Fig 2: ZNF32 promotes breast cancer stem cell-like properties associated with drug resistance partially by up-regulating GPER expression.(a) OCT4, KLF4 and Nanog gene expression were measured by qPCR in ZNF32 over-expressing ZR-75-30 cells with or without GPER knockdown. (b) ALDH1 positive cells were detected by flow cytometry, showed using ALDH1+ gate based on ZR-75-30 cells vector siNC. Bar graph in the right panel was ALDH1 positive cells in vector siNC, ZNF32 siNC, vector siGPER 1#, ZNF32 siGPER 1# cells. (c) The stem cell frequencies were calculated using the online Extreme Limiting Dilutions Assay (ELDA) analysis program. Significant differences in stem cell frequencies were detected between vector siNC (1/16.2), ZNF32 siNC (1/9.3), vector siGPER 1# (1/28.4), and ZNF32 siGPER 1# (1/15.5) cells. (d) ZR-75-30 cells with ZNF32 over-expression were plated in 96-well plates, and GPER expression was knocked down using siRNA. After 24 h of incubation, 10 ng/ml or 20 ng/ml Taxol or DMSO (control) was added, and MTT analysis was used to detect cell viability

Fig 3: The roles of ZNF32 and GPER in tumour formation in xenografts.(a) Western analysis was used to detect the GPER knockdown efficiency in ZNF32 over-expressing ZR-75-30 cells. Quantification of 3 independent experiments is shown in the right panel. A total of 5 × 106 viable cells were implanted subcutaneously into nude mice. 7 days after inoculation, the mice received U0126 or vehicle. A tumour diameter of 3 mm was considered tumour formation, and the tumour formation times were recorded (b). Tumours were collected 3 weeks after implantation (c). Tumour volume (d) and tumour weight (e) were calculated. (f) A total of 1 × 105, 2 × 105, 3 × 105, 4 × 105 or 5 × 105 viable cells were implanted subcutaneously into nude mice. 7 days after inoculation, the mice received vehicle or U0126. A tumour diameter of 3 mm was considered tumour formation, and the tumour formation was recorded 3 weeks after subcutaneous transplantation. (g) Representative images of ZNF32, GPER and ALDH1 expression in breast cancer samples as detected by IHC. Quantification of detected result is shown in the right panel. (H) (I) Overall survival probability of breast cancer patients with the indicated gene expression levels

Fig 4: 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

Fig 5: ZNF32 and GPER regulate breast cancer stem cell populations by activating the ERK pathway.(a) Western analysis was used to detect ERK activation. Quantification of ZNF32 and pERK expression in 3 independent experiments. (b) Western analysis was used to detect ERK activation in ZNF32 over-expressing cells with or without GPER interference. Quantification of ZNF32 and pERK expression levels from 3 independent experiments. (c) ERK inhibitor U0126 (20 μM) or DMSO (control) was added to ZR-75-30 cells over-expressing ZNF32, followed by incubation for 24 h. Western analysis was used to detect ERK activation. Quantification of ZNF32 and pERK levels from 3 independent experiments. (d) qPCR analysis was used to measured ALDH1 gene expression in ZNF32 over-expressing or vector control ZR-75-30 cells treated with or without U0126 or DMSO for 24 h. (e) qPCR analysis was used to detect OCT4, Nanog and KLF4 gene expression in ZNF32 over-expressing or vector control ZR-75-30 cells treated with or without U0126 or DMSO for 24 h. (f) (g) ZR-75-30 cells with ZNF32 over-expression or knockdown were plated in 96-well plates. After 24 h of incubation, pretreatment with 20 μM U0126 was performed for 2 h before the addition of 10 ng/ml or 20 ng/ml Taxol or DMSO (control), and MTT analysis was used to detect cell viability