Journal: Cancers

Article Title: Multiple Components of Protein Homeostasis Pathway Can Be Targeted to Produce Drug Synergies with VCP Inhibitors in Ovarian Cancer.

doi: 10.3390/cancers14122949

Figure Lengend Snippet: Figure 6. Mifepristone activates the HRI kinase pathway. (A): Mouse embryonic fibroblast generated from PERK knockout mice (PERK-KO-DR) and mouse embryonic fibroblast generated from GCN2 knockout mice (GCN2-KO-DR) were incubated with 20 µM of mifepristone for 6 and 12 h. Vehicle (V) and TN (2 µM-Tunicamycin) treatments were performed for 12 h and lysates were probed with indicated antibodies. β-actin was used as loading control. (B): RMG1 cells were transiently transfected with scrambled (Scr) and small interfering RNAs (siRNAs) targeting HRI (si-HRI) and PKR (si-PKR) for 48 h followed by treatment of 20 µM of mifepristone for 6 and 10 h. Protein lysates were then subjected to immunoblotting with indicated antibodies. β-actin was used as loading control. All experiments were performed in at least two biological replicates. Uncropped immunoblots can be found in Figure S12.

Article Snippet: The primary antibodies, purchased from Cell Signaling Technology (Danvers, MA, USA), included PARP (#9542), total caspase-3 (#9665), cleaved caspase-3 (#9661), Grp78 (#3177), CHOP (#5554), β-actin (#3700), ubiquitin (#3933), ATF6 (#65880), PERK (#3192), ATF4 (#11815), IRE1α (#3294), XBP1 (#12782), GCN2 (#3302) and PKR (#12297), except p-eIF2α (ab32157, Abcam) and p-IRE1α (nb100-2323, Novus, Centennial, CO, USA).

Techniques: Generated, Knock-Out, Incubation, Control, Transfection, Western Blot

Journal: Cancers

Article Title: Multiple Components of Protein Homeostasis Pathway Can Be Targeted to Produce Drug Synergies with VCP Inhibitors in Ovarian Cancer.

doi: 10.3390/cancers14122949

Figure Lengend Snippet: Figure 8. Putative mechanism for synergistic cytotoxicity between CB-5083 and mifepristone. Treat- ment with VCP inhibitor-CB-5083 activates the activating transcription factor 6 (ATF6) branch and the protein kinase R-like endoplasmic reticulum kinase (PERK) branch. Activation of ATF6 can result in the induction of glucose-regulated protein 78 (Grp78). Induction of Grp78 is considered a pro-survival response. Similarly, PERK activation can ultimately induce CCAAT/enhancer-binding protein homologous protein (CHOP). Enhanced expression of CHOP can be a pro-apoptotic response. Mifepristone treatment inhibits the ATF6 branch and enhances CHOP expression through the acti- vation of the heme-regulated inhibitor (HRI) kinase pathway. The combination of these two effects could result in the enhanced cytotoxicity seen with CB-5083 and mifepristone combination.

Article Snippet: The primary antibodies, purchased from Cell Signaling Technology (Danvers, MA, USA), included PARP (#9542), total caspase-3 (#9665), cleaved caspase-3 (#9661), Grp78 (#3177), CHOP (#5554), β-actin (#3700), ubiquitin (#3933), ATF6 (#65880), PERK (#3192), ATF4 (#11815), IRE1α (#3294), XBP1 (#12782), GCN2 (#3302) and PKR (#12297), except p-eIF2α (ab32157, Abcam) and p-IRE1α (nb100-2323, Novus, Centennial, CO, USA).

Techniques: Activation Assay, Binding Assay, Expressing

Journal: Oncogene

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

doi: 10.1038/onc.2010.153

Figure Lengend Snippet: (A) PERK protein levels were measured by immunoprecipitation (IP) followed by Western blot analysis in the following cell lines: MCF10A (1), MCF7 (2), T47D (3), MDA-MB231 (4), MDA-MB468 (5), TE3 (6), TE7 (7), KYSE 520 (8). (B) PERK protein levels following shRNA targeting of PERK. (C) Parental MDA-MB468 cell line, shPERK-transduced cells (shPERK), and shPERK-transduced cells reconstituted with mouse Myc-PERK (+mPERK) were treated with 2μg/ml tunicamycin for the indicated intervals. Western analysis for ATF4, CHOP, or β-actin. (D) Volume of orthotopic tumors formed from the mouse mammary tumor-derived cells transduced in vitro with empty vector virus (Neu/PERK loxP/loxP ) or Cre-expressing retrovirus (Neu/PERK Δ/Δ ) 28 days post-transplant (n=4). Representative image of tumors are provided. All p-values determined by Student t-test. (E) Western analysis of transgenic ErbB2 and PERK expression following infection of mouse mammary tumor-derived cells with control (Neu/PERK loxP/loxP ) or Cre-expressing retrovirus (Neu/PERK Δ/Δ ). Thapsigargin treatment (50nM, 1h) was used to demonstrate that PERK is functional.

