Skip to main content
. 2013 Oct 8;5(12):1821–1834. doi: 10.1002/emmm.201302654

Figure 3.

Netrin-1 silencing sensitizes A549R cells to Doxorubicin and induces apoptotic cell death via UNC5B receptor.

  • A–C. Netrin-1 silencing sensitizes tumour cells to Doxorubicin. A549R cells were transfected with either a scramble siRNA (siCTRL) or with a specific siRNA targeting netrin-1 (siNet). 24 h after transfection, cells were treated with increasing concentrations of Doxorubicin. Cell death rate (A), measured by toxilight kit as described in materials and methods section, and cell survival (B), was evaluated 48 h after treatment. Results were normalized to control, untreated cells. While scramble siRNA-transfected cells showed a general resistance to Doxorubicin treatment, netrin-1 silencing strongly induced cell death and decreased cell survival in presence of Doxorubicin. Evaluation of cell death percentage (C), measured by 4′,6-diamidino-2-phenylindole (DAPI) exclusion as described in the materials and methods section, confirmed that netrin-1 siRNA sensitized A549R cells to 0.5 and 2 µM Doxorubicin treatment.

  • D,E. Netrin-1 silencing triggers apoptosis in combination with Doxorubicin treatment. A549R cells were transfected as in (A–C), and treated with the indicated Doxorubicin concentrations for 24 h. Active caspase-3 (D), normalized to untreated cells, and DNA fragmentation (E) were evaluated as described in the materials and methods section. While Doxorubicin failed to induce apoptosis in A549R cells transfected with a scramble siRNA (siCTRL), cells silenced for netrin-1 showed a strong increase in the apoptotic rate.

  • F. Combination of netrin-1 silencing and Doxorubicin treatment induces cell death through netrin-1 receptor UNC5B. A549R cells were transfected with scramble siRNA (siCTRL), netrin-1-specific siRNA (siNet), UNC5B-specific siRNA (siUnc5B) and with a combination of netrin-1 and UNC5B-targeting siRNA. 24 h after transfection, cells were treated with the indicated Doxorubicin concentrations for 48 h, and cell death rate was measured by toxilight and normalized to control, untreated cells. While netrin-1 silencing (siNet) sensitized A549R cells to Doxorubicin treatment, as compared to siCTRL-transfected cells, the simultaneously silencing of netrin-1 and UNC5B (siNet + siUnc5B) rescued cell death induction by siNet and Doxorubicin treatment. *, p < 0.05; **, p < 0.01. DoxoR, Doxorubicin.

Figure 3