Journal: PLoS ONE

Article Title: Silencing of Doublecortin-Like (DCL) Results in Decreased Mitochondrial Activity and Delayed Neuroblastoma Tumor Growth

doi: 10.1371/journal.pone.0075752

Figure Lengend Snippet: (A) shRNA expression in the different NB cell lines treated with doxycycline (Dox) or vehicle (Veh) for 72 hours. (B) Western blotting results of DCL and α-tubulin expression in Dox-inducible NB cells treated for 72 hours with doxycycline (Dox) or vehicle (Veh). (C) Quantification and normalization of DCL expression to α-tubulin. (D) Cell proliferation 72 hours after starting adding Dox or Veh to the growth medium. N1E-115 cells, mouse NB cell line used to develop the Dox-inducible NB cell lines. NC, negative control Dox-inducible NB cells. shDCL-2 and shDCL-3, Dox-inducible NB cell lines that express shRNA against DCL. RQ, relative quantification. Error bars, s.e.m. **, P <0.01; ***, P <0.001.

Article Snippet: Doxycycline (Dox) and G418 were purchased from Sigma-Aldrich Chemie B.V (Zwijndrecht, The Netherlands).

Techniques: shRNA, Expressing, Western Blot, Negative Control, Quantitative Proteomics

Journal: PLoS ONE

Article Title: Silencing of Doublecortin-Like (DCL) Results in Decreased Mitochondrial Activity and Delayed Neuroblastoma Tumor Growth

doi: 10.1371/journal.pone.0075752

Figure Lengend Snippet: (A) DCL and α-tubulin expression in Dox-inducible tumors from nude mice treated with doxycycline- (Dox) or vehicle (Veh)-diet. (B) DCL expression normalized to α-tubulin. (C) Immunostaining of Ki67 (green) positive cells in the Dox-inducible NB tumors. (D) Average of positive Ki67 cells per high-power field (HPF). Immunostaining (E) and quantification (F) of cleaved caspase-3 positive cells (green) per HPF. Images (C and E) are representative of the average expression of each group. For each section, 5-10 different fields were analyzed. NC, negative control Dox-inducible NB tumors. shDCL-2 and shDCL-3, Dox-inducible NB tumors that express shRNA targeting DCL. Blue, Hoechst staining. Red, α-tubulin staining. Arrows, examples of Ki67 positive cells (C) or cells with cleaved caspase-3 (E). Error bars, s.e.m. *, P <0.05. Scale bars, 25 µm (C) or 50 µm (D).

Article Snippet: Doxycycline (Dox) and G418 were purchased from Sigma-Aldrich Chemie B.V (Zwijndrecht, The Netherlands).

Techniques: Expressing, Immunostaining, Negative Control, shRNA, Staining

Journal: PLoS ONE

Article Title: Silencing of Doublecortin-Like (DCL) Results in Decreased Mitochondrial Activity and Delayed Neuroblastoma Tumor Growth

doi: 10.1371/journal.pone.0075752

Figure Lengend Snippet: DCL (green) and mitochondria (red) staining in Dox-inducible NB cells treated with doxycycline revealed less colocalization (yellow) in NB cells with DCL knockdown (shDCL-2 and shDCL-3). Colocalization scores and images of Dox-inducible NB cells treated with vehicle are shown in Figure S4. Mitochondria were stained with 100 nM MitoTraker Orange CMTM Rosamine. Blue, Hoechst staining. NC, negative control Dox-inducible NB cell lines that express scramble shRNA. shDCL-2 and shDCL-3, Dox-inducible NB cell lines that express shRNA against DCL. Scale bars, 10 µm.

Article Snippet: Doxycycline (Dox) and G418 were purchased from Sigma-Aldrich Chemie B.V (Zwijndrecht, The Netherlands).

