Fig 2: Prosurvival signaling in luminal-C cells of Pten(i)pe-/- prostates in response to Gemini-72.(A) Venn diagram depicting the number of genes of the NF-?B, TNF, and IL-17 pathways downregulated in luminal-C cells of a Pten(i)pe-/- mouse by a 1-week Gemini-72 treatment. (B) Violin plot showing the Nfkbia transcript levels in luminal-C cells of a Pten(i)pe-/- mouse treated for 1 week with vehicle or Gemini-72. ***P < 0.001. Comparisons between vehicle- and Gemini-72–treated mice were performed using a Wilcoxon rank sum test. (C) Western blot analysis of phosphorylated IKKa/ß(S176/S180) (pIKKa/ß) in pooled lysates of FACS-sorted luminal-C cells of three Pten(i)pe-/- mice treated for 1 week with vehicle or Gemini-72. (D) Schematic representation of NF-?B activation. (E) Immunoblot of I?Ba levels in whole prostates of Pten(i)pe-/- mice treated with vehicle or Gemini-72 for 1 week. Lysates of three prostates per condition were pooled. (F) RelA/p65 (red) immunostaining in PINs of Pten(i)pe-/- mice treated for 1 week with vehicle or Gemini-72. Green and yellow arrows point to cytosolic and nuclear p65 staining, respectively. Representative images of the DL lobes are shown. n = 3 mice per condition. Scale bars, 100 µm. (G) Western blot analysis of Bcl-xL in whole prostates (top) and FACS-sorted luminal-C cells (bottom) of Pten(i)pe-/- mice treated for 1 week with vehicle or Gemini-72. n = 3 prostates per lane. (H) Heat map of cytokine levels in FACS-sorted luminal-C cells from Pten(i)pe-/- mice treated at 9 months AGI with Gemini-72 for 1, 2, and 3 weeks. Lysates were obtained from a pool of three prostates per condition. (I) Phosphorylated Akt(S473) (red) and Trop2 (green) immunostaining in PINs of Pten(i)pe-/- mice treated for 3 weeks with vehicle or Gemini-72. Representative images of the DL lobes are shown. n = 3 mice per condition. Scale bars, 100 µm.

Fig 3: NF-?B represses ANT1 gene transcription and protein expression.(A) RT-PCR shows the ANT1, TNFa, and I?Ba mRNA expression in HEK 293 cells after 24 hours transfection with IKKß and NF-?B/p65. The histogram depicts the mean ratios of ANT1 mRNA to ß-actin ± SEM (n = 3); *p < 0.05, Student’s t test. (B) ANT1 and I?Ba mRNA expression in HEK293 were examined in the presence of NF-?B activity inhibitor JSH-23 (15 µM) and the NF-?B activator H2O2 (50 µM Values represent means ± SEM (n = 3); **p < 0.01, Student’s t test. (C) The endogenous ANT1 protein in HEK293 was affected by NF-?B/p65 transfection and NF-?B inhibitor JSH-23 (15 µM). Cropped blots were displayed. Anti-ANT1 (ab110322, Abcam) and anti-NF-?B/p65 (#8242, CST) antibodies were used in WB. ß-actin was used as loading controls. Values represent means ± SEM (n = 3); *p < 0.05, Student’s t test. (D) The IRDye 800-labelled consensus NRE oligo was used as probes in EMSA. Nuclear extracts in EMSA were extracted from T98G cells treated with TNFa (10 ng/ml) for 0, 90, 180 and 360 minutes. NRE, NF-?B responsive elements (E) ANT1 mRNA levels and NF-?B activities in TNFa (10 ng/ml) induced T98G cells for 0, 90, 180 and 360 minutes were depicted in line graph; *p < 0.05, **p < 0.01, One-way ANOVA test, as compared with initial time point. (F) T98G cells stimulated by TNFa (10 ng/ml) for 0, 90, 180 and 360 minutes were harvested and lysed for WB of endogenous ANT1, I?Ba and COX-IV. Anti-ANT1 (ab110322; Abcam), anti-I?Ba (#4814, CST) and anti-COX-IV (#4850, CST) antibodies were used in WB. ß-actin was used as a loading control. Cropped blots were displayed. Values represent means ± SEM (n = 3); **p < 0.01, One-way ANOVA test, as compared with initial time point. (G) ANT1 over-expression plasmid (p3xflagANT1) was transfected into T98G cells and stimulated by TNFa for 0, 90, 180 and 360 minutes. Anti-flag (M2, F1804, Sigma-Aldrich) was used to detect exogenous ANT1 expression. Cropped blots were displayed. Values represent means ± SEM (n = 3); NS, no significant difference, One-way ANOVA test, as compared with initial time point.

