Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK is highly expressed in and required for Th17 cells. (A) Naïve CD4 T cells were cultured under each subset differentiation conditions for 3 days. The transcript level of Fak was measured by RT-qPCR (left) and the protein level of FAK was measured by immunoblot analysis (right). (B, C) Naïve CD4 T cells from Fak fl/fl mice were introduced with a control empty vector (control) or a CRE recombinase-expressing vector (RV-Cre) to induce Fak deletion and cultured under Th17-polarizing conditions for 3 days (B) or various subset-polarizing conditions (C) for 3 days. (B) GFP+ cells were sorted. The transcript level of Fak was measured by RT-qPCR (left) and the protein level of FAK was measured by immunoblot analysis (right). (C) The expression of GFP, IFN-γ, IL-4, IL-17A, and FOXP3 was analyzed by flow cytometry (top). The statistical analysis was performed on pooled data from five independent experiments (bottom). Error bars represent the standard deviation. The significance of differences between groups was determined by one-way ANOVA (A) and Student t test (B, C) . ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Control, Plasmid Preparation, Expressing, Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK affects Th17 cell differentiation program. (A, B) Naïve CD4 T cells from Fak fl/fl mice were cultured and sorted as Figure 1B . (A) IL-17A+ and FOXP3+ cells were measured by flow cytometry. (B) Transcript levels of Il17a , Rorc , Il23r , and Foxp3 were measured by RT-qPCR. (C, D) Naïve CD4 T cells were introduced with either the control vector (MSCV-LMP) or Fak shRNA vectors (#1, #2, and #3) and cultured under Th17-polarizing conditions for 3 days. (C) The transcript level of Fak was measured by RT-qPCR (left) and protein level of FAK was measured by immunoblot analysis (right). (D) IL-17A+ cells among the vector-transduced cells (GFP+) were measured by flow cytometry (left). GFP+ cells were sorted and the transcript level of Il17a was measured by RT-qPCR (right). All of RT-qPCR data were normalized to Gapdh . (E–G) Naïve CD4 T cells were transduced with control or RV-Cre and cultured under Th17-polarizing conditions for 3 days. GFP+ cells were sorted and subjected to RNA-seq analysis. (E) Scatter plot of RNA-seq data. (F) Gene ontology analysis of differentially expressed genes (DEGs) from control and RV-Cre-transduced Th17 cells. (G) Heatmap of immune/inflammatory response-related genes among the DEGs from control and RV-Cre-transduced Th17 cells. All of RT-qPCR data were normalized to Gapdh . Data in (A–D) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Differentiation, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Control, Plasmid Preparation, shRNA, Western Blot, Transduction, RNA Sequencing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK deficiency ameliorates the severity of EAE. (A, B) Naïve CD4 T cells from WT and Fak fl/fl Rorc cre mice were cultured under polarizing conditions toward each different subset for 3 days. Transcript levels of Ifng for Th1 cells, Il4 for Th2 cells, Il17a for Th17 cells, and Foxp3 for Treg cells were measured by RT-qPCR (A) and the percentage of IL-17A+ cells under Th17-polarizing conditions was measured by flow cytometry (B) . (C) EAE was induced in control (WT, n = 10) and Fak fl/fl Rorc cre (n = 10) mice as described in the Materials and Methods section. The symptoms of EAE were monitored every day and clinical scores were evaluated after EAE induction. (D, E) Histopathological analysis of lumbar spinal cords from control and Fak fl/fl Rorc cre mice at the peak of the disease. (D) H&E stained sections of spinal cords. Arrows indicate the inflammatory foci. (E) Immunohistochemical staining of myelin basic protein (MBP). Arrows indicate the more preserved myelin in Fak fl/fl Rorc cre mice compared with control mice. (F) IL-17A+ and IFNγ+ cells (left) and FOXP3+ cells (right) among CNS-infiltrating CD4 T cells were measured by flow cytometry. (G–J) The percentage and absolute number of CNS-infiltrating mononuclear cells were measured. (G) CD4+ cells, (H) IL-17A+ cells, (I) IFNγ+ cells, and (J) FOXP3+ cells. (K) Transcript levels of Il17a , Rorc , Il23r , Ifng , and Foxp3 in CNS-infiltrating mononuclear cells were measured by RT-qPCR. Data were normalized to Gapdh . Data in (F–K) are pooled from ten independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Flow Cytometry, Control, Staining, Immunohistochemical staining, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK regulates the STAT3 signaling pathway in Th17 cells. (A) Naïve CD4 T cells were cultured under various differentiation conditions for the indicated time periods and the transcript levels of Fak , Il17a , Rorc , and Il17f were measured by RT-qPCR. (B) Naïve CD4 T cells were introduced with control vector or Rorc -expressing vector ( Rorc O/X) and cultured in Th17-polarizing conditions for 3 days. Transcript levels of Rorc , Il17a , and Fak were measured by RT-qPCR. (C) Naïve CD4 T cells were cultured under Th17-polarizing conditions with dose-dependent treatment of GSK805 for 3 days. Transcript levels of Il17a and Fak were measured by RT-qPCR. (D, E) Naïve CD4 T cells from Fak fl/fl mice were cultured as described in Figure 1B and GFP+ cells were sorted. (D) Each protein level was measured by immunoblot analysis (left) and the ratios of pSTAT3/STAT3 and pSTAT5/STAT5 were calculated by densitometry (right). (E) Transcript level of Il2 was measured by RT-qPCR (left) and the protein level of IL-2 from the supernatants was measured by ELISA (right). All of RT-qPCR data were normalized to Gapdh . Data in (A, B, D, E) were pooled from three independent experiments, and data in (C) from five. