BM stroma-derived soluble factors promoted ALCAM-knocked down myeloma cell clonogenicity. (A) Flow cytometry of surface ALCAM expression in CTR-KD, AL-KD1 (shRNA sequence 1), and AL-KD2 (shRNA sequence 2) MM cells. The number indicates mean fluorescence intensity (MFI). (B) Top: western blotting of ALCAM expression in CTR-KD vs AL-KD MM cells in RPMI8226 and MM.1S; bottom: result quantification. (C) Expression of ALCAM messenger RNA in CTR-KD vs AL-KD MM cells (RPMI8226 and MM.1S) analyzed by qRT-PCR. (D) Imaging of colony formation assays in RPMI8226/BMSC cocultured condition (top) and BMSC conditioning medium (BMSC-M) (bottom). BMSCs from healthy individuals. The results are quantified in (E) left panel and right panel, respectively. The data were obtained from 3 independent experiments and shown as the mean ± SD. (F) Immunofluorescence microscopy of cells (RPMI8226-CTR vs RPMI8226-ALCAM-KD) under 3D printing. The data were obtained from 10 randomly chosen fields and shown as the mean ± SD. The results are summarized in the right panel. (G) Soft agar assay showing that soluble ALCAM-Fc (0.5 mg/mL) attenuated BMSC-M conferred RPMI8226-CTR cells and RPMI8266-ALCAM-KD clonogenicity. The data are shown as the mean ± SD. (B,E-G) Two-tailed Student t test or (C) 1-way ANOVA with multiple comparisons. *P < .05; **P < .01; ***P < .001.