Ishikawa

Endometrial carcinoma cell lines HEC-1-B (ATCC® HTB-113™), HEC-1-A (ATCC® HTB112™) and KLE (ATCC® CRL1622™) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) and Ishikawa was purchased from Sigma-Aldrich (St. Louis, MO, USA). HEC-1-B cell line was maintained in MEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 2% penicillin/streptomycin (PAN-Biotech GmbH, Aidenbach, Germany). HEC-1-A cell line was maintained in Mc Coy’s 5A (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS and 2% penicillin/streptomycin. The Ishikawa cell line was maintained in MEM supplemented with 5% FBS and 2% penicillin/streptomycin. KLE was maintained in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS and 2% penicillin/streptomycin. All cells were grown at 37 °C in 5% CO₂.

Endometrial carcinoma cell lines HEC-1-B, AN3CA and KLE were purchased from ATCC (Manassas, VA, USA) and Ishikawa was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cells were obtained directly from the cell banks that perform cell line characterizations utilizing short tandem repeat profiling and passaged in the authors’ laboratory for fewer than 6 months after resuscitation. HEC-1-B cell line was maintained in MEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10 % fetal bovine serum (FBS), Ishikawa cell line was cultured in MEM (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 5 % FBS, KLE was maintained in DMEM, and AN3CA was cultured using EMEM (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10 % FBS. All cell lines were maintained with supplementation of 2 % penicillin/streptomycin and incubated in humidified chamber at 37 °C in 5 % CO₂ atmosphere.

A human endometrial adenocarcinoma cell line, Ishikawa (Sigma-Aldrich), was used for ChIP-seq and gene expression experiments. Ishikawa cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) and incubated at 37°C with 5% CO₂. The cells were transferred to hormone-depleted media (phenol red-free RPMI [Gibco] with 10% charcoal-dextran stripped fetal bovine serum [Sigma-Aldrich]), at least 5 d before transfection by gRNA and dCas9 fusion plasmids. Ishikawa deletion lines were previously created and verified. All deletions are homozygous deletions selected through single cell cloning. Deletions were cultured in the same conditions as parental Ishikawa cells, again being transferred to hormone-depleted media at least 5 d before transfection.
The cells were transfected using the FuGENE HD Reagent (Promega) according to the manufacturer’s protocol for unlisted cells. dCas9 fusions (dCas9-VP16(10x) or dCas9-p300(core)) plasmids were transfected at a mass ratio of 3:2 to pooled gRNA plasmids. Mass ratio of dCas9 fusions to tomato reporter plasmid (Addgene 30530, gift from Gerhart Ryffel) was 6:1. Plasmid solutions were prepared in Opti-MEM.