Figure 3: Characterization of ESR1–CCDC170 protein products and their transforming activity in MCF10A breast epithelial cells.

(a) Schematic of ESR1–CCDC170 fusion variants and encoded proteins identified in breast cancer cell lines. ORFs are depicted in dark shades. The exon numbers are based on reference sequence NM_001122742 for ESR1 and NM_025059 for CCDC170. (b) Immunoblot analysis of MCF10A and T47D cells expressing ΔCCDC170 ORFs, the fusion-positive HCC1428 cell line and fusion-negative control cell lines, using an anti-CCDC170 polyclonal antibody. Arrows indicate the ΔCCDC170 bands. To enhance the detection of differentially sized ΔCCDC170 protein variants, the blot region pertaining to the molecular weight of each respective ΔCCDC170 variant was cut and then probed with the antibody. A longer exposure time is required to enhance the visualization of the E2–E8 fusion protein. The identity of the 14 kDa band detected in HCC1428 is verified by an siRNA against the E2–E10 fusion expressed by this line. Overexpression of ΔCCDC170 variants in MCF10A cells significantly enhances (c) cell migration, (d) matrigel invasion and (e) clonal expansion. (f) Cell cycle analysis of the MCF10A cell models. Error bars represent the s.d. of at least three replicate measurements per condition. The results shown are representative of experiments performed at least twice. ***P<0.001 (t-test). ‘Vector’ indicates MCF10A cells transduced with pLenti7.3 vector containing an YFP ORF.