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1.
Figure 6

Figure 6. ROS accumulation triggers DNA double strand breaks in PERK-deficient breast cancer cells and tumors. From: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

(A) Immunofluorescent staining of γ-H2AX foci in control or PERK knockdown MDA-MB468 cells. (B) Quantification of γ-H2AX positive (>5 foci) cells under standard tissue culture conditions. Error bars represent S.D. from 3 independent experiments performed in triplicate. (C) Quantification of the COMET tail moment in control or PERK knockdown cells under standard tissue culture conditions. Error bars indicate S.D. from 3 experiments.

Ekaterina Bobrovnikova-Marjon, et al. Oncogene. ;29(27):3881-3895.
2.
Figure 4

Figure 4. Increased levels of Reactive Oxygen Species (ROS) in PERK knockdown cells contribute to reduced kinetics of cell growth. From: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

(A) DCF fluorescence measured by FACS using CM-H2DCFDA dye. (B) Mean values of ROS determined from three independent experiments. Uninfected (black), stably infected with empty vector (blue) or shPERK (red) in both (A) and (B). (C) Growth analysis of MDA-MB468 parental cell line, cells transduced with control shRNA, shPERK, or shPERK and reconstituted with mouse Myc-PERK in the presence of ROS scavenger N-acetylcysteine (NAC).

Ekaterina Bobrovnikova-Marjon, et al. Oncogene. ;29(27):3881-3895.
3.
Figure 3

Figure 3. PERK knockdown triggers DNA damage response signaling pathway. From: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

(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).

Ekaterina Bobrovnikova-Marjon, et al. Oncogene. ;29(27):3881-3895.
4.
Figure 5

Figure 5. ROS accumulation triggers oxidative DNA lesions in PERK-deficient breast cancer cells and tumors. From: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

(A) Detection of 8-oxoguanine oxidized DNA adduct (8-OxoG) using a FITC conjugated 8-OxoG binding peptide in parental MDA-MB468 cells, MDA-MB468 cells transduced with control shRNA, shPERK, or shPERK and reconstituted with mouse Myc-PERK. (B) Quantification of 8-oxoG positive cells from 3 independent experiments. (C) 8-OxoG in paraffin sections from tumors formed by parental MDA-MB468 cells, MDA-MB468 cells transduced with control shRNA, or shPERK. (D) Quantification of 8-OxoG positive cells shown in (C) is provided and error bars indicate S.D. from 4 animals. (E) Detection of 8-OxoG in paraffin sections from orthotopic tumors formed by mouse mammary tumor-derived cells transduced with empty vector (Neu/PERKloxP/loxP) or retrovirus expressing Cre recombinase (Neu/PERKΔ/Δ). (F) Quantification of 8-OxoG positive cells from (E); error bars indicate S.D. from 4 animals. All p-values determined by Student t-test.

Ekaterina Bobrovnikova-Marjon, et al. Oncogene. ;29(27):3881-3895.
5.
Figure 2

Figure 2. PERK knockdown triggers a G2/M delay. From: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

(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 (PERKloxP/loxP) and mammary gland-specific PERK knockout mice (PERKΔ/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.

Ekaterina Bobrovnikova-Marjon, et al. Oncogene. ;29(27):3881-3895.
6.
Figure 1

Figure 1. PERK expression is maintained in cancer cells wherein it regulates tumor expansion in vivo. From: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

(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/PERKloxP/loxP) or Cre-expressing retrovirus (Neu/PERKΔ/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/PERKloxP/loxP) or Cre-expressing retrovirus (Neu/PERKΔ/Δ). Thapsigargin treatment (50nM, 1h) was used to demonstrate that PERK is functional.

Ekaterina Bobrovnikova-Marjon, et al. Oncogene. ;29(27):3881-3895.
7.
Figure 8

Figure 8. PERK loss attenuates MMTV-Neu-driven mammary tumorigenesis in mice, but promotes spontaneous mammary tumor formation in aged mammary gland-specific PERK knockout mice. From: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

(A) Kaplan-Meier analysis of tumor-free survival for cohorts of MMTV-Neu/PERKloxP/loxP (n=21) and MMTV-Neu/PERKΔ/Δ Δ/Δ (n=27) mice. (B) Hematoxylin and eosin staining demonstrating histology of control (Neu/PERKloxP/loxP) and PERK knockout (Neu PERKΔ/Δ) mammary gland tumors. (C) Western analysis for PERK, ErbB2, and eIF4E levels on whole protein extracts from control (Neu/PERKloxP/loxP) and PERK knockout (Neu/PERKΔ/Δ) mammary gland tumors or mammary gland from lactating PERKloxP/loxP dam (L10). (D) Nrf2 was precipitated from tumor lysates prepared from MMTV-Neu/PERKΔ/Δ or control mice and blotted for phospho-Thr and Nrf2. PERK expression was determined by immunoblot. (E) PERK excision delays development of Neu-driven hyperplastic lesions. Representative mammary glands from 9- to 14-months old control (Neu/PERKloxP/loxP) and PERK knockout (Neu/PERKΔ/Δ) mice revealing pre-malignant lesions are shown. (F) Hematoxylin and eosin staining on lungs from control (Neu/PERKloxP/loxP, n=24) and PERK knockout (Neu/PERKΔ/Δ, n=27) mice revealing metastatic lesions. (G) Troma-1 (cytokeratin-8) staining on lung specimens containing metastatic foci. LT=lung tissue; Met=metastasis; OL=overlay. (H) Hematoxylin and eosin staining for tumor histology and whole mount of hyperplastic lesions in mammary glands of PERKΔ/Δ aged females. (I) qRT-PCR for ErbB2 on genomic DNA from PERKΔ/Δ tumors and FISH analysis on paraffin sections from the same animals. Levels of ErbB2 in tumors were compared to matched spleen tissues.

Ekaterina Bobrovnikova-Marjon, et al. Oncogene. ;29(27):3881-3895.
8.
Figure 7

Figure 7. Reduced activity of Nrf2 causes increased oxidative stress in PERK knockdown cells. From: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

(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.

Ekaterina Bobrovnikova-Marjon, et al. Oncogene. ;29(27):3881-3895.

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