Figure 7. Apoptotic pathways in CDH2 knockdown cancer stem cells (CSCs).
(A) IPA output shows down-regulation of apoptotic pathways in control (scramble) CSCs relative to CDH2 and Cx43 knockdown CSCs. (B) Western blot for caspase 3 and 7 with whole cell extracts from CSCs with scramble shRNA or, CDH2 or Cx43 knockdown. The normalized band densities are shown at right. *P < 0.05 versus CDH2 or Cx43 knockdown. (C) Apoptotic activity was analyzed with Apo-ONE Homogeneous caspase 3/7 assay kit and the relative fluorescence unit presented for MDA-MB-231, knockdown for CDH2 or Cx43, or scramble shRNA. In parallel, the assay was performed with cells, treated with carboplatin (220 mg/ml) or doxorubicin (1 mM) for 4 h. (D) Western blot for Pgp with whole-cell extracts from CSCs from MDA-MB-231, knockdown for CDH2 or Cx43, or control/scramble shRNA. Bands were normalized with β-actin and presented at right, n = 3. **P < 0.05 versus Cx43 or CDH2 knockdown. (E) Cell viability was determined for MDA-MB-231, knockdown for CDH2 or Cx43, or control with scramble shRNA, treated with carboplatin (220 mg/ml). The analyses were performed at 24 h intervals up to 120 h using cell titer blue. The data are presented as % viable cells ± SD, n = 3. The percentage of viable cells at 120 h is zoomed on the right. *P < 0.05 versus knockdown cells at the 120 h time point. (E, F) The studies in “(E)” were repeated, except with doxorubicin (1 mM). *P < 0.05 versus Cx43 and CDH2 knockdown. (G) Protocol used to inject NSG mice, i.v. with 5 × 105 CSCs isolated from MDA-MB-231-Oct4-GFP, knockdown for CDH2 (RFP), Cx43 (RFP), or scrambled shRNA (RFP). Mice were injected intraperitoneally with 5 mg/ml carboplatin at days 3 and 5. (H) At day 7, sections from paraffin embedded femurs were labeled with anti–pan-cytokeratin-Texas Red (red) and anti–Ki67-AF405 (blue). Tissues were imaged with EVOS FL Auto 2 at magnification of 200×. Images represent six mice/group. (I) The number of BC cells (red) in mouse femur from “(H)” was counted using ImageJ and presented as mean BC cells ± SD, n = 6. (J) Cartoon summarizes the data in this Figure: Decreased apoptotic pathways in CSCs impart chemoreistance. Cx43/CDH2 knockdown cells reversed the resistance to chemosensitization.
Source data are available for this figure.
