Doxorubicin

Mouse melanoma (B16F0, ATCC; mycoplasma free) or mouse lung carcinoma (CMT19T, CR-UK Cell Production; mycoplasma free) cells (1 × 10⁶) were injected subcutaneously in the flank of Pdgfb-iCre^ER;Fak^fl/fl mice and wild-type control mice (Pdgfb-iCre^ER;non-floxed or Fak^fl/fl). Simultaneously, animals were given a soy-free diet (Harlan) to reduce oestrogen levels and increase tamoxifen sensitivity. At days 7 and 8 after tumour inoculation, once tumour growth had begun, all mice were injected intraperitoneally (i.p.) with 150 μl of 10 mg ml⁻¹ of tamoxifen (Sigma, T5648) diluted in10% ethanol in peanut oil (Sigma) to induce endothelial-cell FAK deletion. From day 8 onwards, all animals were fed with tamoxifen-containing diet (TAM400, Harlan). All animals with B16F0 subcutaneous tumours were injected i.p. with 8 mg kg⁻¹ of doxorubicin (Accord Healthcare) or PBS as a negative control on days 9, 11 and 13 after tumour-cell inoculation. Alternatively, animals with subcutaneous CMT19T tumours were irradiated with 5Gy of γ-irradiation on day 10 after tumour injection. For both tumour types calliper measurements were taken over time and animals were killed when tumours reached the maximum size allowed by UK Home Office regulations.

Wild-type and FAK-null endothelial cells or transfected wild-type endothelial cells were grown in normal MLEC media supplemented or not with 0.125 μM doxorubicin (Accord Healthcare) or irradiated (5Gy). After 48 h of serum starvation, whole-cell lysates were extracted at 48 h (for doxorubicin-treated endothelial cells) and 72 h (for irradiated endothelial cells). Mouse cytokine arrays (Proteome Profiler ARY006, R&D Systems) were processed according to the manufacturer's instructions using 100 μg of lysates (in 3% SDS, 60 mM sucrose, 65 mM Tris-HCl pH 6.8) per membrane. Pixel analysis was used for quantification with Image J software.