Fig. 4: JNK pathway plays a key role in breast cancer progression mediated by NMT1 knockdown.

a The Human Phospho-Kinase Array was used to detect multiple phosphorylated kinases in Shctrl and ShNMT1-infected SUM149 cells. Template showing the location of kinase antibodies spotted onto the Human Phospho-Kinase Array kit and relevant kinases were indicated by numbers (left). Quantification of mean spot pixel densities of the indicated kinases (right). b The indicated kinases were detected in Shctrl and ShNMT1-infected SUM149 cells by western blot. c The indicated kinases were detected in Shctrl and ShNMT1-infected MDA-MB-231 cells by western blot. d Autophagy related protein expression was determined by western blot in Shctrl and ShNMT1-infected SUM149, MDA-MB-231 and HCC1937 cells. e Representative images showing the formation of GFP-LC3 puncta in Shctrl and ShNMT1-infected SUM149 cells (left). GFP-LC3 puncta per cell was quantified (right). f Shctrl and ShNMT1-infected SUM149 and MDA-MB-231 cells were treated with SP600125 (20 uM) or same volume of DMSO for 48 h. The expression of JNK, LC3 and p21 were then detected by western blot. g Representative images of ALDH-positive cells in the cells from f (left). ALDH-positive cells were quantified (right). h Quantification of Mammosphere formation from the cells in f. i MTT assay was used to measure the proliferation of cells in f. j, k Wound healing assay (j) and Transwell assay (k) were used to measure migration and invasion ability of the cells in f as described in methods. Data represent the mean ± SD of 3 independent experiments where *P < 0.05, **P < 0.01 and ***P < 0.001