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. **P < 0.01; ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Control, Plasmid Preparation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK regulates Th17 cell differentiation through the NF-κB pathway. (A) Naïve CD4 T cells from Fak fl/fl mice were transduced with control vector or RV-Cre and cultured under Th17-polarizing conditions for 3 days. GFP+ cells were sorted and rested in normal media for 2 days and serum-free medium for an additional 8 hours. The cells were restimulated with anti-CD3/CD28 antibodies for the indicated time periods, and nuclear/cytoplasmic extracts were prepared. Each protein level was measured by immunoblot analysis (right). The ratios of IκB/β-Actin, pIκB/β-Actin, and nuclear RelA/cytoplasmic RelA were calculated by densitometry (right). (B, C) Naïve CD4 T cells from Fak fl/fl mice were transduced with RV-Cre and cultured under Th17-polarizing conditions for 3 days (B) and 16 hours (C) and GFP+ cells were sorted. (B) Transcript level of Socs3 was measured by RT-qPCR. (C) Relative RelA binding to each indicated locus was measured through ChIP assay. Nuclear extracts from cultured cells were reacted with an anti-RelA antibody, and precipitated DNA fragments were measured by qPCR. Isotype-matching IgG was used as a negative control. (D–F) Il17a promoter activity was measured through luciferase assay. (D) EL4 cells were transfected with pGL3- Il17a promoter vector and control or Fak siRNA, and the cells were rested for 20 hours. After transfection, the cells were divided into non-stimulated or stimulated groups, with the stimulated groups received 4 hour stimulation with PMA/ionomycin. (E) EL4 cells were transfected with the pGL3- Il17a promoter vector and rested for 20 hours with or without PDTC treatment (1 μM). (F) EL4 cells were transfected as described in (D) and rested for 20 hours with 1 μM PDTC treatment. (G–I) Naïve CD4 T cells from Rela fl/fl mice were introduced with a control empty vector (WT) or a Cre recombinase expressing vector (p65 KO) to induce Rela deletion and cultured under Th17-polarizing conditions for 3 days. (G) IL-17A+ and FOXP3+ cells were measured by flow cytometry. (H) GFP+ cells were sorted and transcript level of Rela , Il17a , Rorc , and Il23r were measured by RT-qPCR. (I) Naïve CD4 T cells from Rela fl/fl mice were cultured as described in (G) and additionally treated with vehicle (control) or FAK inhibitor (PND1186, 1 μM). IL-17A+ and FOXP3+ cells were measured by flow cytometry. RT-qPCR data in (B, H) were normalized to Gapdh . Data in (A–I) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Differentiation, Transduction, Control, Plasmid Preparation, Cell Culture, Western Blot, Quantitative RT-PCR, Binding Assay, Negative Control, Activity Assay, Luciferase, Transfection, Expressing, Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: A FAK inhibitor blocks differentiation of Th17 cells in vitro. (A–E) Naïve CD4 T cells were cultured under Th17- or Treg-polarizing conditions with dose-dependent treatment of PND1186 for 3 days. IL-17A+ and FOXP3+ cells were measured by flow cytometry (A) and transcript levels of Il17a and Foxp3 were measured by RT-qPCR (C) in Th17 cells. FOXP3+ cells were measured by flow cytometry (B) and transcript level of Foxp3 was measured by RT-qPCR (D) in Treg cells. (E–G) Naïve CD4 T cells were cultured under Th17-polarizing conditions with vehicle (control) or PND1186 (1 μM) treatment for 3 days (E, G) and the indicated time periods (F) . (E) pSTAT3 and pSTAT5 levels were measured by flow cytometry. (F) Nuclear or cytoplasmic extracts were prepared from cultured cells, and each protein level was measured by immunoblot analysis (right). The ratios of IκB/β-Actin, pIκB/β-Actin, and nuclear RelA/cytoplasmic RelA were calculated by densitometry (right). (G) Relative RelA binding to each indicated locus was measured through ChIP assay. Nuclear extracts from cultured cells were incubated with an anti-RelA antibody and the precipitated DNA fragments were measured by qPCR. An isotype-matching IgG was used as a negative control. RT-qPCR data in (C, D) were normalized to Gapdh . Data in (A–G) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: In Vitro, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Control, Western Blot, Binding Assay, Incubation, Negative Control, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Different Roles of Negative and Positive Components of the Circadian Clock in Oncogene-induced Neoplastic Transformation *

