Fig. 1: NMT1 knockdown inhibits breast cancer initiation, growth, and metastasis.

a Heatmap from Microarray analysis representing the up- and down-regulated genes in Mir100 overexpression versus Ctrl cells both in SUM159 and MDA-MB-231. b NMT1 expression in clinical breast tumor tissues and adjacent noncancerous tissues were detected by IHC (left). Semi-quantitative IHC scores of NMT1 in breast tumor tissues and adjacent tissues (right). c NMT1 was knocked down via ShNMT1 lentiviral infection (Shctrl was the control). NMT1 expression was detected by qPCR and western blot in SUM149 and MDA-MB-231. d ALDEFLUOR assay of the Shctrl and ShNMT1-infected SUM149 and MDA-MB-231 cells (left). Quantification of ALDH-positive cells in these cells (right). e Mammosphere formation assay in Shctrl and ShNMT1-infected SUM149 and MDA-MB-231 cells. Number of mammospheres was calculated (n > 3). f, g MTT assay (f) and Colony formation assay (g) were used to measure cell proliferation activity as described in methods. h Cell cycle distribution analyzed by FACS in Shctrl and ShNMT1-infected SUM149 and MDA-MB-231 cells. i Cell cycle related proteins were detected by western blot in Shctrl and ShNMT1-infected SUM149 and MDA-MB-231 cells. j, k Wound healing assay (j) and Transwell assay (k) were used to measure cell migration and invasion ability as described in methods. l For each group, 500k SUM149 or 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. m Tumor weight from l. n The representative images for Ki67 IHC staining in tumors from l (left) and quantification of Ki67 positive cells (right). o Tumors from l were collected and cells were isolated from each tumor. ALDH was accessed by the ALDEFLUOR assay on viable dissociated cells. p HE staining of lung sections from Shctrl and ShNMT-infected MDA-MB-231 tumor group and the numbers of metastatic lesions per lung section were counted. Black arrows represent metastatic nodules in lungs. Data represent the mean ± SD of 3 independent experiments where *P < 0.05, **P < 0.01 and ***P < 0.001