Journal: Cancer cell

Article Title: Stress Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas

doi: 10.1016/j.ccell.2017.10.011

Figure Lengend Snippet: (A) Gross morphology of pancreata from KrasG12D and KrasG12D;IkkαΔpan mice, whose age is indicated. Arrow – pancreas. (B) H&E and amylase stained pancreatic sections from above mice. Scale bars: 100 µm. (C) Tumor latency (n = 6). (D) Kaplan-Meier survival curves (n = 6). (E) IB analysis of pancreatic lysates from 5-week-old mice. (F) Q-RT-PCR of Sqstm1 mRNA in 5-week-old mice of indicated genotypes (n = 5). (G) Q-RT-PCR of NRF2 target genes in RNA isolated from acinar and ductal cells of 5-week-old mice of indicated genotypes (n = 5). (H, I) Quantification of lesions per high power (200×) field (HPF) in 5- and 8-week-old mice of indicated genotypes (n = 5) for ADM (H) and PanIN (I). (J) Quantification of Ki67 staining in pancreata of 5-week-old mice. (K) IB analysis of pancreatic lysates. Loading control (ERK) is in panel E. (L) IHC of Sox9 and CK19 in pancreata of 5-week-old mice. Scale bars: 25 µm. (M) IHC of NQO1, MDM2 and HES1 in ductal progenitor lesions of 5-week-old mice. Scale bars: 25 µm. (N) p53 IB of pancreatic lysates from 5-week-old mice. Results in C and F–J are mean ± SEM; **, p < 0.01; ***, p < 0.001; ND, not detected. Statistical significance was calculated using Student’s t test (C, F–J) or log-rank test (D). See also Figure S2.

Article Snippet: MIA PaCa-2 cells seeded in 24-well plates at a 2 × 10 4 cells/well were transfected with Lipofectamine 3000 reagent (Thermo Fisher Scientific, L3000015) with the following plasmids and sh-RNAs: pGL3Basic-Mdm2-T1 (Addgene, Plasmid# 32365) ( Chang et al., 2004 ) and its corresponding ARE mutant plasmid PGL3Basic-Mdm2-ARE-mut obtained by site-directed mutagenesis, pRL-TK control plasmid (Promega, E2241), WT p62 expression vector ( Umemura et al., 2016 ), pCDNA3-Myc3-Nrf2 (Addgene, Plasmid# 21555) ( Furukawa and Xiong, 2005 ) , Flag-p53/pRK5 (Addgene 39237) ( Jiang et al., 2011 ) , NRF2-shRNA (Santa Cruz, sc-37030-SH), Control-shRNA (Santa Cruz, sc-108060) or treated with 10 μM sulforaphane (Millipore Sigma, S4441) or DMSO as control.

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Isolation, Control

Journal: Cancer cell

Article Title: Stress Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas

doi: 10.1016/j.ccell.2017.10.011

Figure Lengend Snippet: (A) Quantification of ADM and PanIN1 density, amylase and Ki67 staining of pancreatic sections of indicated 5-week-old mice (n = 5). (B) H&E staining, amylase IHC, amylase and CK19 co-IF, and Sox9 IHC of pancreatic sections from indicated 5-week-old mice. Scale bars: 25 µm. (C) Kaplan-Meier survival curves of indicated mouse strains (n = 10). (D) Q-RT-PCR analysis of mRNA in acinar and ductal cell fractions from indicated 5-week-old mice (n = 5). (E) NQO1, MDM2 and HES1 IHC of pancreatic sections from indicated 5-week-old mice. Scale bars: 25 µm. (F) p53 IB analysis of pancreatic lysates from 5-week-old mice of indicated genotypes. (G) Frequency of ALDH expression in EpCAM+ cells from 8-week-old KrasG12D (n = 3), KrasG12D;IkkαΔpan (n = 7), and KrasG12D;Ikkα/p62Δpan (n = 4) mice. (H) Sphere-forming capacity of isolated ALDH+ cells from indicated genotypes (n = 3 per group). (I) Sphere formation of control and p62-ablated MIA PaCa-2 and Capan-2 cells with or without NICD1 overexpression. (J) Representative images and quantified SA-β-gal staining and γ-H2AX and ERK1/2 IB of cells as in I. Scale bars: 100 µm. Results in A, D, and G–J are mean ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Statistical significance was calculated using Student’s t test (A, D, G–J) or log-rank test (C). See also Figure S3.

Article Snippet: MIA PaCa-2 cells seeded in 24-well plates at a 2 × 10 4 cells/well were transfected with Lipofectamine 3000 reagent (Thermo Fisher Scientific, L3000015) with the following plasmids and sh-RNAs: pGL3Basic-Mdm2-T1 (Addgene, Plasmid# 32365) ( Chang et al., 2004 ) and its corresponding ARE mutant plasmid PGL3Basic-Mdm2-ARE-mut obtained by site-directed mutagenesis, pRL-TK control plasmid (Promega, E2241), WT p62 expression vector ( Umemura et al., 2016 ), pCDNA3-Myc3-Nrf2 (Addgene, Plasmid# 21555) ( Furukawa and Xiong, 2005 ) , Flag-p53/pRK5 (Addgene 39237) ( Jiang et al., 2011 ) , NRF2-shRNA (Santa Cruz, sc-37030-SH), Control-shRNA (Santa Cruz, sc-108060) or treated with 10 μM sulforaphane (Millipore Sigma, S4441) or DMSO as control.

