Journal: bioRxiv

Article Title: The p53 Protein is a Suppressor of Atox1 Copper Chaperon in Tumor Cells Under Genotoxic Effects

doi: 10.1101/2023.07.25.550476

Figure Lengend Snippet: Dependence of Atox1 and p53 level in HCT116 and A549 cell lines with different TP53 status: A - immunoblotting with antibodies to p53, p21, and Atox1; beta-actin was used as a normalization. A densitometric analysis of the obtained data is shown below. B – RT-qPCR analysis with primers for TP53, CDKN1A, and ATOX1 genes; GAPDH gene was used as a reference. C - immunofluorescence staining with primary antibodies to Atox1, secondary antibodies with AlexaFluor488. DAPI was used for nuclei staining. WT – wild type cells, TP53 -/- – cells without TP53. For all experiments: n = 3, mean +/− SEM, paired Student t-test, p < 0,05.

Article Snippet: Transformed human cell lines used: HCT116 (colon adenocarcinoma) with intact p53; HCT116p53 -/- with a deletion of both alleles of the TP53 genes, as well as the A549 line with wild (A549) and knockout p53 (A549p53 -/- ) by the CRISPR-Cas9 method, acquired at ATCC.

Techniques: Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The p53 Protein is a Suppressor of Atox1 Copper Chaperon in Tumor Cells Under Genotoxic Effects

doi: 10.1101/2023.07.25.550476

Figure Lengend Snippet: Influence of cytotoxic agents on the activity of Atox1 at different status (WT and KO) of the TP53 gene in A549 and HCT116 cell lines, 24h after drugs exposure. A - immunoblotting with antibodies to p53, p21, and Atox1; beta-actin was used as a normalization. A densitometric analysis of the obtained data is shown below. B – RT-qPCR analysis with primers for TP53, CDKN1A and ATOX1 genes; GAPDH gene was used as a reference, C - immunofluorescence staining with primary antibodies to Atox1, secondary antibodies with AlexaFluor488. DAPI was used for nuclei staining. DOX – doxorubicin (0,1μM), CIS – cisplatin (35μM), PMA – phorbol-12-myristate- 13-acetate (80nM), H 2 O 2 – hydrogen peroxide (450μM), BLE – bleomycin (10μM). WT – wild type cells, TP53 -/- – cells without TP53. For all experiments: n = 3, mean +/− SEM, two-way ANOVA, p < 0,05.

Article Snippet: Transformed human cell lines used: HCT116 (colon adenocarcinoma) with intact p53; HCT116p53 -/- with a deletion of both alleles of the TP53 genes, as well as the A549 line with wild (A549) and knockout p53 (A549p53 -/- ) by the CRISPR-Cas9 method, acquired at ATCC.

Techniques: Activity Assay, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The p53 Protein is a Suppressor of Atox1 Copper Chaperon in Tumor Cells Under Genotoxic Effects

doi: 10.1101/2023.07.25.550476

Figure Lengend Snippet: Influence of ionizing radiation on the activity of Atox1 at different status (WT and KO) of the TP53 gene in A549 and HCT116 cell lines, 24h after ionizing irradiation (10Gy) exposure. A - immunoblotting with antibodies to p53, p21, and Atox1; beta-actin was used as a normalization. A densitometric analysis of the obtained data is shown below. B – RT-qPCR analysis with primers for TP53, CDKN1A and ATOX1 genes; GAPDH gene was used as a reference. The value of WT 0Gy (control) was taken as a 1.0 for all genes and is not shown in the graphs. For all experiments: n = 3, mean +/− SEM, two-way ANOVA, p < 0,05.

Article Snippet: Transformed human cell lines used: HCT116 (colon adenocarcinoma) with intact p53; HCT116p53 -/- with a deletion of both alleles of the TP53 genes, as well as the A549 line with wild (A549) and knockout p53 (A549p53 -/- ) by the CRISPR-Cas9 method, acquired at ATCC.

Techniques: Activity Assay, Irradiation, Western Blot, Quantitative RT-PCR, Control

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

Journal: Journal of Bacteriology

Article Title: Utilizing Whole Fusobacterium Genomes To Identify, Correct, and Characterize Potential Virulence Protein Families

doi: 10.1128/JB.00273-19

Figure Lengend Snippet: Quantitation of cell-associated F. nucleatum and F. necrophorum to HCT116 cancerous colonocytes. (A) Superresolution imaging of surface bound and intracellular F. nucleatum 23726 in HCT116 CRC cells. (B) Quantitation of genes in six potential virulence gene families in F. nucleatum 23726, F. necrophorum 25286, and F. necrophorum 1_1_36S. (C) HCT116 binding assays where percent invasion is determined as the number of bacteria recovered as live CFU after incubation and washing compared to the number of input bacteria.

Article Snippet: In (top panel), a confluent monolayer of healthy fetal human colonocytes (FHC; ATCC CRL-1381) were grown in Dulbecco modified Eagle medium (DMEM)–F-12 medium supplemented with 10% FBS (Atlanta Biologicals) in 1.5 coverslip glass-bottom plates.

Techniques: Quantitation Assay, Imaging, Binding Assay, Bacteria, Incubation