Journal: Current Issues in Molecular Biology

Article Title: Silencing LY6K Suppresses CD44 + EpCAM + HCT116 Human Colon Cancer Stem Cells Growth: Insights from In Vitro and In Vivo Evidence

doi: 10.3390/cimb46120840

Figure Lengend Snippet: Knockdown of LY6K inhibits migration and invasion of CCSCs in vitro. Optical micrographs and the number of migrated ( A ) and invaded ( B ) CCSCs following siLY6K or siNC transfection for 48 h according to Transwell migration and invasion assay. n = 3. ( C ) Western blot analyses showing the protein levels of MMP-2, MMP-9, and TIMP-1 in CCSCs after treatment with siLY6K or siNC for 48 h. Values are the mean ± SE (n = 3). ** p < 0.01, *** p < 0.001, or **** p < 0.0001 indicates significant differences from the Mock and siNC group as assessed by one-way ANOVA with Tukey–Kramer multiple comparisons tests. LY6K, lymphocyte antigen 6, locus K; CCSC, colon cancer stem cells; MMP, matrix metalloproteinase; TIMP-1, tissue inhibitor of MMP-1; n.s., not significant.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies [LY6K (1:750, #PIPA572689, Invitrogen), MMP-2 (3:1000, #BM0569, Boster, Pleasanton, CA, USA), MMP-9 (1:300, #BM4089, Boster), tissue inhibitor of MMPs-1 (TIMP-1) (3:1000, #BM4980, Boster), cyclin-dependent kinase (CDK) 4 (3:1000, #BM4672, Boster), cyclin D1 (3:1000, #bs-0623R, Bioss), CDK2 (3:1000, #bs-0757R, Bioss), cyclin E1 (3:1000, #bs-0573R, Bioss), caspase-3 and cleaved caspase-3 (1:1000, #14220, CST), β-actin (1:1500, #AF0003, Beyotime), and β-tubulin (1:1000, #AC008, Beyotime)], followed by incubation with secondary antibodies [horseradish peroxidase-conjugated goat anti-rabbit (1:2000, #A0208, Beyotime) and anti-mouse (1:2000, #A0216, Beyotime)] for 1 h at room temperature.

Techniques: Knockdown, Migration, In Vitro, Transfection, Invasion Assay, Western Blot