doi: 10.1074/jbc.M115.706481

Figure Lengend Snippet: Primer sets for PCR analysis

Article Snippet: The membranes were reacted with antibodies against activating transcription factor 4 (ATF4; sc-200, Santa Cruz Biotechnology), p16INK4a (sc-1207), p19ARF (sc-22784), retinoblastoma protein (pRB; sc-69791), p53 (sc-6243), PER2 (sc-25368), BMAL1 (sc-48790) ACTIN (sc-1616), or anti-phospho-pRB-Thr826 (44–576; BIOSOURCE International, Camarillo, CA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Different Roles of Negative and Positive Components of the Circadian Clock in Oncogene-induced Neoplastic Transformation *

doi: 10.1074/jbc.M115.706481

Figure Lengend Snippet: Different oncogenic phenotypes of Per2m/m and Bmal1−/− cells after introduction of H-rasV12 and SV40LT. A–C, representative locomotor activity records of wild-type (A), Per2m/m (B), or Bmal1−/− (C) mice. Light regime: 10 days in a 12-h light and 12-h dark cycle followed by 10 days in a constant dark cycle. D, schematic experimental procedure for evaluating the oncogenesis of clock gene-deficient cells. MEFs were prepared from wild-type, Per2m/m, or Bmal1−/− mice. Cells were infected with retroviral vectors expressing H-rasV12 and SV40LT. E, the time course of mRNA expressions of H-rasV12 and SV40LT in wild-type, Per2m/m, or Bmal1−/− cells after infection with retrovirus vectors expressing oncogenes. F, anchorage-independent growth of wild-type, Per2m/m, and Bmal1−/− cells after the concomitant introduction of H-rasV12 and SV40LT. Cells infected with oncogenes were subjected to a soft-agar colony assay, and their colony formation and viability were assessed 14 days after seeding. Control cells were infected with mock vectors. The left panels show representative microscopic photographs of colony formation in each type of cells. The scale bars indicate 100 μm. The right panel shows viability of cells. Values are shown as means ± S.E. (n = 3–6). Mean values of mock-transfected wild-type cells were set at 1.0. ##, p < 0.01 significantly different from the mock-transfected group. **, p < 0.01 significantly different from oncogene-transfected wild-type and Per2m/m cells. G, tumor formation by oncogene-introduced wild-type, Per2m/m, or Bmal1−/− cells. Equal numbers of cells (5 × 106 cells) were implanted into the dorsal air sacs of NOD/SCID mice. Values are the means ± S.E. (n = 4). **, p < 0.01; *, p < 0.05 significantly different from the other groups at the corresponding time points.

Article Snippet: The membranes were reacted with antibodies against activating transcription factor 4 (ATF4; sc-200, Santa Cruz Biotechnology), p16INK4a (sc-1207), p19ARF (sc-22784), retinoblastoma protein (pRB; sc-69791), p53 (sc-6243), PER2 (sc-25368), BMAL1 (sc-48790) ACTIN (sc-1616), or anti-phospho-pRB-Thr826 (44–576; BIOSOURCE International, Camarillo, CA).

