GEPs from the MMRF CoMMpass dataset 21 ( ) and Zhan’s dataset, 14 2 independent datasets, both showed that lower ALCAM expression had shorter overall survival (OS) than those with higher expression ( Figure 1A). 12 Published GEPs of patients with MM were downloaded. To examine the clinical significance of ALCAM expression in MM, we performed microarray-based analyses as previously described. Analysis of Student t test with a false discovery rate < 0.05 after Benjamini-Hochberg correction was used for the screening of significantly changed proteins. The median normalization strategy was used in data analysis.
#Mendeley data software#
Subsequent data analysis was performed using R statistical software version 3.6.0. The peptides were searched by MaxQuant (Version 1.6.2.6) using Uniprot database to identify target proteins. Activity type was higher energy collisional dissociation. The auto gain control target, maximum injection time, and collision energy were 10 000 ms, 30 ms, and 30%, respectively. MS2 was analyzed with an isolation window (m/z) of 1.6 at an orbitrap resolution of 60 000. The radio frequency lens, auto gain control target, maximum injection time, and exclusion duration were 30%, 4.0 e 5, 50 ms, and 30 seconds, respectively. MS1 was analyzed with a scan range (m/z) of 350 to 1700 at an orbitrap resolution of 60 000. In brief, samples separated on a C18 capillary column over a 120-minute gradient. All samples were analyzed by SCE higher energy collisional dissociation tandem mass spectometry (MS) using an Orbitrap Fusion Lumos Tribrid (Thermo Fisher Scientific). After electrophoresis, the entire sample lane was excised, and digested with tryptic, followed by desalting with C18 stagetip column. Precipitated proteins were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 2 groups included immunoglobulin G pull-down and ALCAM pull-down, with triplicate samples in each group.
RPMI8226 cell lysate was used for immunoprecipitation.
Mass spectrometry was used to identify ALCAM interacting molecules. In this study, we showed that ALCAM suppressed MM clonogenic within the BM microenvironment. However, the function of ALCAM in MM is largely unknown. 12 MIF knockdown repressed the expressions of a panel of adhesion molecules on MM cells, including ALCAM, therefore reducing MM cell adhesion to the BM. 7, 9-11 In our previous work, we showed that macrophage migration inhibitory factor (MIF) regulated MM homing to the BM and that ALCAM was downstream of MIF-initiated cell signaling in MM. 8 Furthermore, the expression of ALCAM correlates with the disease progression in prostate cancer, breast cancer, colorectal carcinoma and melanoma. 5-7 As a cell adhesion molecule, ALCAM is known to interact with its ligand CD6 and mediate intercellular adhesion and migration. 4,5 ALCAM regulates various fundamental biological functions, such as cell adhesion, cell migration, autophagy and apoptosis, angiopoiesis, and hematopoiesis. Activated leukocyte cell adhesion molecule (ALCAM), a type I transmembrane glycoprotein, is a member of the immunoglobulin superfamily highly expressed by neuronal, endothelial, hematopoietic, and epithelial cells.