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