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Fig. 3 | BMC Cancer

Fig. 3

From: Aspirin inhibits epithelial-to-mesenchymal transition and migration of oncogenic K-ras-expressing non-small cell lung carcinoma cells by down-regulating E-cadherin repressor Slug

Fig. 3

Slug up-regulation in A549 cells occur conjointly with oncogenic K-ras and p65NFκB pathways. a Schematic representations (upper panels) and RT-PCR data (lower panel) of p65NFκB-occupied region on SLUG promoter 1 (−329 to −129; primer sets 3 and 4) and SLUG promoter 2 (−496 to −384 and −181 to −40; primer sets 1 and 5, respectively) in A549 cells as determined by ChIP analysis. Lane 1 denotes input control and parallel immunoprecipitation with control IgG antibody is shown in lane 2. b Chromatin from oncogenic K-ras expressing control A549 cells, control-cDNA-/ DN-K-ras-transfected A549 cells was immunoprecipitated with p65NFκB antibody. PCR amplification was performed on p65NFκB binding regions on the SLUG promoter 1 (upper left panel) and SLUG promoter 2 (upper right panel). Lane 1, 2 and 3 denotes input control and parallel immunoprecipitation with control IgG antibody is shown in lane 4, 5 and 6. (c) RT-PCR analysis for Slug mRNA levels in untransfected, ERK-siRNA-/ IκBα-SR-cDNA-transfected A549 cells and in A549 cells transfected with both ERK-siRNA and IκBα-SR-cDNA. The inset shows the immunoblot analysis of nuclear p65NFκB levels for the transfection efficiency of IκBα-SR-cDNA. d ChIP assay for p300 binding on the SLUG promoters 1 and 2 in untransfected, DN-K-ras-/IκBα-SR-cDNA transfected A549 cells and in A549 cells transfected with both DN-K-ras and IκBα-SR-cDNA. e Nuclear lysates of oncogenic K-ras expressing A549 cells and DN-K-ras-transfected A549 cells was subjected to immunoprecipitation using anti-p300 antibody or with control IgG. The same blot was sequentially probed with p-Elk-1 and p300-specific antibodies. To confirm comparable protein input of the respective samples following, 20 % of lysates used for immunoprecipitation were subjected to western blot analysis with anti-α-Actin. Data are presented as mean ± SEM or representative of three independent experiments

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