Fig. 1: ZNF32 increases breast cancer stem-like cell populations.

(a) Representative images of mammosphere formation, and bar graph showing the fold change in the number of mammospheres per field in the shNC, shZNF32, vector, ZNF32. shZNF32 and ZNF32 groups compared to the shNC or vector groups. (b) (c) ALDH1 and ZNF32 expression levels were measured in normal and suspension-cultured ZR-75-30 cells by flow cytometry using ALDH1⁺ or ZNF32⁺ gates based on normal cultured cells. The bar graph in the right panel shows ALDH1 or ZNF32 expression in normal or suspension-cultured cells. (d) Correlation analysis between ZNF32 and Nanog expression levels in breast cancer cell lines using CCLE. (e) (f) OCT4, Nanog and KLF4 expression levels were detected by qPCR in ZNF32 over-expressing or knockdown ZR-75-30 cells. (g) ALDH1-positive cells were detected by flow cytometry using an ALDH1⁺ gate based on shNC or vector cells. The bar graph in the right panel shows the number of ALDH1-positive cells in the shNC, shZNF32, vector, and ZNF32 ZR-75-30 groups. (h) Stem cell frequency was calculated using the online Extreme Limiting Dilutions Assay (ELDA) analysis program. A significant difference in stem cell frequency was detected between vector (1/18.3) and ZNF32 (1/10.7) ZR-75-30 cells. (i) ZR-75-30 cells with ZNF32 knockdown or over-expression were plated in 96-well plates and incubated for 24 h, and then, 10 ng/ml or 20 ng/ml Taxol or DMSO as a solvent control was added. MTT analysis was used to assess cell viability