skbr3 cells  (AcceGen Biotechnology)

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Estradiol dose-response and recovery of spiked estradiol from tampon extracts. (A) T47D-luc cells were treated with an 11-point estradiol dilution series starting at 30nM, followed by 10nM, 3.3nM, 1.11nM, 370pM, 123pM, 41pM, 14pM, 4.6pM, 1.5pM, and 0.5pM). For each concentration, 2 μL of steroid dilution was added to 198 μL assay medium, and cells were incubated for 24 hours. Estrogen-responsive luciferase activity was quantified using BrightGlo substrate. Luminescence values represent the mean of 4 technical replicates, and a sigmoidal dose-response curve with EC 50 determination was generated using GraphPad v10. (B) Recovery of estradiol from tampon brands A-D following spiking and extraction. One gram of each tampon from Brands A, B, C, and D were spiked with 2nM estradiol, soaked in a 1:1 water:ethanol solution for 4 hours, extracted, evaporated to dryness, and reconstituted in methanol. Extracts were diluted 1:100 in cell culture medium and then serially diluted 1:2 to generate 4 assay points. T47D-luc cells were incubated with a further 1:10 dilution of each tampon extract for 24 hours and luciferase activity measured using BrightGlo. Each point represents mean ± SEM of 4 technical replicates corrected for the blank control sample (1:1 ethanol:water solution SPE-extracted as per samples). The data for the spiked tampons are plotted alongside estradiol standards run in parallel.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Concentration Assay, Incubation, Luciferase, Activity Assay, Generated, Extraction, Cell Culture, Control

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Estrogenic activity detected in extracts from 8 commercially available tampon brands (A-H). T47D-luc cells were exposed to methanolic extracts prepared from 1 g of core tampon material (string removed) from 8 tampon brands sold in New Zealand. Each 1 g sample was soaked in a 1:1 ethanol:water solution for 4 hours, sonicated, filtered, extracted, and then evaporated to dryness, and reconstituted in methanol. The final extract was diluted into assay medium at 1:1000 (1), followed by serial dilutions to generate 1:2000 (2), 1:4000 (3), and 1:8000 (4) exposure concentrations. Cells were incubated with each dilution for 24 hours, and estrogen-responsive luciferase activity was quantified using a luminescence plate reader. Data are presented as the mean ± SEM of 2 independently extracted tampons per brand, each tested as 2 portions of 1 g each, and each dilution tested with 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Activity Assay, Sonication, Incubation, Luciferase, Microplate Reader Luminescence Measurement

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Independent batches of Tampon Brand D show estrogenic activity. T47D-luc cells were treated with methanolic extracts generated from 1 g of core tampon material (string removed) from 3 independent batches of Brand A and Brand D. For each batch, 3 separate tampons were extracted independently. Each 1 g sample was soaked in 1:1 ethanol:water solution for 4 hours, sonicated, filtered, extracted, and evaporated to dryness, and reconstituted in methanol. Reconstituted extracts were diluted into assay medium at 1:1000 (1), followed by serial 1:2 dilutions to produce 1:2000 (2), 1:4000 (3), and 1:8000 (4) exposure concentrations. Cells were incubated for 24 hours, and estrogen-responsive luciferase activity was quantified by using a luminescence plate reader. Data are shown as mean ± SEM of 3 independently extracted tampons per batch, tested as 2 portions of 1 g each with each dilution tested as 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Activity Assay, Generated, Sonication, Incubation, Luciferase, Microplate Reader Luminescence Measurement

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Estrogenic activity of internationally sourced tampon brands. T47D-luc cells were treated with methanolic extracts prepared from intact tampons (string removed) from a panel of internationally sourced tampon brands (Brands I-R). For each brand, 2 or 3 independent tampons were extracted separately on independent days. Each tampon was soaked in a 1:1 ethanol:water solution for 4 hours, sonicated, filtered, extracted, evaporated to dryness, and reconstituted in methanol. Reconstituted extracts were diluted into assay medium at 1:1000, and cells were incubated for 24 hours before estrogen-responsive luciferase activity was quantified using a luminescence plate reader. Data are shown as mean ± SEM of 2 or 3 independently extracted tampons per brand with each dilution tested as 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Activity Assay, Sonication, Incubation, Luciferase, Microplate Reader Luminescence Measurement

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Effect of sonication on estrogenic activity of tampon extracts. T47D-luc cells were exposed to methanolic extracts prepared from 2 independent tampons per brand (Brands A and D, string removed), comparing extraction with and without sonication. Each tampon was soaked in a 1:1 ethanol:water solution for 4 hours, either sonicated or left nonsonicated, then filtered, extracted, evaporated to dryness, and reconstituted in methanol. Extracts were diluted into assay medium at 1:1000 (1), followed by serial 1:2 dilutions to generate 1:2000 (2) and 1:4000 (3) exposure concentrations. Cells were incubated for 24 hours, and estrogen-responsive luciferase activity was quantified. Data represent mean ± SEM of t2wo independently extracted tampons per condition with each dilution tested as 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Sonication, Activity Assay, Extraction, Incubation, Luciferase

