DCL knockdown results in a delayed NB tumor growth.

<p>The normalized tumor growth is shown for shDCL-2 (A), shDCL-3 (B) and NC (C) tumors developed from the correspondent Dox-inducible NB cells lines injected subcutaneously in BALB/c athymic nude mice. Tumor growth (A-C) was normalized to the maximum tumor size per group. (D) Average of tumor volume 9, 12 and 14 days after injecting the Dox-inducible NB cells. NC, negative control Dox-inducible NB tumors. shDCL-2 and shDCL-3, Dox-inducible NB tumors that express shRNA against DCL. Error bars, s.e.m. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

NB cells with DCL knockdown proliferate less in an energetic challenged environment.

<p>(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. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

DCL plays a role in mitochondrial activity.

<p>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. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

DCL silencing leads to smaller tumor formation with necrotic areas.

<p>(A) Representative mice of the different groups 14 days after inoculation of the Dox-inducible NB cells. (B) Mice weight over time. (C) Tumor histology showing high vascularization (arrows) and necrotic areas (<b>*</b> in blue). The quantification of the vascular structures and necrotic areas is shown in Figure S3. Error bars, s.e.m. *, <i>P</i><0.05. Scale bars, 50 µm.</p

DCL knockdown results in less mitochondrial colocalization in NB cells.

<p>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.</p

DCL knockdown in Dox-inducible NB cells results in less NB proliferation.

<p>(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. **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Serine/Proline (S/P)-rich and the second doublecortin (DCX)-domains of DCL are required for mitochondrial activity regulation.

<p>Cytochrome c oxidase activity (A) and ATP synthesis (B) in COS-1 cells transfected with empty vector, with DCL full-length or with one of the different DCL fragments subcloned into pDsRed2-N1 vector is shown. The structure of DCL full-length and the different DCL mutants is represented, indicating the DCX-domains and S/P-rich domain (SP). Error bars, s.e.m. *, <i>P</i><0.05.</p

DCL knockdown in Dox-inducible NB tumors correlates with less proliferation and enhanced apoptosis.

<p>(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. *, <i>P</i><0.05. Scale bars, 25 µm (C) or 50 µm (D).</p

DCL knockdown leads to a lower expression of mitochondrial-related genes.

<p>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. **, <i>P</i><0.01; ***, <i>P</i><0.001.</p