**2.2 mRNA analysis by qRT-PCR**

Total RNA was extracted from tissue samples (30 mg), ground in liquid N2 with a pestle and mortar and from cell lines (104 cells) using the RNeasy Mini kit method (QIAGEN, UK) as per manufacturer's protocol. 300 ng of total RNA was reverse transcribed with (+RT) or without (-RT) reverse transcriptase (RT) using the high-capacity cDNA reverse transcription kit (Life Technologies, Paisley, UK). 2 μl of cDNA were amplified by real time PCR with commercially available TaqMan assays (Life Technologies, Paisley, UK) for *ESR1* (Hs00174860\_m1), *ESR2* (Hs01100353\_m1), and the reference genes *GAPDH* (Hs02758991\_g1), *PGK1* (Hs00943178\_g1), and *ACTB* (Hs01060665\_g1) in a Chromo 4 thermal cycler (Bio-Rad Laboratories LTD, Hemel Hempstead, UK). Expression of *ESR1* and *ESR2* was quantified relative to the geometric mean of three reference genes and reported as relative to max using the GenEX software Version 5 (MultiD, DE) in accordance with MIQE guidelines [26].

## **2.3 Immunohistochemistry**

Immunohistochemistry (IHC) slides were prepared in the Histopathology Department at the Royal Derby Hospital. Normal mucosa and EC samples were stained using ERα and ERβ antibodies (NCL-L-ER-6F11 and 6007907, respectively, Novacastra, Newcastle, UK). ERα and ERβ positive breast cancer samples were used as positive controls. The 'H-score method was used to measure the strength of ER-staining in normal esophageal mucosa and matched tumour samples [27]. Positive staining was defined as an H-score ≥ 10 in this study.
