Figure 5. Angiogenic capabilities of HUVECs suspended in conditioned media (CM) from cisplatin- or vehicle-treated A549 and H358 cells in the presence or absence of TIMP-1 siRNA.

A549 and H358 cells were incubated with transfection reagent in the absence of any siRNA (first and second triplets or bands), transfected with TIMP-1 siRNA (TIMP-1 si; third and fourth triplets or bands), or non-silencing siRNA (non si, fifth and sixth triplet or bands) for 24 h in DMEM containing 10% FCS. Subsequently, cells were washed and treated with vehicle or 1 µM cisplatin for 48 h in serum-free DMEM prior to collection of CM. (A) and (C), HUVECs suspended in CM from A549 (A) or H358 (C) cells were subjected to the upper chambers to quantify migration (black bars) or were used in the viability assay (WST-1, white bars) following a further 24-h incubation. Tube formation assays were performed after a 2-h incubation of HUVECs with the indicated CM (grey bars). (B) and (D), Monitoring of TIMP-1 was performed in parallel using CM obtained from A549 (B) or H358 (D) cells. Western blot images are representative of each experiment. Values above the blots are the mean ± SEM and represent alterations in TIMP-1 protein levels in CM in comparison with vehicle-treated cells (set as 100%), according to densitometric analyses. Western blotting analysis of TIMP-1 in CM was supplemented with β-actin analysis of the respective cell lysates. Values are the mean ± SEM of n = 3 (A, migration and tube formation, B, C), n = 4 (D) or n = 6 (A, viability). ^**p < 0.01, ^***p < 0.001 vs. corresponding vehicle control; ^## p < 0.01, ^### p < 0.001 vs. the respective cisplatin-treated group without siRNA, one-way ANOVA plus a post-hoc Bonferroni test.