EMT-Related Gene Expression in MCF-7 Cells Treated with Monocarbonyl Analogs of Curcumin C66 and B2BrBC Following EMT Induction – RT-qPCR Array Dataset

Monocarbonyl analogs of curcumin (MACs), C66 [(2E,6E)-2,6-bis[(2-trifluoromethyl)benzylidene]cyclohexanone] and B2BrBC [(2E,6E)-2,6-bis(2-bromobenzylidene)cyclohexanone], were synthesized at the Institute of Chemistry, Ss. Cyril and Methodius University in Skopje, North Macedonia, following the previously published method (DOI: 10.17632/vdgz7pk6vh.1). MCF-7 human breast cancer cells (ATCC, Manassas, VA, USA; Cat. # HTB-22) were cultured in DMEM/F12 (50:50) medium (Corning Life Sciences, Corning, NY, USA; Cat. # 10-092-CM) supplemented with 10% FBS (Avantor, Radnor, PA, USA; Cat. # 89510-186) and antibiotic/antimycotic mixture (Corning Life Sciences, Corning, NY, USA; Cat. # 30-004-Cl) at 37 °C in 5% CO2 with 95% atmospheric air. For experiments, cells were seeded into 6-well plates (0.3 x 106 cells per well) in complete tissue culture media, supplemented with StemXVivo epithelial-to-mesenchymal transition (EMT)‑inducing media supplement (Bio‑Techne, Minneapolis, MN, USA; Cat. # CCM017), containing TGFβ1, Wnt5a, and antibodies against E‑cadherin, sFRP1, and Dkk‑1. After 48 h of incubation, the medium was replaced with pre‑warmed complete medium, and fresh EMT supplement was added along with either vehicle (DMSO), C66, or B2BrBC (100 µM each) for an additional 72 h. MCF-7 cells cultured in medium without a StemXVivo EMT Inducing Media Supplement, but with vehicle alone, served as the control. Total RNA was isolated using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA; Cat. # 15596026) and quantified with a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA samples were normalized to 1 µg total RNA and reverse transcribed into cDNA using qScript cDNA SuperMix (Quantabio, Beverly, MA, USA; Cat. # 95048) on a SimpliAmp Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA, USA). qPCR was carried out using the Human Epithelial to Mesenchymal Transition PCR Array kit (QIAGEN, Hilden, Germany; Cat. # 330231 PAHS‑090ZA) with PerfeCTa SYBR Green FastMix (Quantabio, Beverly, MA, USA; Cat. # 95072) on a QuantStudio 3 Real‑Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA, USA) under the following conditions: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C, and a final dissociation stage of 15 s at 95 °C, 1 min at 60 °C, and 15 s at 95 °C. Cycle threshold (Ct) values were determined using QuantStudio Design & Analysis Software (Applied Biosystems, Thermo Fisher Scientific).姜黄素单羰基类似物（Monocarbonyl analogs of curcumin, MACs）C66[(2E,6E)-2,6-双[(2-三氟甲基)亚苄基]环己酮]与B2BrBC[(2E,6E)-2,6-双(2-溴亚苄基)环己酮]，由北马其顿斯科普里圣西里尔与美多德大学化学研究所按照已发表的方法（DOI: 10.17632/vdgz7pk6vh.1）合成。 MCF-7人乳腺癌细胞（美国模式培养物集存库ATCC，美国弗吉尼亚州马纳萨斯；货号HTB-22）培养于DMEM/F12(50:50)培养基（康宁生命科学Corning Life Sciences，美国纽约州康宁；货号10-092-CM），该培养基添加10%胎牛血清（Fetal Bovine Serum, FBS；Avantor，美国宾夕法尼亚州拉德诺；货号89510-186）及抗生素-抗真菌混合液（康宁生命科学Corning Life Sciences，美国纽约州康宁；货号30-004-Cl），培养条件为37℃、5%CO₂及95%大气环境。 实验前，将细胞以每孔0.3×10⁶个的密度接种于6孔板，使用完全组织培养基，并添加StemXVivo上皮间质转化（epithelial-to-mesenchymal transition, EMT）诱导培养基添加剂（Bio-Techne，美国明尼苏达州明尼阿波利斯；货号CCM017），该添加剂包含转化生长因子β1（TGFβ1）、Wnt5a以及靶向E-钙粘蛋白、sFRP1和Dkk-1的抗体。孵育48小时后，更换预热的完全培养基，加入新鲜EMT添加剂，同时分别添加溶剂对照（二甲基亚砜，DMSO）、C66或B2BrBC（终浓度均为100μM），继续孵育72小时。以仅添加DMSO溶剂对照、未添加StemXVivo EMT诱导培养基添加剂的培养细胞作为空白对照组。 采用TRIzol试剂（TRIzol reagent；Invitrogen，赛默飞世尔科技Thermo Fisher Scientific，美国马萨诸塞州沃尔瑟姆；货号15596026）分离总RNA，使用NanoDrop One分光光度计（赛默飞世尔科技Thermo Fisher Scientific，美国特拉华州威尔明顿）对RNA进行定量。将RNA样品均一化至1μg总RNA，使用qScript cDNA SuperMix（Quantabio，美国马萨诸塞州贝弗利；货号95048）将总RNA反转录为cDNA，反转录反应在SimpliAmp热循环仪（应用生物系统Applied Biosystems，赛默飞世尔科技Thermo Fisher Scientific，美国加利福尼亚州卡尔斯巴德）上完成。 采用人类上皮间质转化PCR芯片试剂盒（凯杰QIAGEN，德国希尔德；货号330231 PAHS-090ZA），搭配PerfeCTa SYBR Green FastMix（Quantabio，美国马萨诸塞州贝弗利；货号95072），在QuantStudio 3实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qPCR）系统（应用生物系统Applied Biosystems，赛默飞世尔科技Thermo Fisher Scientific，美国加利福尼亚州卡尔斯巴德）上进行qPCR反应，反应程序如下：50℃孵育2分钟，95℃孵育10分钟，随后40个循环：95℃变性15秒、60℃退火延伸1分钟，最后进行溶解曲线分析：95℃15秒、60℃1分钟、95℃15秒。采用QuantStudio Design & Analysis软件（应用生物系统Applied Biosystems，赛默飞世尔科技Thermo Fisher Scientific）计算循环阈值（Cycle threshold, Ct）值。