Fig 1.
Low expression levels of NDUFB9 in highly metastatic breast cancer cells.
(A) Schematic overview of iTRAQ. (B) The normalized median density of MDA-MB-231 and MDA-MB-231HM. (C) The spectrum of NDUFB9 obtained from MS. (D) The fold change ratio in the MDA-MB-231 and MDA-MB-231HM breast cancer cell lines by iTRAQ labeling based on quantitative proteomic analyses. (E,F) Protein and mRNA expression of NDUFB9 in breast cancer cell lines. Results are representative of 3 independent experiments.
Fig 2.
Knockdown of NDUFB9 promoted MDA-MB-231 proliferation.
(A) Knockdown efficiency of NDUFB9 in MDA-MB-231 and MCF-10A cells analyzed by Western blot and the below panel shows histograms of the results. (B) CCK-8 proliferation assays in MDA-MB-231 shCon and shNDUFB9 cells. (C) Cell cycle analysis in MDA-MB-231 shCon and shNDUFB9 cells at 6h and 12h after cells were synchronized at the G1/S boundary. (D, E) Histograms of the S and G2/M phases of the cell cycle for 6 h and 12 h respectively. Results above are repeated at least 3 times. Data were presented as the mean ± SD (Student’s t-test, n≥3; *P < 0.05, **P < 0.01 and ***P < 0.001).
Fig 3.
Loss of NDUFB9 expression resulted in enhancement of MDA-MB-231 cell migration and invasion.
(A) The migration and invasion ability of MDA-MB-231 and migration of MCF-10A were evaluated with a transwell assay. The left panels show images of representative fields (100× magnification) of invasive cells, and the right panel shows histograms of the results. (B) Effects of NDUFB9 on the migration of MDA-MB-231 cells in a wound-healing assay. The red lines indicate the initial scratch wound location, and the yellow area shows the scratch wound mask. Images were captured at the indicated times after wounding. (C) The effect of NDUFB9 on the percent of relative wound density at the indicated time point. Results above are repeated at least 3 times. Data were presented as the mean ± SD (Student’s t-test, n≥3; **P < 0.01, and ***P < 0.001).
Fig 4.
Depletion of NDUFB9 altered mitochondrial metabolism.
(A) FACS analysis (left) and statistical data (right) of mitochondrial ROS generation in MDA-MB-231 and MCF-10A cells. (B) Intracellular NAD+, NADH, total NAD+/NADH and the ratio of NAD+/NADH were analyzed independently in extracts from whole cells in the NDUFB9 knockdown and control groups. (C) mt-DNA content was determined by quantitative real-time PCR of mtDNA extracted from MDA-MB-231 cells, using three pairs of mtDNA primers (primer-1, primer-2, and primer-3). (D, E) Glucose uptake and lactate secretion were assessed in NDUFB9 knockdown or control MDA-MB-231 cells. Results above are repeated at least 3 times. Data were presented as the mean ± SD (Student’s t-test, n≥3; **P < 0.01, and ***P < 0.001).
Fig 5.
Suppression of NDUFB9 expression induced EMT and activated the AKT/mTOR/p70S6K signaling pathway.
(A-B) Immunoblotting analysis of E-cadherin, Fibronectin-1 (FN-1), Vimentin, phosphorylated SMAD3, SMAD3 and SMAD4 in MCF-10A and MDA-MB-231 cells transfected with NDUFB9-specific or control shRNA. GAPDH was used to normalize for equal loading. (C) Expression levels of phosphorylated Akt (S129), total Akt, phosphorylated mTOR(S2448), total mTOR, phosphorylated p70S6K and p70S6K were examined by immunoblotting in MDA-MB-231. Results above are representative of 3 independent experiments. (D-F) Histograms of the results from A, B and C respectively. Data were presented as the mean ± SD (Student’s t-test, n≥3; **P < 0.01, and ***P < 0.001).