; smurf2, Smad ubiquitination regulatory factor 2; AD, adenovirus; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Fig 5: TM1‐3P alleviated synovial tissue damage and inflammation in OA rats. (A,B) Histopathological changes in knee joints were observed by HE and Annona Red O/Fast Green staining. (C,D) MMP13, ADAMTS5, CollagenII, and Aggrecan expressions in synovial tissue were examined by immunohistochemistry. (E) The expressions of ONECUT2, Smurf2, and Rap1 in synovial tissue were tested by western blot. (F) IL‐1β, IL‐6, and TNF‐α levels in synovial tissue were evaluated by qRT‐PCR and western blot. *P < 0.05 vs Sham group, #P < 0.05 vs LV‐sh‐LncRNA NC group.