Fig 2: OTUD5 negatively regulates TAK1 activation and inflammation in podocytes.MPC5 cells transfected with Flag-OTUD5 (a) or si-OTUD5 (b) were stimulated with HG/PA for 30 min. Representative western blot analysis of P-TAK1. (n = 3 independent experiments). c, d Representative western blot analysis of P-TAK1 in kidney tissues of each group. (n = 6 samples). MPC5 cells transfected with Flag-OTUD5 (e) or si-OTUD5 (f) were stimulated with HG/PA for 30 min. Representative western blot analysis of phosphorylated and total protein levels of ERK, P38, and JNK. (n = 3 independent experiments). MPC5 cells transfected with si-OTUD5 were pretreated with 10 μM Takinib (TAK1 inhibitor) for 1 h before exposure to HG/PA. g Levels of P-TAK1, P-ERK, P-P38, and P-JNK were detected by western blot. h Real-time qPCR showing mRNA levels of Il6 and Tnfα. (n = 3 independent experiments; P values were determined by one-way ANOVA with Bonferroni’s correction and data are presented as mean ± SD). i His-TAK1 was transfected into NIH/3T3 with or without Flag-OTUD5 (WT or C224A). Co-IP was performed with an anti-His antibody, followed by a western blot of TAK1 and TAB2. (n = 3 independent experiments). j MPC5 cells transfected with Flag-OTUD5 (WT or C224A) were stimulated with HG/PA for 30 min. Representative western blot analysis of phosphorylated and total protein levels of TAK1, ERK, P38, and JNK. (n = 3 independent experiments). k His-TAK1(WT or K158R) was transfected into NIH/3T3 with or without Flag-OTUD5. Co-IP was performed with an anti-His antibody, followed by a western blot of TAK1 and TAB2. (n = 3 independent experiments). l His-TAK1 (WT or K158R) and Flag-OTUD5 were transfected into MPC5 cells for 24 h and then stimulated by HG/PA for 30 min. Representative western blot analysis of phosphorylated and total protein levels of TAK1, ERK, P38, and JNK. (n = 3 independent experiments).

Fig 3: Network of miRNA target genes in nickel-treated cells. (a) Octagonal shapes represent mRNAs and diamond shape represents miR-4417. The figure was generated using CytoScape®. (b) BEAS-2B (1 × 106 cells/6 cm dish) and A549 (5 × 105 cells/6 cm dish) cells were treated with NiCl2 (0, 0.25, 0.5 mM and 0, 0.5, 1 mM, respectively) for 48 h. The mRNA levels were analyzed on RT-PCR. β-actin was used as the internal control. The relative ratios of ENOSF1/β-actin, NAP1L5/β-actin and TAB2/β-actin are shown. (c) BEAS-2B (1 × 106 cells/6 cm dish) and A549 (5 × 105 cells/6 cm dish) cells were treated with NiCl2 (0, 0.25, 0.5 mM and 0, 0.5, 1 mM, respectively) for 72 h and the protein levels were determined on Western blot. β-actin was used as the internal control. The relative ratios of ENOSF1/β-actin, RAB6A/β-actin, p-TAB2/β-actin and TAB2/β-actin are shown.

Fig 4: Pacritinib inhibits the association of IRAK1 with TAK1. Macrophages were pretreated with pacritinib or controls for 2 h and then treated for 6 h with 5 μg mL−1 GU-rich ssRNA. n = 4. Cells were lysed, and IRAK1 was immunoprecipitated (IP). The presence of phosphorylated, ubiquitinated, and total IRAK1, TAK1, TAB2, TRAF6, and ACTB was assayed by western blot. N = 4.

Fig 5: Effects of miR-4417 on mesenchymal marker fibronection. (a) A549 and BEAS-2B cells were transfected with miR-vector (vector control) or miR-4417/pcDNATM 6.2-GW for 48 h. Quantitative real-time PCR analysis was used to detect miR-4417 expression. All values are normalized to the level of RNU6B. Each bar represents the mean ± SD of triplicate experiments; #p < 0.05 (compared to BEAS-2B miR-vector cells) and **p < 0.01 (compared to A549 miR-vector cells). (b) Left, the mRNA levels of miR-4417-targeted genes, including NAP1L5 and TAB2, were determined on RT-PCR. β-actin was used as the internal control. The relative ratios of NAP1L5/β-actin and TAB2/β-actin are shown. Right, the TAB2 mRNA levels were determined on real-time PCR. **p < 0.01 (c) Top, the predicted hsa-miR-4417 seed sequence binding site of the TAB2 3′UTR using miRDB. Bottom, BEAS-2B cells were cotransfected with psiCHECK-2/TAB2 3′UTR and miR-vector or hsa-miR-4417 with scramble inhibitor or miR-4417 inhibitor plasmid. After 48 h, the transcriptional activity of each reporter plasmid was normalized relative to β-galactosidase activity. Each bar represents the mean ± SD of triplicate experiments; *and #p < 0.01 (d) BEAS-2B and A549 cells were transfected with negative control or miR-4417 mimic (100 nM) for 48 h, and analyzed by Western blot. β-actin was used as the internal control. The relative ratios of Fibronectin/β-actin and E-cadherin/β-actin are shown. (e) BEAS-2B cells were transfected with or without microOFFTM miR-4417 inhibitor (100 nM) or scrambled miRNA inhibitor (scrambled inhibitor, 100 nM) followed by exposure to NiCl2 (0 and 0.25 mM) for 48 h. (f) BEAS-2B cells were co-transfected with miR-4417/pcDNATM 6.2-GW (5 μg) and miR-4417 inhibitor (100 nM) or scrambled miRNA inhibitor (scrambled inhibitor, 100 nM), and analyzed by Western blot. (g) BEAS-2B cells were infected with lentivirus carrying shTAB2 #442 or #452 or vector control (shLuc) and analyzed by Western blot. β-actin was used as the internal control. The relative ratios of TAB2/β-actin, Fibronectin/β-actin and E-cadherin/β-actin are shown.