Fig. 1

E-cadherin regulates cell growth and CSC-like phenotypes in human MM-derived cells. E-cadherin was depleted in RPMI 8226 and NCI-H929 cells using the CRISPR/Cas9 system, designated as CDH1-KO RPMI 8226 and CDH1-KD NCI-H929 cells, respectively (Additional file 2: Figure S2). A Cell viability was evaluated by MTT assay to monitor cell proliferation at 24, 48, 72, and 96 h of culture. Data are mean ± SD (n = 3). ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 versus WT control cells; two-tailed Student’s t-test. B Cell surface expression of CD138 was analyzed by flow cytometry. The proportion of CD138-positive (CD138⁺) and CD138-negative (CD138⁻) cells is shown. Data are mean ± SD (n = 3). ^***p < 0.001, ^****p < 0.0001 versus WT cells; two-tailed Student’s t-test. C Western blot analysis of prosurvival Bcl-2 and Mcl-1 proteins. β-actin was used as a loading control. The significant decrease in Bcl-2, but not Mcl-1, level was detected in CDH1-KO RPMI 8226 and CDH1-KD NCI-H929 cells compared to WT cells (^**p < 0.01; two-tailed Student’s t-test). D (upper) Cell cycle analysis based on DNA content was analyzed by flow cytometry using propidium iodide staining. (lower) Quantitative real-time PCR (RT-qPCR) analysis of mRNA expression of cell cycle regulator genes. GAPDH served as the internal control. Data are mean ± SD (n = 3). ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001 versus WT cells; two-tailed Student’s t-test. E Western blot analysis of Akt and MAPK family proteins. The significant decrease in phosphorylated (p)-Akt, p-p38, and p-p44/42 levels was detected in CDH1-KO RPMI 8226 and CDH1-KD NCI-H929 cells compared to WT cells (^*p < 0.05; two-tailed Student’s t-test). F Representative micrographs showing MM colonies under clonogenic assay (see also Additional file 2: Figure S3 for quantitative analysis of colony number and size). Scale bar = 200 μm. G SP subpopulation analysis using flow cytometry based on Hoechst 33342 dye efflux. SP cells (box) were determined by their disappearance in the presence of fumitremorgin C (see also Additional file 2: Figure S5 for quantitative analysis). H Western blot analysis of ALDH1A1 and ABCG2. A significant decrease in ABCG2, but not ALDH1A1, level was detected in CDH1-KO RPMI 8226 and CDH1-KD NCI-H929 cells compared to WT cells (^*p < 0.05; two-tailed Student’s t-test). I RT-qPCR analysis of mRNA expression of stemness-regulated genes. Data are mean ± SD (n = 3). ^****p < 0.0001 versus WT cells; two-tailed Student’s t-test. J Western blot analysis of SOX9 level in CDH1-KO RPMI 8226 and CDH1-KD NCI-H929 cells (see also Additional file 2: Figure S6 for quantitative analysis)