Journal: Nature cell biology

Article Title: ATF4 couples MYC-dependent translational activity to bioenergetic demands during tumor progression

doi: 10.1038/s41556-019-0347-9

Figure Lengend Snippet: a. Immunoblot of B cells isolated from WT (n=3), pre-lymphoma Eμ-myc (n=3) or lymphoma bearing Eμ-myc mice (n=6), lysates from different mice per group were used. b . Immunoblot from two normal human B cells (NHBC) and Burkitt’s lymphoma cell lines. c. Immunoblot from normal human colonocytes (NHC) or colon cancer cell lines. d. Immunoblot from MCF10 (non-tumorigenic breast epithelial cell line) or breast cancer cell lines. e. Pearson correlation between EIF4EBP1 and ATF4 target gene expression in Diffused Large B cell Lymphoma (DLBCL, n=562), Colorectal Adenocarcinoma (COAD, n=329), Breast Cancer (BRCA, n=1218) and Sarcoma (SARC, n=265). The center lines depict linear regression lines and shaded regions are 95% confidence intervals for regression lines. Datasets analyzed are listed in the section. Previously known ATF4 target gene list used in this analysis is shown in table. f. Kaplan-Meier plots of progression free survival (left) and overall survival (right) of DLBCL patients with high or low EIF4EBP1 expression. The survival analysis using Kaplan-Meier and two-sided log-rank test between high and low EIF4EBP1 expression groups was performed on all the patients with records of progression-free survival (left, n= 173) and overall survival (right, n=171), respectively. g. Proposed model of ATF4 and MYC co-operation in tumor progression. All immunoblots are from n=1 and unprocessed scans of blots are shown in .

Article Snippet: Normal human colonocytes were purchased from Cell Applications Inc (732Cn-10a).

Techniques: Western Blot, Isolation, Targeted Gene Expression, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Modulation of Nuclear Factor E2-related Factor-2 (Nrf2) Activation by the Stress Response Gene Immediate Early Response-3 ( IER3 ) in Colonic Epithelial Cells

doi: 10.1074/jbc.M113.490920

Figure Lengend Snippet: IER3 interferes with Nrf2 activation in human NCM460 colonocytes. NCM460 cells were transfected with an IER3 expression vector or the empty vector (mock) and then analyzed for Nrf2 activation. a, nuclear and cytoplasmic extracts from untreated or tBHQ- (50 μm, 16 h) or SFN- (10 μm, 16 h) treated cells were analyzed by Nrf2 Western blotting (lamin A/C and tubulin, respectively, served as loading control). Three of six biological replicate experiments are shown from which n-fold band intensities of Nrf2 (100 kDa) were determined by densitometry analysis normalizing to the corresponding loading controls (mean ± S.D. (error bars), n = 6). b, ARE-Luc assays were conducted in IER3-transfected or untransfected cells subject of treatment (16 h) with 50 μm tBHQ or 10 μm SFN or not. Data are expressed as ARE-specific relative luciferase units (RLU) and represent the mean ± S.D., n = 4. c, NQO1 and GCLC mRNA levels were analyzed by qPCR (using TATA-binding protein (TBP) mRNA for normalization) in IER3-transfected or untransfected cells subjected to treatment (16 h) with 50 μm tBHQ or 10 μm SFN or not. Data represent the mean ± S.D., n = 4. d, total cell lysates from untreated or tBHQ- (50 μm, 24 h) or SFN- (10 μm, 24 h) treated cells were analyzed by Western blotting for NQO1 and GCLC as well as IER3 expression (Hsp90 served as loading control). Three of four biological replicate experiments are shown from which n-fold band intensities were determined by densitometry analysis normalizing to Hsp90 (mean ± S.D., n = 4). * indicates statistical significance between IER3 and mock transfectants.

Article Snippet: Cell Lines and Animals Human NCM460 colonocytes ( 41 ) were purchased from INCELL Corp. (San Antonio, TX) and cultured as described ( 12 ).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Luciferase, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Modulation of Nuclear Factor E2-related Factor-2 (Nrf2) Activation by the Stress Response Gene Immediate Early Response-3 ( IER3 ) in Colonic Epithelial Cells

