Journal: Neoplasia (New York, N.Y.)

Article Title: A Preclinical Study Combining the DNA Repair Inhibitor Dbait with Radiotherapy for the Treatment of Melanoma 1

doi: 10.1016/j.neo.2014.08.008

Figure Lengend Snippet: Effect of Dbait on H2AX phosphorylation. (A) SK28 and 501mel melanoma cells were transfected with an inactive control oligonucleotide or Dbait ± NU7026 (DNA-PK inhibitor). Immunofluorescence of γ-H2AX (red) and chromatin (DAPI; blue) was visualized. Dbait treatment led to non-localized pan-nuclear H2AX phosphorylation evidencing Dbait activity. This activity was dependent on DNA-PK activation. Bar, 50 μm. (B) SK28 melanoma cells were transfected with an inactive control oligonucleotide or Dbait. Immunofluorescence of γ-H2AX (red) and chromatin (DAPI; blue) was visualized immediately after irradiation and/or Dbait treatment. Irradiation alone resulted in localized γ-H2AX foci representing radio-induced DNA DSBs; Dbait treatment with or without irradiation led to non-localized pan-nuclear H2AX phosphorylation evidencing Dbait activity. Bar, 30 μm. (C) SK28 cells were transduced with lentiviruses that express either control, non-targeting shRNA, or shRNA targeting DNA-PKcs. After Dbait transfection, cells were immunostained with mouse monoclonal anti–DNA-PKcs or anti–γ-H2AX. Dbait activity was not detected in cells transduced with shRNA targeting DNA-PKcs. Bar, 50 μm.

Article Snippet: Subconfluent SK28 cells were transduced with lentiviruses that expressed either the control, non-targeting shRNA (shCTL; sc-108080; Santa Cruz Biotechnology, (Dallas, Texas, USA)), or shRNA targeting DNA-PKcs (shDNA-PK; sc-35200-V; Santa Cruz Biotechnology) at a multiplicity of infection of 3 using polybrene (5 μg/ml).

Techniques: Phospho-proteomics, Transfection, Control, Immunofluorescence, Activity Assay, Activation Assay, Irradiation, Transduction, shRNA

Journal: PloS one

Article Title: Lactate activates HIF-1 in oxidative but not in Warburg-phenotype human tumor cells.

doi: 10.1371/journal.pone.0046571

Figure Lengend Snippet: Figure 2. Lactate inhibits PHD2 activity in oxidative tumor cells. (A) HIF-1a and b-actin protein expression was detected using Western blotting in the lysates of SiHa TCs incubated during 24-h with 10 mM lactate or not and increasing doses of 2-oxoglutarate. The upper panels show representative experiments and the graph HIF-1a protein expression normalized to b-actin levels. Data are expressed as % of lactate induction. **p,0.01, ***p,0.005 versus 10 mM lactate without 2-oxoglutarate; n = 8. (B) ODD-driven luciferase activity was measured in SiHa TCs treated during 24-h with 10 mM lactate or not. *p = 0.0206; n = 6. (C) HIF-1a and b-actin protein expression was detected using Western blotting in the lysates of SiHa TCs transfected with a specific siRNA against PHD2 (siPHD2) or with a control siRNA (siCTR) and incubated during 24-h with 10 mM lactate or not. The upper panels show representative experiments and the graph HIF-1a protein expression normalized to b-actin levels. ns, p.0.05, *p,0.05; n = 3. doi:10.1371/journal.pone.0046571.g002

Article Snippet: Control shRNA (shCTR) was Addgene plasmid 1864.

Techniques: Activity Assay, Expressing, Western Blot, Incubation, Luciferase, Transfection, Control

Journal: PloS one

Article Title: Lactate activates HIF-1 in oxidative but not in Warburg-phenotype human tumor cells.

doi: 10.1371/journal.pone.0046571

Figure Lengend Snippet: Figure 5. Targeting MCT1 inhibits lactate-induced but not basal HIF-1 activity in tumor cells. (A) SiHa TCs were cultured during 24-h in fresh medium containing 10 mM lactate, lactate +5 mM a-cyano-4-hydroxycinnamate (CHC), or none of the drugs. HIF-1a and b-actin were detected using Western blotting. The upper panels show a representative experiment and the graph shows HIF-1a protein expression normalized to b-actin. ***p,0.005 versus control; ###p,0.005 versus lactate alone; n = 3–8. (B) As in (A) but with WiDr TCs. ns, p.0.05 versus control; n = 3–8. (C–F) TCs were infected with a control shRNA (shCTR, left panels) or with a specific shRNA targeting MCT1 (shMCT1-1, right panels). The cells were then cultured during 24-h in the presence of 10 mM lactate or not (control), after which HIF-1 activity was quantified using a dual reporter luciferase assay. The assay was performed using (C) SiHa (n = 5–7), (D) HeLa (n = 3–4), (E) FaDu (n = 5–8), and (F) WiDr (n = 4–5) TCs. ns, p.0.05, *p,0.05, ** p,0.01, ***p,0.005 versus control. doi:10.1371/journal.pone.0046571.g005

Article Snippet: Control shRNA (shCTR) was Addgene plasmid 1864.