Techniques: Staining, Knockdown, Negative Control, shRNA

Journal: PLoS ONE

Article Title: Silencing of Doublecortin-Like (DCL) Results in Decreased Mitochondrial Activity and Delayed Neuroblastoma Tumor Growth

doi: 10.1371/journal.pone.0075752

Figure Lengend Snippet: Fold change of Cox7c (A), Cox6a2 (B), Ndufa1 (C) and Ndufa13 (D) mRNA expression in Dox-inducible NB cells (shDCL-2 or shDCL-3). Cells were treated with doxycycline (Dox) or vehicle (Veh) for 72 hours. Fold change was calculated by normalization to the negative control (NC) Dox-inducible NB cell line treated with Dox or Veh respectively. Error bars, s.e.m. **, P <0.01; ***, P <0.001.

Article Snippet: Doxycycline (Dox) and G418 were purchased from Sigma-Aldrich Chemie B.V (Zwijndrecht, The Netherlands).

Techniques: Expressing, Negative Control

Journal: PLoS ONE

Article Title: Silencing of Doublecortin-Like (DCL) Results in Decreased Mitochondrial Activity and Delayed Neuroblastoma Tumor Growth

doi: 10.1371/journal.pone.0075752

Figure Lengend Snippet: Fold change in cytochrome c oxidase activity (A) and ATP synthesis (B) in shDCL-2 and shDCL-3 Dox-inducible NB cells treated for 72 hours with doxycycline (Dox) or vehicle (Veh). (C) DCL and α-tubulin expression in Dox-inducible NB cells treated with either Dox or Veh for 72 hours and subsequently transfected with DCL or empty plasmids. Cells were lysed for western blotting analysis 48 hours after transfection. (D) DCL expression normalized to α-tubulin. DCL expression is recovered in shDCL-2 and shDCL-3 cells after transfection with DCL plasmid (C-D). Fold change of cytochrome c oxidase activity (E) and ATP synthesis (F) in shDCL-2 and shDCL-3 cells treated with either Dox or Veh (72 hours) and then transfected with DCL or empty plasmid (48 hours). Fold changes were determined by normalizing to the negative control (NC) Dox-inducible NB cell line treated with Dox or Veh respectively. Cytochrome c oxidase activity was investigated in isolated mitochondria. Error bars, s.e.m. *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: Doxycycline (Dox) and G418 were purchased from Sigma-Aldrich Chemie B.V (Zwijndrecht, The Netherlands).

Techniques: Activity Assay, Expressing, Transfection, Western Blot, Plasmid Preparation, Negative Control, Isolation

Journal: PLoS ONE

Article Title: Silencing of Doublecortin-Like (DCL) Results in Decreased Mitochondrial Activity and Delayed Neuroblastoma Tumor Growth

doi: 10.1371/journal.pone.0075752

Figure Lengend Snippet: (A-D) Cell proliferation in Dox-inducible NB cell lines in the presence of low glucose (A) or high glucose (B) medium and treated with doxycycline (Dox) or vehicle (Veh) for 72 hours. Cell proliferation in shDCL-2 (C) and shDCL-3 (D) cells 72 hours after starting the treatment with either Dox or Veh and in the presence of low or high glucose medium. (E) Ratio in cell proliferation between Dox-inducible NB cells that grew in low and high glucose medium. (F) Cell proliferation in shDCL-2 and shDCL-3 cells grown in high glucose medium, treated with Dox or Veh for 72 hours and subsequently transfected with empty plasmid or DCL plasmid to recover DCL expression. NC, negative control Dox-inducible NB cell lines. shDCL-2 and shDCL-3, Dox-inducible NB cell lines that express shRNA against DCL. Error bars, s.e.m. *, P <0.05; **, P <0.01.

Article Snippet: Doxycycline (Dox) and G418 were purchased from Sigma-Aldrich Chemie B.V (Zwijndrecht, The Netherlands).

Techniques: Transfection, Plasmid Preparation, Expressing, Negative Control, shRNA