Techniques: Activity Assay, Infection, Expressing, Colony Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Different Roles of Negative and Positive Components of the Circadian Clock in Oncogene-induced Neoplastic Transformation *

doi: 10.1074/jbc.M115.706481

Figure Lengend Snippet: Induction of cellular senescence in oncogene-introduced Bmal1−/− cells. A, β-gal staining of wild-type, Per2m/m, or Bmal1−/− cells infected with H-rasV12 and SV40LT. Arrows in the microscopic photograph indicate β-gal-positive cells. The scale bars indicate 50 μm. Values are the means ± S.E. (n = 3). **, p < 0.01 significantly different from other oncogene-introduced cells. B, the mRNA levels of p16Ink4a and p19Arf in wild-type, Per2m/m, or Bmal1−/− cells infected with oncogenes. Values are the means ± S.E. (n = 3). ##, p < 0.01 significantly different from mock-transfected group. **, p < 0.01 significantly different from other oncogene-introduced cells. C, the protein abundance of p16INK4a, p19ARF, p53, phosphorylated pRB (phos-pRB), and pRB in wild-type, Per2m/m, or Bmal1−/− cells infected with oncogenes.

Article Snippet: The membranes were reacted with antibodies against activating transcription factor 4 (ATF4; sc-200, Santa Cruz Biotechnology), p16INK4a (sc-1207), p19ARF (sc-22784), retinoblastoma protein (pRB; sc-69791), p53 (sc-6243), PER2 (sc-25368), BMAL1 (sc-48790) ACTIN (sc-1616), or anti-phospho-pRB-Thr826 (44–576; BIOSOURCE International, Camarillo, CA).

Techniques: Staining, Infection, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Different Roles of Negative and Positive Components of the Circadian Clock in Oncogene-induced Neoplastic Transformation *

doi: 10.1074/jbc.M115.706481

Figure Lengend Snippet: The overexpression of p53 in oncogene-introduced Bmal1−/− cells overrides the binding capacity of SV40LT. A, the oncogene-introduced wild-type, Per2m/m, or Bmal1−/− cells were lysed and were then subjected to immunoprecipitation with anti-SV40LT antibodies. The amounts of p53, pRb, and SV40LT in cell lysates, supernatants, and immune complexes were detected by Western blotting (WB). B, the mRNA levels of p53 target genes, p21 and Bax, in wild-type, Per2m/m, or Bmal1−/− cells infected with oncogenes. Values are the means ± S.E. (n = 3). *, p < 0.05 significantly different from other oncogene-introduced cells. C, influence of p53 inhibitor Pifithrin-α (PFTα) on the anchorage-independent growth of wild-type and Bmal1−/− cells after the concomitant introduction of H-rasV12 and SV40LT. Cells infected with oncogenes were subjected to a soft agar colony assay in the presence or absence of 30 μm pifithrin-α. The colony formation of cells and their viability were assessed 14 days after seeding. Control cells were infected with mock vectors. The upper panels show representative microscopic photographs of colony formation in each type of cells. The scale bars indicate 100 μm. The lower panel shows viability of cells. Values are shown as the means ± S.E. (n = 4). Mean values of mock-transfected wild-type cells were set at 1.0. ##, p < 0.01; **, p < 0.01 significantly different between the two groups.

Article Snippet: The membranes were reacted with antibodies against activating transcription factor 4 (ATF4; sc-200, Santa Cruz Biotechnology), p16INK4a (sc-1207), p19ARF (sc-22784), retinoblastoma protein (pRB; sc-69791), p53 (sc-6243), PER2 (sc-25368), BMAL1 (sc-48790) ACTIN (sc-1616), or anti-phospho-pRB-Thr826 (44–576; BIOSOURCE International, Camarillo, CA).