doi: 10.1074/jbc.M113.490920

Figure Lengend Snippet: Increased Nrf2 activation in human NCM460 colonocytes with suppressed IER3 expression. NCM460 stably transfected with an IER3-shRNA or control-shRNA vector were analyzed for Nrf2 activation. a, nuclear and cytoplasmic extracts from untreated or tBHQ- (50 μm, 16 h) or SFN- (10 μm, 16 h) treated cells were analyzed by Nrf2 Western blotting (lamin A/C and tubulin, respectively, served as loading control). Three of six biological replicate experiments are shown from which n-fold band intensities of Nrf2 (100 kDa) were determined by densitometry analysis normalizing to the corresponding loading controls (mean ± S.D. (error bars), n = 6). b, ARE-luciferase assays were conducted in IER3 shRNA- or control shRNA-transfected cells subjected to treatment (16 h) with 50 μm tBHQ or 10 μm SFN or not. Data are expressed as ARE-specific relative luciferase units (RLU) and represent the mean ± S.D., n = 4. c, NQO1 and GCLC as well as IER3 mRNA levels were analyzed by qPCR (using TATA-binding protein (TBP) mRNA for normalization) in IER3 shRNA- or control shRNA-transfected cells subjected to treatment (16 h) with 50 μm tBHQ or 10 μm SFN or not. Data represent the mean ± S.D., n = 6. d, total cell lysates from untreated or tBHQ- (50 μm, 24 h) or SFN- (10 μm, 24 h) treated cells were analyzed by Western blot for NQO1 and GCLC expression (Hsp90 served as loading control). Three of six biological replicate experiments are shown from which n-fold band intensities were determined by densitometry analysis normalizing to Hsp90 (mean ± S.D., n = 6).* indicates statistical significance between IER3 shRNA- and control shRNA-expressing cells.

Article Snippet: Cell Lines and Animals Human NCM460 colonocytes ( 41 ) were purchased from INCELL Corp. (San Antonio, TX) and cultured as described ( 12 ).

Techniques: Activation Assay, Expressing, Stable Transfection, Transfection, shRNA, Control, Plasmid Preparation, Western Blot, Luciferase, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Modulation of Nuclear Factor E2-related Factor-2 (Nrf2) Activation by the Stress Response Gene Immediate Early Response-3 ( IER3 ) in Colonic Epithelial Cells

doi: 10.1074/jbc.M113.490920

Figure Lengend Snippet: Decreased ROS level in human NCM460 colonocytes with suppressed IER3 expression depending on Nrf2. a–c, NCM460 cells overexpressing IER3 or not (mock) (a) or NCM460 cells stably transfected with control or IER3 shRNA (b and c) were subjected to tBHQ treatment (24 h), or not, and then stained with cH2DCFdA to detect intracellular ROS (a and b) or with MitoSOX Red to detect mitochondrial ROS (c). Fluorescence was determined 4 h later; data represent the mean ± S.D. (error bars), n = 4. d, NCM460 stably transfected with control or IER3 shRNA were treated with control or Nrf2 siRNA for 40 h. Then, tBHQ was added, or not, for 24 h followed by cH2DCFdA staining and fluorescence measurement 4 h later; data represent the mean ± S.D., n = 6. * indicates statistical significance between the IER3 shRNA- and control shRNA-expressing cells.

Article Snippet: Cell Lines and Animals Human NCM460 colonocytes ( 41 ) were purchased from INCELL Corp. (San Antonio, TX) and cultured as described ( 12 ).

Techniques: Expressing, Stable Transfection, Transfection, Control, shRNA, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Modulation of Nuclear Factor E2-related Factor-2 (Nrf2) Activation by the Stress Response Gene Immediate Early Response-3 ( IER3 ) in Colonic Epithelial Cells

doi: 10.1074/jbc.M113.490920

Figure Lengend Snippet: Greater Nrf2 activity in IER3-deficient human NCM460 colonocytes confers apoptosis protection. a, NCM460 cells stably transfected with control or IER3 shRNA and subjected to tBHQ treatment (24 h), or not, were left untreated or treated with either 10 ng/ml TRAIL (8 h) or 20 μg/ml etoposide (24 h). Caspase assays were conducted, and apoptosis was expressed as n-fold of untreated, mean ± S.D. (error bars); n = 4. b, IER3 shRNA or control shRNA NCM460 cells were treated with control or Nrf2 siRNA. After 24 h, tBHQ was added, or not, followed by TRAIL or etoposide treatment 24 h later. Caspase assays were conducted and apoptosis was expressed as n-fold of untreated, mean ± S.D.; n = 4. * indicates statistical significance between the IER3 shRNA- and control shRNA-expressing cells.

Article Snippet: Cell Lines and Animals Human NCM460 colonocytes ( 41 ) were purchased from INCELL Corp. (San Antonio, TX) and cultured as described ( 12 ).

Techniques: Activity Assay, Stable Transfection, Transfection, Control, shRNA, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Modulation of Nuclear Factor E2-related Factor-2 (Nrf2) Activation by the Stress Response Gene Immediate Early Response-3 ( IER3 ) in Colonic Epithelial Cells

doi: 10.1074/jbc.M113.490920

Figure Lengend Snippet: Increased clonal growth of IER3-deficient NCM460 cells. NCM460 cells stably transfected with control or IER3 shRNA were seeded at a density of 200 or 500 cells/well on a 6-well plate and cultured for 1–2 weeks in the absence or presence of 50 μm tBHQ. Then, cells were fixed and stained with crystal violet. Visualized colonies with a diameter of >0.25 mm were counted, and the plating efficiency was calculated. Representative results (left panel) of four independent experiments performed in duplicates are shown, and the evaluation was carried out using the mean values ± S.D. (error bars, right panel) from these duplicate experiments. * indicates statistical significance between the IER3 shRNA- and control shRNA-expressing cells.

