1. Volume 32, Issue 5, Pages 561-573.e6 (November 2017)
iASPP Is an Antioxidative Factor and Drives Cancer Growth and Drug Resistance by Competing with Nrf2 for Keap1 Binding 
Wenjie Ge, Kunming Zhao, Xingwen Wang, Huayi Li, Miao Yu, Mengmeng He, Xuting Xue, Yifu Zhu, Cheng Zhang, Yiwei Cheng, Shijian Jiang, Ying Hu 
Cancer Cell 
Volume 32, Issue 5, Pages e6 (November 2017)
DOI: /j.ccell
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2. Cancer Cell 2017 32, 561-573.e6DOI: (10.1016/j.ccell.2017.09.008)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1 Cytoplasmic iASPP Lowers ROS in a p53-Independent Manner
(A and B) Representative western blot (WB) images showing the efficiency of iASPP overexpression/KD or iASPP expression recovery in CCF-RC1 (A) and T24 (B) cells. Protein loading is indicated by β-actin. Quantification of bands was conducted by ImageJ.
(C–I) ROS levels, as indicated by DCFH-DA fluorescence, were measured by flow cytometry in CCF-RC1 (C and E), T24 (D and F), H1299 (G), and HCT116 (H and I) cells after iASPP overexpression, KD, 5-FU treatment (E and F), or p53 KD (H and I), as indicated.
(J and K) The distribution of iASPP was determined by nuclear and cytoplasmic fraction in T24 cells after iASPP overexpression (J) or KD (K).
Data are derived from three independent experiments and presented as means ± SEM in the bar graphs (A–I). Values of controls were normalized to 1. ∗p < 0.05; ∗∗p < 0.01 (A–F); #p < 0.05, ##p < 0.01, compared with 5-FU-treated controls (E and F). Vect., vector control; Ctl, control.
See also Figure S1.
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4. Figure 2
iASPP-Mediated ROS Inhibition Is Dependent on Nrf2 Antioxidative Signaling
(A) Representative WB images showing the efficiency of iASPP overexpression/KD or Nrf2 KD in T24 cells.
(B) ROS was determined in T24 cells after ectopic iASPP expression with or without siRNA-mediated Nrf2 KD.
(C–E) pGL3-ARE-luc activity (C and D) and expression of NQO1, HMOX1, and FTH1 (E) were measured by luciferase reporter assay and real-time RT-PCR, respectively, in T24 cells with the indicated treatments.
Data are derived from three independent experiments and represented as means ± SEM in the bar graphs. Values in control cells were normalized to 1. ∗∗p < 0.01; N.S., not significant (B–E). See also Figure S2.
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5. Figure 3 iASPP Inhibits Ubiquitin-Proteasome Degradation of Nrf2
(A and B) Nrf2 protein and mRNA levels were analyzed after iASPP overexpression (A) or KD (B). The quantification results derived from at least three independent experiments are presented as means ± SEM in the bar graphs. ∗∗p < 0.01; N.S., not significant.
(C and D) Representative images of Nrf2 protein determined by WB (C) and quantification of the Nrf2 band intensity by ImageJ (D) in the control and iASPP KD T24 cells in the presence of 100 μg/mL CHX for the indicate time periods. Nrf2 levels in the untreated cells were normalized to 1 (D).
(E) Nrf2 protein levels were determined by WB in control and iASPP KD T24 cells in the presence or absence of proteasome inhibitor MG132.
(F and G) Ubiqutin-Nrf2 (Ub-Nrf2) was determined by immunoprecipitation (IP) of Nrf2 with a subsequent WB assay with anti-ubiquitin antibody in iASPP overexpressed (F) or KD (G) T24 cells.
See also Figure S3.
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6. Figure 4
iASPP Promotes Nrf2 Stabilization by Competitive Binding to Keap1
(A and B) Nrf2 protein level determined by WB (A) and pGL3-ARE-luc activity determined by luciferase reporter assay (B) after ectopic iASPP expression in control and Keap1 KD T24 cells. Bar graphs (means ± SEM) represent the relative Nrf2 protein levels (A) and relative pGL3-ARE-luc activity (B) derived from three independent experiments.
(C) The interaction between iASPP and Nrf2 was determined by a co-immunoprecipitation (co-IP) assay with the treatment of quercetin (80 μM) or 5-FU (5 μM) for 24 hr in T24 cells. Bar graphs (means ± SEM) represent the relative iASPP or Nrf2 proteins that bound with Keap1 derived from three independent experiments.
(D) The in vitro binding assay was performed to evaluate the interaction of Keap1-Nrf2 with iASPP full-length, truncated, or DLK28, DLT241, or DLD256 mutants, as indicated in the diagram. DLX mutants were generated by replacing the DLX residues with AGE.
