Fig. 1: SNIP1 methylation promotes its oncogenic functions.
From: KMT5A-methylated SNIP1 promotes triple-negative breast cancer metastasis by activating YAP signaling
a A schematic workflow of IAP LC-MS/MS experiments. BT549 and MDA-MB-231 cell lysates were digested to perform IAP LC–MS/MS assays. b MS analysis showing potential methylation sites in SNIP1 after the immunoprecipitation (IP) of SNIP1 in BT549 and MDA-MB-231 cells. c Schematic representation of SNIP1 showing the c-MYC binding domain (FHA) and its amino acid sequence with all lysine (K) residues highlighted in red. NLS, nuclear localization signal. FHA, Forkhead associated domain. d Immunoblot (IB) analysis of the lysine methylation of wildtype (WT) and KR mutant SNIP1 in MDA-MB-231 and BT549 cells transfected with the indicated plasmids; lysates were assessed by IP with anti-HA and IB with anti-Pan-Me-Lys and anti-HA (n = 3). e LC-MS/MS spectrum of the tryptic peptide IADIPIDHPSCSK identified a monomethylated residue at K301, carrying a mass of +14.016 Da. f, g Cells derived from MDA-MB-231/sgSNIP1 cells re-overexpressed with indicated SNIP1-WT or SNIP1-K301R and selected with hygromycin (200 μg/ml) for 72 h before the collection was subjected to mouse xenograft assays by orthotopic injection. Tumor sizes were monitored and analyzed (f, g) (n = 6 mice per group). h Representative photos of Ki-67 positive staining cells in murine primary tumor sections of indicated tumor tissues. Scale bar, 100 μm (IHC). i Quantification of the number of cells positive for Ki-67 staining per field of vision in tumor sections from (h) (n = 6 for each group). FOV field of view. j–m Cells generated in (f) were subjected to mouse xenograft through tail vein injection. Lung metastases were monitored (j, l) and calculated (k, m) (n = 8 mice per group). n Kaplan–Meier survival of mice in (j) (n = 8 mice per group). Data information: In (g, i, k, m), statistical analysis was performed by a two-tailed t-test. In (n), by log-rank test. ***P < 0.001. Data are represented as mean ± SEM. Panels (d) and (f–n) show one experiment representative of three independent experiments with similar results.
