Journal: eLife

Article Title: Trait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction

doi: 10.7554/eLife.56697

Figure Lengend Snippet: ( A ) Basal activity of reference alleles of RE candidates in HL1 (light grey bars) and COS7 cells (dark grey bars). Luciferase activity for each RE candidate is normalized to the activity of the empty pGL2-SV40 vector. ( B ) Luciferase activity of reference alleles of RE candidates upon stimulation with Smad/Gata- (SG4) and Tcf+LiCl- (Wnt) factors in COS7 cells. Luciferase activity of each RE candidate is normalized to the activity of each RE candidate upon co-transfection with pcDNA (control). Transfections were performed in duplicates and replicated twice. Error bars represent standard deviation. *: p<0.05.

Article Snippet: Amplified sequences were ligated in the XcmI site of a modified pGL2-SV40 vector (Promega) using T4 DNA Ligase (ThermoFisher Scientific; 15224–090).

Techniques: Activity Assay, Luciferase, Plasmid Preparation, Cotransfection, Transfection, Standard Deviation

Journal: eLife

Article Title: Trait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction

doi: 10.7554/eLife.56697

Figure Lengend Snippet: ( A ) Overview of the murine Tbx3 locus with ATAC_AVJ, EMERGE and STARR_SG4 data. Fragments selected for luciferase assay, selected either on STARR-seq activity or EMERGE/ATAC peaks are depicted below. ( B ) Correlation between luciferase reporter activity (fold change of reporter activity over activity of empty pGL2-SV40 vector; threshold FC >2) and STARR-seq (log2FC of SG4 over pcDNA control; threshold log2FC >0.58 (FC >1.5)) of selected fragments.

Article Snippet: Amplified sequences were ligated in the XcmI site of a modified pGL2-SV40 vector (Promega) using T4 DNA Ligase (ThermoFisher Scientific; 15224–090).

Techniques: Luciferase, Activity Assay, Plasmid Preparation

Journal: Biology of Reproduction

Article Title: Endocrine disruptor exposure during development increases incidence of uterine fibroids by altering DNA repair in myometrial stem cells †

doi: 10.1093/biolre/ioy097

Figure Lengend Snippet: DNA end-joining efficiency is decreased in DES vs. VEH MSCs. Luciferase gene reporter assay detected lesser DNA end-joining activity in DES vs. VEH MSCs following co-transfection with linearized HindIIIpGL2-control luc and CMV-β-gal plasmids. Means of combined experimental and biological replicates were compared using a two-tailed Student unpaired t-test. Lines represent the mean normalized end-joining activity ± SD. *P < 0.0001

Article Snippet: Plasmid DNA preparation Briefly, SV40- luc pGL2-control vector plasmid DNA ( Supplemental Figure S1A , Promega, Madison, WI) was used to transform DH5α Escherichia coli cells for plasmid amplification and was then isolated using PureYield Plasmid Midiprep System (Promega).

Techniques: Luciferase, Reporter Assay, Activity Assay, Cotransfection, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Estradiol Regulates mRNA Levels of Estrogen Receptor Beta 4 and Beta 5 Isoforms and Modulates Human Granulosa Cell Apoptosis

doi: 10.3390/ijms22095046

Figure Lengend Snippet: E2 induces transactivation of ERs in hGCs. Cells were transiently transfected with Firefly luciferase reporter plasmid containing or not (pGL2) estrogen-response elements (EREs) upstream of luciferase (ERE-Luc). An internal control vector containing Renilla luciferase was also included. Twenty-four hours post-transfection, cells were treated with either solvent vehicle (V) or 10 nM E2, in the presence or absence of 1 µM Fulvestrant (F), an ER degrader (SERD). Twenty-four hours later, cells were lysed and analyzed for luciferase activity. Firefly luciferase activity was normalized relative to Renilla luciferase activity, reported as relative transactivation activities arbitrarily set at 1 for the solvent vehicle (V) condition in ERE-Luc-transfected cells. Each point represents the mean ± SEM of 14 experiments (from each patient) performed in six replicates. *** p ≤ 0.001 vs. solvent vehicle (V) by two sides Wilcoxon signed-ranks test. A.U., Arbitrary Units.

