Fig 1: IKK promoted oxidative stress injury caused by obesity through CYLD phosphorylation. The protein levels of p-CYLD, CYLD, and p-Nrf2. Nrf2, HO-1 and NQO1 were measured using western blotting in treated HK-2 cells. IKK, IκB kinase; CYLD, cylindromatosis; Nrf2, NF-E2-related factor 2; HO-1, haem oxygenase 1; NQO1, NAD(P)H dehydrogenase quinone 1; HK-2, human kidney 2. Significance was assessed by an ANOVA test. The experimental data are shown as the mean ± SD. N = 3. *P < 0.05, **P < 0.01, ***P < 0.001
Fig 2: Schematic representation of the proposed mechanism by which the phosphorylation of IKK and CYLD mediated the activation of the Nrf2/ARE pathway, thereby inducing oxidative stress in human kidney cells. The imbalance between the increase in ROS and/or the decrease in antioxidant activity promotes oxidative stress injury to tissues or cells. Regulation of mitochondrial production of ROS is required for the proper adaptation of renal cells to metabolic stress. The phosphorylation of IKK induced phosphorylation of CYLD results in the inactivation of its deubiquitination activity and is thus unable to prevent the ubiquitination of Nrf2, which promoted the production of ROS and the depolarization of mitochondrial membrane potential and ultimately facilitated oxidative stress injury in vitro, which is conducive to the development of ORN
Fig 3: Oxidative stress injury was observed in kidney tissues of ORN model mice. A The production of ROS in the kidneys of ORN model mice and control mice was measured using DHE staining. B–E MAD level, SOD activity, CAT activity and GSH-Px activity were measured in mouse kidneys. F Representative IHC staining of Nrf2 and HO-1 in mouse kidneys. G Western blot analysis of p-IKK, IKK, p-CYLD, CYLD, Nrf2 and HO-1 expression in mouse kidneys. ORN, obesity-related nephropathy; ROS, reactive oxygen species; DHE, dihydroethidium; MAD, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; IHC, immunohistochemistry; Nrf2, NF-E2-related factor 2; HO-1, haem oxygenase 1; IKK, IκB kinase; CYLD, cylindromatosis. Significance was assessed by a Student's t test. The experimental data are shown as the mean ± SD. N = 10. *P < 0.05, **P < 0.01, ***P < 0.001
Fig 4: Knockdown of CYLD attenuated the effect of an IKK inhibitor on oxidative stress injury in an in vitro ORN model. Cells were transfected with sh-CYLD to silence CYLD expression and divided into four groups: ox-LDL + sh-NC, ox-LDL + TPCA1 + sh-NC, ox-LDL + sh-CYLD, and ox-LDL + TPCA1 + sh-CYLD. A and B The knockdown efficiency of sh-CYLD was assessed using qRT–PCR and western blotting. C Effect of CYLD knockdown on TG levels in treated HK-2 cells. D Lipid deposition in different groups was measured using Oil Red O staining. E and F MAD level and SOD activity of treated HK-2 cells. G and H ROS levels and ??m of treated HK-2 cells were measured by DHE staining and JC-1 assay, respectively. CYLD, cylindromatosis; IKK, I?B kinase; ORN, obesity-related nephropathy; ox-LDL, oxidized low-density lipoprotein; NC, normal control; TG, triglyceride; HK-2, human kidney 2; MAD, malondialdehyde; SOD, superoxide dismutase; DHE, dihydroethidium; ROS, reactive oxygen species; ??m, mitochondrial membrane potential. Significance was assessed by an ANOVA test. The experimental data are shown as the mean ± SD. N = 3. *P < 0.05, **P < 0.01, ***P < 0.001
Fig 5: Functional studies in cHL-derived and DLBCL-derived cell lines(A) Western blot analysis of total BTK, CYLD truncation, NFKB (p52 isoform), and phospho-IkBa. (B and C) Activity of BTK selective inhibitors in cHL-derived and DLBCL-derived cell lines. IC50 range calculation of HBL1, HDLM2, L540, and L1236 cell lines in the presence of different concentrations of Ibrutinib or AVL-292, after 48 hours.
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