Article Snippet: Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19 ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore).

Techniques: Immunoprecipitation, Western Blot, shRNA, Derivative Assay, In Vitro, Plasmid Preparation, Virus, Expressing, Transgenic Assay, Infection, Control, Functional Assay

Journal: Oncogene

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

doi: 10.1038/onc.2010.153

Figure Lengend Snippet: (A) MDA-MB468 cells were infected with control shRNA or anti-PERK shRNA for the indicated intervals. Cells were pulsed with BrdU 45 min prior to harvest for FACS analysis. (B) Kinetics of growth of the MDA-MB468 parental cell line, control shRNA-(shControl) or shPERK-transduced cells (shPERK), and shPERK-transduced cells reconstituted with mouse Myc-PERK (+mPERK). PERK protein levels following expression of shRNA targeting human PERK and reconstitution with mouse Myc-PERK are shown. (C) Proliferation rates in mammary gland sections from control (PERK loxP/loxP ) and mammary gland-specific PERK knockout mice (PERK Δ/Δ ) on pregnancy day 16 (P16) and lactation day 3 (L3) were determined by immunohistochemistry for BrdU (animals were injected with BrdU 1 h prior to being euthanized). (D) Quantification of BrdU-positive cells from (C) is shown; error bars indicate S.D. among 3 animals, 5 acini were counted per animal.

Article Snippet: Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19 ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore).

Techniques: Infection, Control, shRNA, Expressing, Knock-Out, Immunohistochemistry, Injection

Journal: Oncogene

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

doi: 10.1038/onc.2010.153

Figure Lengend Snippet: (A) Immunofluorescence staining for DNA damage-induced foci containing phospho-ATM and phospho-Chk2 following acute PERK knockdown (72 h after infection) in MDA-MB468 cells. (B) Quantification of phospho-ATM positive cells (>3 foci) is shown; error bars indicate S.D. from 3 slides, 5 fields were counted per slide. p-value was determined by Student t-test. (C) Western analysis of DNA damage response-associated markers following PERK knockdown. (D) IP/kinase assays assessing CDK2-dependent phosphorylation of histone H1 (bottom panel). CDK2 complexes were immunoprecipitated from MDA-MB 468 cells treated as indicated. PERK levels were assessed by IP/immunoblot and CDK2 recovery in precipitates was assessed by CDK2 immunoblot (middle panel).

Article Snippet: Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19 ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore).

Techniques: Immunofluorescence, Staining, Knockdown, Infection, Western Blot, Phospho-proteomics, Immunoprecipitation

Journal: Oncogene

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

doi: 10.1038/onc.2010.153

Figure Lengend Snippet: (A) Quantitative real time PCR analysis of Nrf2 target genes NQO1 and GCLC in the indicated cell lines asynchronously proliferating under standard conditions. (B) Purified recombinant Nrf2-Neh2 domain of WT, T80A, S40A or T80A/S40A, was incubated with purified recombinant ΔN-PERK in the in vitro kinase assay. Phosphorylated Nrf2-Neh2 was detected by autoradiography (upper panel). (C) 293T cells were transfected with WT Nrf2 or Nrf2-T80A. 24 hours after transfection, cells were left untreated (C) or treated with tunicamycin (Tu) for 2 hours followed by immunoprecipitation with anti-Nrf2 antibody. Threonine phosphorylation was detected using a phospho-Thr reactive antibody. Nrf2 in the IP and the whole cell lysate (WCL) was detected with Nrf2 specific antibody. (D) Proliferation of the indicated cell lines was assessed by a 6-day growth curve under standard tissue culture conditions as described in materials and methods. PERK levels were detected by IP/Western blot analysis. (E) Oxidized guanine in damaged DNA was detected by a FITC-conjugated 8-OxoG binding peptide in PERK knockdown cells infected with pBabe control vector (shPERK) or Nrf2-HA (shPERK Nrf2-HA). Quantification of 8-OxoG positive cells is provided. Error bars represent S.D. from 3 experiments. (F) 8-OxoG was detected in PERK knockdown cells transfected with scramble siRNA (Scrm), or keap1 siRNA (sikeap1). Error bars in graphs represent S.D. from 3 experiments. Western blot panels demonstrate levels of Nrf2-HA and Keap1 in PERK knockdown cells.

Article Snippet: Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19 ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore).

Techniques: Real-time Polymerase Chain Reaction, Purification, Recombinant, Incubation, In Vitro, Kinase Assay, Autoradiography, Transfection, Immunoprecipitation, Phospho-proteomics, Western Blot, Binding Assay, Knockdown, Infection, Control, Plasmid Preparation