Article Snippet: An empty pGL2-Firefly luciferase vector (pGL2), a pGL2 vector containing three copies of vitellogenin estrogen-response element sequence (ERE-Luc) upstream of luciferase (Addgene, Watertown, MA 02472, USA), and a pRL-SV40-Renilla luciferase (pRL-Renilla) vector (Promega, Charbonnières les bains, France) were used in the reporter assays.

Techniques: Transfection, Luciferase, Plasmid Preparation, Control, Solvent, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Estradiol Regulates mRNA Levels of Estrogen Receptor Beta 4 and Beta 5 Isoforms and Modulates Human Granulosa Cell Apoptosis

doi: 10.3390/ijms22095046

Figure Lengend Snippet: Absence of influence of ERβ2/β4/β5 on E2-induced ERβ1 transactivation in HGrC1-transfected cells. ( A ) The presence of ERα and ERβ (1, 2, 4, and 5) isoforms mRNA in hGCs ( n = 49) and HGrC1 cells ( n = 6) was evaluated by RT-qPCR (expression normalized to GAPDH). Values are represented as means ± SEM from three identical wells per patient, measured in triplicate. * p ≤ 0.05; ** p ≤ 0.01 by the Mann–Whitney test. A.U., Arbitrary Units. HGrC1 cells express low levels of endogenous ERα and ERβ isoforms, when compared to those expressed in hGCs. ( B ) HGrC1 cells were transiently cotransfected with the control Flag vector (C) alone or with the Flag-ERβ1 vector (β1), together with the Firefly luciferase reporter plasmid containing or not (pGL2) estrogen-response elements (EREs) upstream of luciferase (ERE-Luc). Flag-ERβ1 was also cotransfected (1:1 ratio) with either Flag-ERβ2 (β1+β2), Flag-ERβ4 (β1+β4), or Flag-ERβ5 (β1+β5). Renilla luciferase reporter plasmid was included as a normalizing transfection control. Twenty-four hours after solvent vehicle (V) or 10 nM E2 treatment, cells were lysed and analyzed for luciferase activity. Firefly luciferase activity was normalized to that of Renilla luciferase and reported as relative transactivation activities arbitrarily set at 1 for the control (C) solvent vehicle (V) condition in ERE-Luc-transfected cells. Each point represents the mean ± SEM in six replicates performed four times. * p ≤ 0.05; ** p ≤ 0.001 vs. vehicle (V) for each group of cotransfection, and ## p ≤ 0.001 vs. control (C) vehicle (V) using the Mann–Whitney test. A.U., Arbitrary Units. ( C ) HGrC1 cells were transiently transfected with either Flag-ERβ1 (β1), Flag-ERβ2 (β2), Flag-ERβ4 (β4), or Flag-ERβ5 (β5). Twenty-four hours post-transfection, cells were fixed, permeabilized, and the localization of Flag-ERβ proteins was monitored by immunofluorescence using an anti-Flag antibody (green). Nuclei were stained with DAPI (blue). Representative images at 20x magnification are presented.

Article Snippet: An empty pGL2-Firefly luciferase vector (pGL2), a pGL2 vector containing three copies of vitellogenin estrogen-response element sequence (ERE-Luc) upstream of luciferase (Addgene, Watertown, MA 02472, USA), and a pRL-SV40-Renilla luciferase (pRL-Renilla) vector (Promega, Charbonnières les bains, France) were used in the reporter assays.

Techniques: Transfection, Quantitative RT-PCR, Expressing, MANN-WHITNEY, Control, Plasmid Preparation, Luciferase, Solvent, Activity Assay, Cotransfection, Immunofluorescence, Staining