Fig 4: Toxic APAP exposure induces proinflammatory NF?B signaling in the distal lung. (a) Representative Western blot and densitometric analysis of NF?? inhibitory proteins p105, I?Ba, and I?Bß in distal lung cytosolic extracts after APAP exposure (2, 4, and 8 hours; 280 mg/kg, IP). GAPDH is shown as loading control. Data is expressed as mean ± SEM (N = 4-6 per time point). *p < 0.05 vs. unexposed control. (b) Representative Western blot and densitometric analysis of NF?? subunits p65 and p50 in distal lung nuclear extracts after APAP exposure (2, 4, and 8 hours; 280 mg/kg, IP). HDAC is shown as a loading control. GAPDH is shown to confirm the purity of the nuclear sample. Data is expressed as mean ± SEM (N = 4-6 per time point). *p < 0.05 vs. unexposed control. (c) Fold change in NF?? target gene mRNA expression: Mmp9, Ccl2, Il1b, Nfkbia, Gclc, and Ptgs2 following APAP exposure (8 hours; 280 mg/kg, IP). Data expressed as mean ± SEM (N = 4-6 per time point). *p < 0.05 5 vs. unexposed control.

Fig 5: Usp26 deficiency facilitates osteoclastic differentiation of BMMs by decreasing IκBα.A Volcano plots of differentially expressed genes in Usp26−/− osteoclasts (Usp26 KO) as compared with WT controls (WT). The blue and red dots represent the down- and upregulated genes, respectively. B KEGG enrichment analysis of the correlated signal pathway to the differently expressed mRNAs between Usp26−/− and WT osteoclasts. C Western blot analysis of P-p65 in BMMs obtained from Usp26−/− mice and littermate controls with or without osteoclastic differentiation. D, E NF-κB transcriptional activity in BMMs induced by osteoclastic differentiation with or without different doses of Usp26 overexpression. F Representative of P-p65 IHC staining in femurs of 5-month-old Usp26−/− mice and their WT littermates (n = 5). Red and black bars represent 25 and 100 μm, respectively. G Western blot analysis of IKKα, IKKβ, IKKγ, and IκBα expression in mouse BMMs. H Co-immunoprecipitation of USP26 with endogenous IKKα, IKKβ, IKKγ, and IκBα in mouse BMMs. I Representative of IκBα IHC staining in femurs of 5-month-old Usp26−/− mice and their WT littermates (n = 5). Red and black bars represent 25 and 100 μm, respectively. J Western blot analysis of IκBα expression in BMMs isolated from Usp26−/− mice and their WT littermates with or without osteoclastic differentiation. K Western blot analysis of IκBα and P-p65 expression in Usp26−/− BMMs with or without Nfkbia overexpression. L, M The osteoclastic genes expression and TRAP staining of Usp26−/− osteoclasts with or without Nfkbia overexpression. N The schematic graph reflects that decreased IκBα results in increased NF-κB activation and enhanced osteoclastic differentiation in Usp26−/− BMMs. O, P Co-immunoprecipitation of USP26 with ectopically expressed Flag-tagged IκBα in 293 T cells. Q Overexpression of USP26 decreases the level of ubiquitinated IκBα. R Western blot analysis of ectopically expressed IκBα protein in 293 T cells with or without USP26 overexpression and treated with cycloheximide (CHX) for indicated time intervals. S The schematic graph reflects the underlying mechanisms of USP26 in decreasing IκBα degradation by reducing the level of ubiquitinated IκBα in 293 T cells. **P < 0.01, ***P < 0.001. P values were analyzed by one-way ANOVA. All data are representative of two to three independent experiments.