Additional file 2 of Novel 3D embryo implantation model within macroporous alginate scaffolds

Additional file 2: Supplementary data 2. ERα transfection of HEC-1A cells. The ERα open reading frame was cloned into a pcDNA6.2/V5 vector (a kind gift from Prof. Carlos Simon, University of Valencia). HEC-1A cells were transfected using Lipofectamine™ 2000 (Invitrogen, Paisley, UK) either with the ERα vector or with an empty vector as control. Following 48 h, medium was replaced with 10 μg/mL blasticidin-containing media (Invitrogen, Paisley, UK) for selection. After 2 weeks, individual colonies were selected. Transfection efficiency was confirmed by ERα mRNA expression levels, evaluated by qPCR and ERα nuclear localization, and evaluated by immunofluorescent staining. Supplementary Fig. 2. Validation of ERα transfection evaluated by ERα expression in ERα transfected HEC-1A cells compared to HEC-1A cells transfected with the empty vector. (A) qPCR analysis: Higher ERα mRNA expression levels in ERα transfected HEC-1A cells, compared to cells transfected with an empty vector (t-test, p < 0.01). Expression levels are relative to RPLP0 levels. (B) Anti-ERα immunofluorescent staining: ERα protein expression in ERα transfected HEC-1A cells (right), compared to cells transfected with an empty vector (left). ERα immunofluorescent staining (green) and 4′,6-diamidino-2-phenylindole (DAPI) staining for nuclei (blue) (Bar: 100 μm).