Article Snippet: Cell Lines and Animals Human NCM460 colonocytes ( 41 ) were purchased from INCELL Corp. (San Antonio, TX) and cultured as described ( 12 ).

Techniques: Stable Transfection, Transfection, Control, shRNA, Cell Culture, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Modulation of Nuclear Factor E2-related Factor-2 (Nrf2) Activation by the Stress Response Gene Immediate Early Response-3 ( IER3 ) in Colonic Epithelial Cells

doi: 10.1074/jbc.M113.490920

Figure Lengend Snippet: Elevated Akt phosphorylation in IER3-deficient murine or human colonocytes and Akt dependence of the IER3 effect on Nrf2 activation. a, tissue sections from DSS-treated Ier3−/− or Ier3+/+mice were submitted to P-Akt immunofluorescence staining and DAPI counterstaining. b, colon organ cultures from untreated Ier3−/− or Ier3+/+ mice (data from two independent experiments are shown) were treated with 50 μm tBHQ, and cell lysates were analyzed by Western blotting detecting phospho-Akt, Akt, and tubulin. n-fold protein band intensities of Akt and P-Akt were determined by densitometry analysis normalizing to tubulin. c, NCM460 cells stably transfected with control or IER3 shRNA were incubated with 50 μm tBHQ or 10 μm SFN for 4 h or not. Cell lysates were analyzed by Western blotting for P-Akt (Hsp90 served as loading control) and three replicate of four experiments are shown. n-fold protein band intensities of P-Akt (lower panel) were determined by densitometry analysis normalizing to the Hsp90 (mean ± S.D. (error bars); n = 4). d and e, ARE-Luc assays were conducted with NCM460 cells stably transfected with control or IER3 shRNA and treated with 50 μm tBHQ or 10 μm SFN for 16 h, or not, either preincubated with 25 μm LY294002 for 1 h or not (d) or following pretreatment with Akt or control (co) siRNA for 48 h (e). The knockdown was verified by Western blotting and protein band densitometry (lower panel). Data are expressed as n-fold ARE-specific relative luciferase units (RLU) and represent the mean ± S.D., n = 4. * indicates statistical significance between the IER3 shRNA- and control shRNA-expressing cells.

Article Snippet: Cell Lines and Animals Human NCM460 colonocytes ( 41 ) were purchased from INCELL Corp. (San Antonio, TX) and cultured as described ( 12 ).

Techniques: Phospho-proteomics, Activation Assay, Immunofluorescence, Staining, Western Blot, Stable Transfection, Transfection, Control, shRNA, Incubation, Knockdown, Luciferase, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Modulation of Nuclear Factor E2-related Factor-2 (Nrf2) Activation by the Stress Response Gene Immediate Early Response-3 ( IER3 ) in Colonic Epithelial Cells

doi: 10.1074/jbc.M113.490920

Figure Lengend Snippet: IER3 deficiency affects the nuclear accumulation of Fyn and its inhibitory effect on Nrf2 activation. a, colon organ cultures from untreated Ier3−/− or Ier3+/+ mice (data from two independent experiments are shown) were left untreated or were treated with 50 μm tBHQ for 8 h. Nuclear extracts were analyzed by Western blotting for Fyn (lamin A/C served as loading control). n-fold protein band intensities were determined by densitometry analysis normalizing to the corresponding loading control. b, nuclear and cytoplasmic extracts from untreated or tBHQ- (50 μm, 8 h) or SFN- (10 μm, 8 h) treated cells were analyzed by Fyn Western blotting (lamin A/C and tubulin, respectively, served as loading control). Three of six biological replicate experiments are shown from which n-fold band intensities of Fyn were determined by densitometry analysis normalizing to the corresponding loading controls (mean ± S.D. (error bars), n = 6). c, after pretreatment with Fyn or control (co) siRNA for 48 h, ARE-Luc assays were conducted with NCM460 cells stably transfected with control or IER3 shRNA and incubated with 50 μm tBHQ or 10 μm SFN for 16 h, or not. The knockdown was verified by Western blotting and protein band densitometry (lower panel). Data are expressed as n-fold ARE-specific relative luciferase units (RLU) and represent the mean ± S.D., n = 4. * indicates statistical significance between the IER3 shRNA- and control shRNA-expressing cells.

Article Snippet: Cell Lines and Animals Human NCM460 colonocytes ( 41 ) were purchased from INCELL Corp. (San Antonio, TX) and cultured as described ( 12 ).

Techniques: Activation Assay, Western Blot, Control, Stable Transfection, Transfection, shRNA, Incubation, Knockdown, Luciferase, Expressing