Fig. 2: ER stress is induced by NMT1 knockdown.

a ER stress related gene expression was determined by qRT-PCR in Shctrl and ShNMT1-infected SUM149 and MDA-MB-231 cells. b ER stress related proteins expression was detected by western blot. c Shctrl and ShNMT1-infected SUM149 and MDA-MB-231 cells were treated with 4-PBA (2uM) or same volume of DMSO for 48 h. ER stress markers were then determined by Western blot. d Quantification of ALDH-positive cells in 4-PBA or DMSO treated cells. e Quantification of Mammosphere formation in 4-PBA or DMSO treated cells. f MTT assay was used to measure cell proliferation activity as described in methods. g, h Wound healing assay (g) and Transwell assay (h) were used to measure cell migration and invasion ability as described in methods. i ER stress related genes were knocked down via lentiviral infection in Shctrl and ShNMT1-infected SUM149 and MDA-MB-231 cells. The expression of PERK, IRE1A and ATF6 was detected by western blot. j Quantification of ALDH-positive cells in h. k Quantification of Mammosphere formation from the cells in h. l MTT assay was used to measure the proliferation of cells in h. m, n Wound healing assay (m) and Transwell assay (n) were used to measure migration and invasion ability of the cells in h as described in methods. o For each group, 1 million of MDA-MB-231 cells were implanted into the mammary glands of 3-week-old to 4-week-old female nude mice and tumor size was monitored weekly (left). The tumor image was shown on the right. p Tumor weight from o. q Tumors from o were collected and cells were isolated from each tumor. ALDH was accessed by the ALDEFLUOR assay on viable dissociated cells. r The representative images for Ki67 IHC staining of tumors and HE staining of lung sections from o (left). Black arrows represent metastatic nodules in lungs. Ki67 positive cells and the numbers of metastatic lesions per lung section were counted (right).Data represent the mean ± SD of 3 independent experiments where *P < 0.05, **P < 0.01 and ***P < 0.001