Fig 2: Expression levels of NFKBIZ and PTEN are positively correlated. (A) NFKBIZ expression was significantly positively correlated with PTEN expression according to the Gene Expression Profiling Interacting Analysis database. (B and C) PTEN expression was increased in transfected T24 and 5637 cells overexpressing NFKBIZ. (D) Representative IHC images of NFKBIZ and PTEN expression in human bladder cancer tissues (magnification, ×200; scale bar, 50 µm). (E) Representative IHC images of PTEN expression in nude mice tumor tissues in the pc-NFKBIZ and pc-NC groups. **P<0.01 vs. NC. NC, negative control; NFKBIZ, NF-?B inhibitor ?; IHC, immunohistochemistry; TPM, transcripts per million.

Fig 3: Increased inflammatory gene expression and cell infiltration in the nephritic kidney of RTEC-specific Regnase-1 deficient mice.Zc3h12afl/fl and Zc3h12aCdh16 mice (n = 3–6) were subjected to AGN. (A–F) At day 7 p.i., renal Cxcl1, Cxcl2, Ccl20 and Ccl2 mRNA (A) expression was measured by qPCR; neutrophil, macrophage, monocyte, B cell, CD4+ T cell, and CD8+ T cell infiltration in the kidney was quantified by flow cytometry (B and C); and Il6 (D), Nfkbiz (E), and Lcn2 (F) transcript expression was evaluated by qPCR. Expression was normalized to Gapdh (A, D, E, and F). The data is pooled from at least 2 independent experiments. Statistical analysis by 2-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: Treatment with the PTEN inhibitor VO enhanced the proliferation, migration and invasion in T24 and 5637 cells overexpressing NFKBIZ. (A and B) Representative images and (C and D) quantitative analyses of T24 and 5637 cell colonies in each group according to colony formation assays. Representative images and quantitative analyses of (E and F) T24 and (G and H) 5637 cell migration rates in each group according to wound healing assays (magnification, ×200). (I and J) Representative images and (K and L) quantitative analyses of invasive T24 and 5637 cells in each group according to Transwell invasion assays (magnification, ×200). **P<0.01. VO, VO-OHpic trihydrate; NC, negative control; NFKBIZ, NF-?B inhibitor ?.

Fig 5: Regnase-1 upregulates IL6 and LCN2 gene expression via NFKBIZ.HK-2 and HK-2?ZC3H12A cells were either treated with siRNA against NFKBIZ or control siRNA. (A) Efficiency of NFKBIZ gene silencing was measured by qPCR. (B and C) Cells were either stimulated with IL-17 or left untreated for 8 hours and analyzed for IL6, LCN2, CCL20, CXCL1, and CXCL2 gene expression by qPCR. Data pooled from at least 3 independent experiments. Statistical analysis by 2-way ANOVA. ***P < 0.001.