Fig. 6

In vitro characterization of inducible EDI3 silencing and its effect on ER-HER2 + tumours in mice. A, Overview of the elements of the inducible lentiviral shRNA vector (SMARTvector™, Dharmacon) containing shRNA oligos under the control of a doxycycline inducible promotor. B, mRNA expression of EDI3 and C, representative Western blot showing EDI3 protein expression in HCC1954 shNEG and HCC1954 shEDI3 (oligos shEDI3 #1, #2, #3) cells treated with 0.01, 0.1 or 1 µg/ml doxycycline for 72 h. D, Representative images (left) and corresponding quantification (right) of colonies formed by HCC1954 shEDI3 cells treated with 0.1 or 1 µg/ml doxycycline. E, Viability (RFU) after treating HCC1954 shEDI3 cells with 0.1 or 1 µg/ml doxycycline. F, Number of EdU positive cells normalized to the control condition (0 μg/ml doxycycline) and representative images (right) after treatment of HCC1954 shNEG and HCC1954 shEDI3 (oligos shEDI3 #1, #2) cells with 10 nM EdU for 2 h followed by labelling with Alexa FluorTM 488 dye (green). Nuclei were stained with Hoechst 33342 (blue), scale bars represent 200 μM. CD1 nude mice were injected subcutaneously with HCC1954 shNEG or HCC1954 shEDI3 cells with (+ Dox) and without (-Dox) doxycycline treatment (0.1 µg/ml) and the effect of silencing EDI3 on tumour G, volume and H, weight were examined. All in vitro data are mean ± SD of three independent experiments. Measurements (G-H) represent the average tumour volume and weight of six to seven mice per condition. (*, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001). EdU, 5-Ethynyl-2′-deoxyuridine; RFU, relative fluorescence units