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Control, Over Expression

Journal: Cancer cell

Article Title: Stress Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas

doi: 10.1016/j.ccell.2017.10.011

Figure Lengend Snippet: (A, B) Q-RT-PCR mRNA analysis primary KrasG12D acinar cells transfected with control or p62 expression vectors with or without p53 (A) or with or without Nutlin-3 (10 µM) treatment (B) (n = 3 each panel). (C) Quantification of duct-like structures formed by KrasG12D primary acinar cells transfected with either empty or p62 and/or p53 expression vectors and cultured for 4 days in Matrigel with or without Nutlin-3 (n = 3). (D) H&E stained pancreatic sections from mice of indicated genotypes treated with DMSO or Nutlin-3 for 21 days (KrasG12D and cerulein-treated KrasG12D) or 14 days (KrasG12D;IkkαΔpan) (n = 7). (E, F) Pancreata of 5-week-old KrasG12D and KrasG12D;IkkαΔpan mice (n = 7 each group), treated as indicated. Pancreas weight (E) and ADM and PanIN lesions (F). (G, H) Sphere formation by Nutlin-3 (10 µM) or DMSO-treated control or Ikkα-ablated KC (G) and KPC (H) cells with or without p62 or NRF2 ablation. (I) Sphere formation of control or IKKα-ablated Capan-2 human PDAC cells with or without p53 overexpression and/or DAPT treatment (10 µM). Results in A–C, E–I are mean ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student’s t test. See also Figure S6.

Article Snippet: MIA PaCa-2 cells seeded in 24-well plates at a 2 × 10 4 cells/well were transfected with Lipofectamine 3000 reagent (Thermo Fisher Scientific, L3000015) with the following plasmids and sh-RNAs: pGL3Basic-Mdm2-T1 (Addgene, Plasmid# 32365) ( Chang et al., 2004 ) and its corresponding ARE mutant plasmid PGL3Basic-Mdm2-ARE-mut obtained by site-directed mutagenesis, pRL-TK control plasmid (Promega, E2241), WT p62 expression vector ( Umemura et al., 2016 ), pCDNA3-Myc3-Nrf2 (Addgene, Plasmid# 21555) ( Furukawa and Xiong, 2005 ) , Flag-p53/pRK5 (Addgene 39237) ( Jiang et al., 2011 ) , NRF2-shRNA (Santa Cruz, sc-37030-SH), Control-shRNA (Santa Cruz, sc-108060) or treated with 10 μM sulforaphane (Millipore Sigma, S4441) or DMSO as control.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Expressing, Cell Culture, Staining, Over Expression

Journal: Cancer cell

Article Title: Stress Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas

doi: 10.1016/j.ccell.2017.10.011

Figure Lengend Snippet: (A) Effects of p53, p62 and NRF2 overexpression, NRF2 silencing and sulforaphane on Mdm2 promoter activity in transfected KPC cells (n = 3). (B) Chromatin immunoprecipitation assays probing NRF2 and small MAF protein recruitment to the MDM2 promoter in WT and NRF2 ablated MIA PaCa-2 (n = 3) cells. (C) Sphere formation of control or MDM2-ablated MIA PaCa-2 and Capan-2 cells with or without NICD1 overexpression. (D) SA-β-gal staining and γ-H2AX IB of cells as in C. (E) Sphere formation of control and p62-ablated MIA PaCa-2 and Capan-2 cells with or without MDM2 CRISPR-mediated activation vector (n = 3). (F) SA-β-gal staining and γ-H2AX IB of cells as in E. (G) Sphere formation of control and NRF2-ablated MIA PaCa-2 and Capan-2 cells with or without MDM2 CRISPR-mediated activation vector (n = 3). (H) SA-β-gal staining and γ-H2AX IB of cells as in G. (I) A scheme explaining how p62 accumulation, operating via the NRF2-MDM2 module leads to dedifferentiation and cell cycle progression in preneoplastic lesions in pancreas. Results are mean ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student’s t test. See also Figure S7.

Article Snippet: MIA PaCa-2 cells seeded in 24-well plates at a 2 × 10 4 cells/well were transfected with Lipofectamine 3000 reagent (Thermo Fisher Scientific, L3000015) with the following plasmids and sh-RNAs: pGL3Basic-Mdm2-T1 (Addgene, Plasmid# 32365) ( Chang et al., 2004 ) and its corresponding ARE mutant plasmid PGL3Basic-Mdm2-ARE-mut obtained by site-directed mutagenesis, pRL-TK control plasmid (Promega, E2241), WT p62 expression vector ( Umemura et al., 2016 ), pCDNA3-Myc3-Nrf2 (Addgene, Plasmid# 21555) ( Furukawa and Xiong, 2005 ) , Flag-p53/pRK5 (Addgene 39237) ( Jiang et al., 2011 ) , NRF2-shRNA (Santa Cruz, sc-37030-SH), Control-shRNA (Santa Cruz, sc-108060) or treated with 10 μM sulforaphane (Millipore Sigma, S4441) or DMSO as control.

Techniques: Over Expression, Activity Assay, Transfection, Chromatin Immunoprecipitation, Control, Staining, CRISPR, Activation Assay, Plasmid Preparation