Techniques: Over Expression, Binding Assay, Immunoprecipitation, Western Blot, Infection, Colony Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Different Roles of Negative and Positive Components of the Circadian Clock in Oncogene-induced Neoplastic Transformation *

doi: 10.1074/jbc.M115.706481

Figure Lengend Snippet: Role of ATF4 in the oncogene-induced transformation of Per2 or Bmal1-defective cells. A, the protein levels of ATF4 in wild-type, Per2m/m, or Bmal1−/− cells infected with H-rasV12 and SV40LT. B, the overexpression of PER2 in oncogene-introduced Per2m/m cells suppresses the expression of ATF4. C, the overexpression of BMAL1 in oncogene-introduced Bmal1−/− cells enhances the expression of ATF4. For panels B and C, the mean value of empty vectors (pcDNA)-transfected cells were set at 1.0. D, influence of siRNA-induced down-regulation of Atf4 on the protein abundance of p16INK4a, p19ARF, p53, phosphorylated pRB (phos-pRB), and pRB in wild-type or Per2m/m cells. E, down-regulation of ATF4 in oncogene-introduced wild-type or Per2m/m cells by siRNA attenuated anchorage-independent growth ability. F, the protein abundance of p16INK4a, p19ARF, p53, phosphorylated pRB (phos-pRB), and pRB in oncogenic wild-type or Bmal1−/− cells transfected with Atf4-expressing vectors. G, overexpression of ATF4 in oncogene-introduced Bmal1−/− cells enhanced the anchorage-independent growth ability. For panels B, C, E, and G, values are shown as the mean ± S.E. (n = 4). **, p < 0.01, *, p < 0.05 significantly different between the two groups.

Article Snippet: The membranes were reacted with antibodies against activating transcription factor 4 (ATF4; sc-200, Santa Cruz Biotechnology), p16INK4a (sc-1207), p19ARF (sc-22784), retinoblastoma protein (pRB; sc-69791), p53 (sc-6243), PER2 (sc-25368), BMAL1 (sc-48790) ACTIN (sc-1616), or anti-phospho-pRB-Thr826 (44–576; BIOSOURCE International, Camarillo, CA).

Techniques: Transformation Assay, Infection, Over Expression, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Different Roles of Negative and Positive Components of the Circadian Clock in Oncogene-induced Neoplastic Transformation *

doi: 10.1074/jbc.M115.706481

Figure Lengend Snippet: Transcriptional regulation of Atf4 gene by clock gene products. A, the time course of mRNA expressions of Per2, Cry1, Cry2, Bmal1, Clock, and Atf4 in wild-type cells after infection with retrovirus vectors expressing H-rasV12 and SV40LT. Mean values of mock-transfected cells on day 0 are set at 1.0. Values are shown as the mean ± S.E. (n = 3). *, p < 0.05 as compared with mock-transfected group at corresponding time points. B, luciferase reporter assay of the mouse Atf4 promoter in oncogene introduced wild-type cells. The upper panel shows a schematic representation of the mouse Atf4 promoter. Numbers below the boxes are nucleotide residues positioned relative to transcription start site (+1). Underlined nucleotide residues indicate a mutated sequence of E-box. Wild-type MEFs and oncogene-introduced wild-type MEFs were transfected with 0.1 μg of each native Atf4::Luc or E-box mutated Atf4::Luc (E-box mut. Atf4::Luc). Cells transfected with reporter constructs were also co-transfected with expressing constructs for Per2, Cry1, Bmal1, or Clock. Values are expressed as relative ratio to the Atf4::Luc activity of wild-type MEFs in the absence of expressing vectors (set at 1.0). All values are shown as the means ± S.E. (n = 4). C, possible mechanisms for the promotion of oncogenicity by disruptions in the molecular circadian clock. When the circadian clock is disrupted due a dysfunction in the transcriptional repressor PER or CRY, ATF4 is markedly expressed by oncogenic stimuli, and cells easily undergo malignant transformation. In contrast, when the circadian clock is disrupted due to a dysfunction in the transcriptional activator CLOCK or BMAL1, ATF4 is hardly expressed by oncogenic stimuli, and p16INK4a and p19ARF induce cellular senescence.

Article Snippet: The membranes were reacted with antibodies against activating transcription factor 4 (ATF4; sc-200, Santa Cruz Biotechnology), p16INK4a (sc-1207), p19ARF (sc-22784), retinoblastoma protein (pRB; sc-69791), p53 (sc-6243), PER2 (sc-25368), BMAL1 (sc-48790) ACTIN (sc-1616), or anti-phospho-pRB-Thr826 (44–576; BIOSOURCE International, Camarillo, CA).

Techniques: Infection, Expressing, Transfection, Luciferase, Reporter Assay, Sequencing, Construct, Activity Assay, Transformation Assay