(E) The interaction between iASPP and the full-length and truncated Keap1, as indicated in the diagram, was determined by co-IP in 293T cells. NTR, N-terminal region; BTB, Bric-a-Brac; IVR, intervening region; DGR, double glycine repeat or Kelch repeat; CTR, C-terminal region.
(F) Interactions among Keap1, Nrf2, and iASPP were determined by co-IP assay using in vitro-translated Keap1 (10 μL), Nrf2 (5 μL), and iASPP (0, 3, or 6 μL). Graph (mean ± SEM) represents the relative iASPP or Nrf2 proteins that bound with Keap1 derived from three independent experiments.
(G) Relative amounts of Nrf2 in the nuclear or cytoplasmic fractions were determined by a cell fraction assay after iASPP overexpression or KD in T24 cells. Bar graphs (means ± SEM) represents the relative Nrf2 proteins in the nucleus or cytoplasm derived from three independent experiments.
∗∗p < 0.01; ∗p < 0.05; N.S., not significant. See also Figure S4.
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7. Figure 5
iASPP/Nrf2 Promotes Cancer Cell Survival Both In Vivo and In Vitro
(A and B) Representative WB (A) and quantification (B) of iASPP, Nrf2, and Keap1 protein levels in 32 human renal cell carcinoma (RCC) (T) and paired adjacent normal controls (N).
(C) Samples in (A and B) were divided into “low” and “high” groups according to the elevation of iASPP (T/N). The increased fold of Nrf2 (T/N) is shown on the y axis.
(D) The survival rate of cells with the indicated treatments was evaluated by MTT assay.
(E–G) ROS levels as indicated by DCFH-DA fluorescence (E), and apoptosis levels revealed by Annexin V staining (F) or caspase-3/-7 activity assay (G) in iASPP KD cells.
(H and I) Apoptosis levels revealed by Annexin V staining (H and I) or caspase (casp.)-3/-7 activity assay (H) in CCF-RC1 cells following the indicated treatments.
(J and K) Tumor volumes at the indicated dates (J) and images as well as tumor weight at day 28 (K) of CCF-RC1 xenografts of si-Ctl and si-iASPP with or without restoring iASPP. The average values are present in the bar graphs (means ± SD) (J) (n = 6 for each pair).
(L) iASPP, Nrf2, Keap1, and cleaved caspase-3 were determined by WB and antioxidative Nrf2 targets were measured by real-time RT-PCR in samples derived from CCF-RC1 xenografts.
Data are derived from three independent experiments and represented as means ± SEM in the bar graphs (D–I and L). ∗p < 0.05; ∗∗p < 0.01, N.S., not significant (B–L). See also Figure S5.
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8. Figure 6
iASPP/Nrf2-Induced ROS Inhibition Correlates with 5-FU Resistance
(A) The IC50 values of 5-FU were determined by MTT assay in control and iASPP KD CCF-RC1 cells.
(B) Bax, cleaved caspase-3, and Bcl-2 were determined by WB after iASPP KD in CCF-RC1 cells in the presence or absence of 5-FU.
(C–F) Apoptosis levels, as revealed by Annexin V staining (C and F) and caspase-3/-7 activity assays (D), and ROS levels, as indicated by DCFH-DA fluorescence (E), were determined in CCF-RC1 cells with the indicated treatments.
(G and H) Tumor volumes at the indicated dates (G) and images as well as tumor weight at day 28 (H) of CCF-RC1 xenografts of si-Ctl and si-iASPP CCF-RC1 with or without 5-FU treatments. The average values are present in the bar graphs (means ± SD) (H) (n = 4 for each group).
(I and J) iASPP, Nrf2, Keap1, Bax, Bcl-2, and cleaved caspase-3 were evaluated by WB (I) and the mRNA levels of antioxidative Nrf2 targets were determined by real-time RT-PCR (J) in the dissected xenografts.
Data are derived from three independent experiments and represented as means ± SEM in the bar graph (A and C–J). ∗p < 0.05; ∗∗p < 0.01; N.S., not significant, relative to controls (A and C–J). ##p < 0.01, relative to 5-FU-treated controls (C–F). See also Figure S6.
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9. Figure 7
Proposed Model of Regulation of Nrf2 Activity by iASPP in Cancer Cells
Under basal conditions, iASPP forms a complex with Keap1 and Nrf2 by binding with Keap1's DGR domain through a DLT motif (middle). Downregulation of iASPP promotes Nrf2 ubiquitination and further degradation (left), suggesting that the recruitment of iASPP to the Keap1-Nrf2 complex is critical for maintaining basal Nrf2 stability. Upon oxidative stresses or iASPP overexpression, iASPP can interfere with Keap1-Nrf2 complex or substitute Nrf2 in binding with Keap1, which leads to Nrf2 nuclear translocation and subsequent transactivation (right). Nrf2 is the master antioxidative factor. iASPP-induced Nrf2 transactivation contributes to iASPP-mediated ROS inhibition, cancer growth, and drug resistance (bottom).
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