Techniques: Activity Assay, Cell Culture, Western Blot, Expressing, Control, Infection, shRNA, Luciferase

Journal: International Journal of Biological Sciences

Article Title: GATA Binding Protein 4 Regulates Tooth Root Dentin Development via FBP1

doi: 10.7150/ijbs.36567

Figure Lengend Snippet: Effect of GATA4 on odontoblasts polarization, cell proliferation and secretion of root dentin matrix. ( A ) H&E-stained sections of the mandibular first molars showed that the odontoblasts have shorter height and flattened morphology in Wnt1-Cre;GATA4 fl/fl mice at P14 and P21 (Bar: 50 μm). ( B-E ) Immunohistochemistry staining images showing expression levels of DSPP (B), COL-1 (C), DCN (D), and PCNA (E) in root of Wnt1-Cre;GATA4 fl/fl mice at P14. ( F ) The height of odontoblasts was measured. Quantitative assessment of the molar root odontoblasts height from (A) at P14 and P21. ( G-J ) Percentages of DSPP (G), COL-1 (H), DCN (I), and PCNA-positive (J) cells in the control and mutant groups were calculated. ( K ) Double-labeled fluorescent immunostaining of DAPI-stained cell nuclei (blue), GFP (green), and merged images in tooth root at P17 after the injetion in vivo (Bar: 50 μm). ( L ) H&E-stained sections of the mandibular first molars showed that root dentin thickness was increased in GATA4 OE group mice at P17 (Bar: 50 μm). ( M ) Quantitative assessment of the molar root dentin thickness at P17. ( N ) Expression of GATA4 after lentivirus injected at P17 (Bar: 100 μm). ( O ) Percentages of GATA4-positive cells in the two groups were calculated. ( P ) Immunohistochemistry staining images showing expression levels of DSPP, COL-1, and DCN in root of mice at P17. ( Q ) Percentages of DSPP, COL-1, and DCN-positive cells in the two groups were calculated. Data expressed as the mean ± standard deviation, n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Recombinant lentivirus of shRNA target GATA4 (shGATA4; 5′-GAATAAATCTAAGACACCA-3′), control lentivirus (shCTRL; 5′-TTCTCCGAACGTGTCACGT-3′), lentivirus to overexpress GATA4 (pcDNA-GATA4) and blank lentivirus were purchased from GenePharma (Shanghai, China).

Techniques: Staining, Immunohistochemistry, Expressing, Control, Mutagenesis, Labeling, Immunostaining, In Vivo, Injection, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: GATA Binding Protein 4 Regulates Tooth Root Dentin Development via FBP1

doi: 10.7150/ijbs.36567

Figure Lengend Snippet: Characterization of DPSCs and expression of GATA4 in DPSCs. ( A ) Flow chart explaining cells isolation, culture and collection for FCM analyses. ( B, C ) Flow cytometry demonstrated that DPSCs expressed mesenchymal markers (CD44 and CD90) at a high level and generated the hematopoietic makers (CD14 and CD45) at a low level. ( D ) After mineralization for 3, 7, and 14 days, GATA4 protein expression was assessed at the indicated time points by western blotting. ( E ) DPSCs infected with lentivirus as assessed by fluorescence microscopy (Bar: 50 μm). ( F ) Efficiency of GATA4 knockdown after infection with lentivirus was analysed by western blotting. ( G ) Quantitative analysis of western blotting bands from (D) is shown as the ratio of GATA4 to GAPDH. ( H ) Quantitative analysis of western blotting bands from (F) is shown as the ratio of GATA4 to GAPDH. Data expressed as the mean ± standard deviation, n = 3. ** P < 0.01.

Article Snippet: Recombinant lentivirus of shRNA target GATA4 (shGATA4; 5′-GAATAAATCTAAGACACCA-3′), control lentivirus (shCTRL; 5′-TTCTCCGAACGTGTCACGT-3′), lentivirus to overexpress GATA4 (pcDNA-GATA4) and blank lentivirus were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Isolation, Flow Cytometry, Generated, Western Blot, Infection, Fluorescence, Microscopy, Knockdown, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: GATA Binding Protein 4 Regulates Tooth Root Dentin Development via FBP1

