Fig. 2.

MICAL3 is a key factor for tumor sphere formation by human BCCs. (A) Comparison of expression levels of MICAL3 mRNA in normal breast (NB) and breast cancer (BC) tissues using Oncomine databases. (B) Kaplan–Meier analysis of relapse-free survival of patients with breast cancer tissues showing low or high MICAL3 expression. Median was used for cutoff values. Gene expression profiles GSE1379 (Upper) and GSE12276 (Lower) were used for analysis. (C) Cells were cultured in SCM that contains several growth factors and hormones (epidermal growth factor, basic fibroblast growth factor, and B27) in DMEM/F12. SCM-induced tumor sphere formation was decreased by MICAL3 knockdown; n = 4. (D) Representative phase contrast images of tumor sphere formation by MICAL3 knockdown in patient-derived BCCs (patients 1 and 2). (Scale bar: 100 µm.) (E) SCM-induced tumor sphere formation was decreased by MICAL3 knockdown in patient-derived BCCs; n = 4. (F) Representative images of tumors generated in mice injected with patient-derived BCCs (patient 4). (Scale bar: 1 cm.) (G) The 10³, 10², or 10 cells per site were s.c. injected into the mammary fat pads of 8-wk-old female immunodeficient mice. Results were obtained 60 d after implantation. Frequency determinations were generated using ELDA software (SI Appendix, Ref. 1). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t tests. For LDAs, the method of Holm was used.