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Fig. 4 | BMC Cancer

Fig. 4

From: Endothelial cell-derived nidogen-1 inhibits migration of SK-BR-3 breast cancer cells

Fig. 4

The STAT3 signal promotes endothelial-dependent cell migration and is blocked by NID1. a Phospho-protein array was performed on cell lysates of SK-BR-3 cells incubated for 4 days in unconditioned medium (ctrl), or conditioned medium derived from confluent HUVECs transfected with siRNA targeting NID1 (siNID1) or control siRNA. b Cell lysates from SK-BR-3 cells, SK-BR-3 cells with control siRNA and SK-BR-3 cells with siRNA knockdown of nidogen-1 were analysed on a phosphoprotein kinase array. The pixel densities of the single dots were quantified by ImageJ software. After subtraction of the background signal, the average of the duplicated spots was plotted (*p ≤ 0.05 versus ctrl by unpaired student’s t-test). c The phosphorylation state of STAT3 (Y705) was evaluated in SK-BR-3 cells after 4 days exposure to unconditioned medium (ctrl) or conditioned medium derived from confluent HUVECs treated without (un), with control siRNA (si ctrl) or with NID1 specific siRNA (siNid1). Total STAT3 expression served as loading control. d Phosphorylation and total levels of STAT3 in SK-BR-3 cells were evaluated by immunoblotting analysis after incubation with conditioned media derived from confluent or subconfluent HUVECs for the time points indicated. GAPDH was used as loading control. e Immunoblotting analysis of phosphorylated (Y705) and total STAT3 levels in SK-BR-3 cells pretreated with increasing concentrations (0 μM, 0.5 μM, and 1 μM) of the STAT3 inhibitor FLLL31 for 18 h and incubation in conditioned medium derived from subconfluent HUVECs. GAPDH served as loading control. f The migratory ability of SK-BR-3 cells was determined using a modified Boyden chamber assay after exposure to conditioned medium derived from subconfluent HUVECs with or without the STAT3 inhibitor FLLL31 (0.5 μM) for 4 days (**p ≤ 0.01 versus ctrl by unpaired student’s t-test)

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