doi: 10.7150/ijbs.36567

Figure Lengend Snippet: Effect of GATA4 on migration, proliferation and odonto/osteogenic differentiation of DPSCs. ( A ) Effect of GATA4 knockdown on cell migration was assessed by wound scratch assays (Bar: 100 μm). ( B ) Effect of GATA4 knockdown on cell migration was assessed by transwell assay (Bar: 100 μm). ( C ) ALP staining observed after 7 days of mineralization (Bar: 100 μm). ( D ) After mineralization for 14 days, alizarin red staining was performed and observed with an image scanner (upper) and under a microscope (lower) (Bar: 100 μm). ( E ) The CCK8 assay was used to analyse the proliferation of DPSCs after infection with GATA4 lentivirus. ( F ) Quantitative assessment of ALP-positive areas. ( G ) Semi-quantitative estimation of calcium. ( H ) Expression levels of odonto/osteogenic-related genes (DSPP, BMP4, RUNX2, OSX, OPN, and OCN) were assessed by western blotting. ( I ) Quantitative analysis of western blotting bands from (H). ( J ) Expressions of odonto/osteogenic markers (Dspp, Dmp1, Col1a1, Bmp4, Runx2, Osx, Ocn, and Alp) were assessed by qRT-PCR. (Bar: 100 μm). Data expressed as the mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Recombinant lentivirus of shRNA target GATA4 (shGATA4; 5′-GAATAAATCTAAGACACCA-3′), control lentivirus (shCTRL; 5′-TTCTCCGAACGTGTCACGT-3′), lentivirus to overexpress GATA4 (pcDNA-GATA4) and blank lentivirus were purchased from GenePharma (Shanghai, China).

Techniques: Migration, Knockdown, Transwell Assay, Staining, Microscopy, CCK-8 Assay, Infection, Expressing, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: GATA Binding Protein 4 Regulates Tooth Root Dentin Development via FBP1

doi: 10.7150/ijbs.36567

Figure Lengend Snippet: Overexpression of GATA4 in DPSCs increased the odonto/osteogenic ability. ( A ) DPSCs infected with lentivirus control and pcDNA-GATA4 were observed under a fluorescence microscope (Bar: 50 μm). ( B ) Protein expression of GATA4 in the DPSCs was tested by western blotting after overexpression of GATA4. ( C ) Quantitative analysis of western blotting bands from (B). ( D ) ALP staining was observed after 7 days of mineralization. ( E ) ARS staining was performed 14 days after mineralization (Bar: 100 μm). ( F ) Quantitative assessment of ALP-positive areas after 7 days of osteogenic induction. ( G ) Semi-quantitative estimation of calcium. Data expressed as the mean ± standard deviation; n = 3. ** P < 0.01.

Article Snippet: Recombinant lentivirus of shRNA target GATA4 (shGATA4; 5′-GAATAAATCTAAGACACCA-3′), control lentivirus (shCTRL; 5′-TTCTCCGAACGTGTCACGT-3′), lentivirus to overexpress GATA4 (pcDNA-GATA4) and blank lentivirus were purchased from GenePharma (Shanghai, China).

Techniques: Over Expression, Infection, Control, Fluorescence, Microscopy, Expressing, Western Blot, Staining, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: GATA Binding Protein 4 Regulates Tooth Root Dentin Development via FBP1

doi: 10.7150/ijbs.36567

Figure Lengend Snippet: GATA4 enhanced glycolysis by negatively regulating FBP1 in DPSCs. ( A ) Co-immunoprecipitated proteins were then separated using SDS-PAGE and stained. ( B ) Mass spectrometry followed by peptide sequencing identified the two proteins as fructose-1,6-bisphosphatase 1 (FBP1) and isoform CRA_d. ( C ) Immunofluorescence staining revealed that GATA4 co-localizes with FBP1 in the nucleus in DPSCs (Bar: 100 μm). ( D ) Expression pattern of GATA4 during tooth development at embryonic day 13.5 (E13.5), E14.5, E15.5, P1, and P14 (Bar: 50 μm). ( E ) FBP1 expression was tested by western blotting after infection with GATA4 lentivirus in DPSCs. ( F ) Quantitative analysis of western blotting bands from (E). ( G ) FBP1 expression was tested by western blotting after GATA4 overexpression in DPSCs. ( H ) Quantitative analysis of western blotting bands from (G). ( I, J ) knockdown of GATA4 resulted in decreased glucose consumption and lactate production. ( K, L ) Overexpression of GATA4 resulted in increased glucose consumption and lactate production. Data expressed as the mean ± standard deviation; n = 3. ** P < 0.01.

Article Snippet: Recombinant lentivirus of shRNA target GATA4 (shGATA4; 5′-GAATAAATCTAAGACACCA-3′), control lentivirus (shCTRL; 5′-TTCTCCGAACGTGTCACGT-3′), lentivirus to overexpress GATA4 (pcDNA-GATA4) and blank lentivirus were purchased from GenePharma (Shanghai, China).

Techniques: Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Sequencing, Immunofluorescence, Expressing, Western Blot, Infection, Over Expression, Knockdown, Standard Deviation