Journal: bioRxiv

Article Title: Discovery and Preclinical Validation of a Clinically Optimized Mitochondrial Complex I Modulator for Alzheimer’s Disease

doi: 10.64898/2026.04.10.717554

Figure Lengend Snippet: a A drug discovery funnel used for the identification of C273. b C273 protects MC65 Tet-Off cells from Aβ toxicity (red line). # indicates cell death in Tet-Off cells treated with vehicle. No toxicity was observed in MC65 Tet-On cells (Aβ not expressed, black line). EC 50 values were calculated using nonlinear regression in GraphPad Prism 10. c Treatment with C273 for 24 h does not induce cytotoxicity in HepG2 human hepatic cells at concentrations up to 100 μM. d C273 increases NADH levels in MC65 Tet-On cells, consistent with inhibition of mtCI. NADH levels were measured over 24 h. e Inhibition of mtCI activity in isolated mouse brain mitochondria by C273 measured using an NADH oxidation assay. f Activities of mitochondrial ETC complexes I–IV were assessed in permeabilized MC65 cells using an electron flow assay in the Seahorse XFe96 Extracellular Flux Analyzer in the presence of C273 (25 and 50 μM). g Injection of rotenone or C273 to monitor OCR kinetics in MC65 Tet-On cells. h Pretreatment with rotenone blocks the protective effect of C273 in MC65 Tet-Off cells (Aβ expressed). Open orange squares: Tet-On cells, no Aβ expression, treated with vehicle (positive control, 100% viability). Blue triangles: Tet-Off cells (Aβ expressed), which undergo extensive cell death after 3 days. Tet-Off cells treated with C273 (2 nM - 5 μM; blue line) show complete protection from Aβ toxicity. Solid red triangles: Tet-On cells treated with 32 nM rotenone, which show no toxicity. Open red triangles: Tet-Off cells treated with 32 nM rotenone alone, resulting in complete cell death. Pretreatment with 32 nM rotenone blocks the protective effect of C273 in Tet-Off cells (red line, grey box), supporting a competitive interaction at mtCI. Partial rescue at higher C273 concentrations reflects incomplete occupancy of the mtCI binding site by rotenone, which inhibits ∼50% of mtCI activity at this dose. The grey box indicates the EC 50 concentration range for C273. P values were calculated using unpaired two-tailed Student’s t -tests; ** P < 0.01. Data are presented as mean ± SD ( n = 4 technical replicates). All experiments were replicated at least twice.

Article Snippet: NF-κB Reporter (Luc) HEK293 cell line and the antioxidant response element (ARE) Luciferase Reporter HepG2 hepatic cell line were purchased from BPS Bioscience (CA, USA) and were cultured in accordance of manufacturer instructions.

Techniques: Drug discovery, Inhibition, Activity Assay, Isolation, Oxidation Assay, Injection, Expressing, Positive Control, Binding Assay, Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Lipid moieties of sonic hedgehog are important for interaction with its inhibitor, WIF1

doi: 10.64898/2026.02.23.707386

Figure Lengend Snippet: Comparison of the signaling activities of rhShh_8908-SH, rhShh_1314-SH and rhShh_1845-SH in the Gli Reporter-NIH3T3 cell line reporter assay. Panel A: rhShh_8908-SH expressed in HEK293 cells was applied at 0–600 nM. EC 50 = 34.22 ± 2.25 nM. Panel B: rhShh_1314-SH expressed in E. coli was applied at 0–2400 nM. EC 50 = 64.81 ± 1.29 nM. Panel C: rhShh_1845-SH expressed in E. coli was applied at 0–2400 nM. EC 50 = 86.09 ± 1.11 nM. In all assays, Gli Reporter-NIH3T3 cells were grown to confluency, the culture medium was removed, and cells were treated with 50 μL aliquots of the indicated Shh concentrations. Data represent one experiment performed in triplicate and are expressed as fold induction relative to luminescence at 0 nM Shh.

Article Snippet: The ONE-StepTM Luciferase Assay System and the Gli Reporter-NIH3T3 cell line were purchased from BPS Bioscience (Inc., San Diego, CA, USA).

Techniques: Comparison, Reporter Assay

Journal: bioRxiv

Article Title: Lipid moieties of sonic hedgehog are important for interaction with its inhibitor, WIF1

doi: 10.64898/2026.02.23.707386

Figure Lengend Snippet: Inhibition of the signaling activities of rhShh_8908-SH, rhShh_1314-SH and rhShh_1845-SH proteins by human WIF1 protein in the Gli Reporter-NIH3T3 cell line reporter assay. Panel A: Inhibition of human rhShh_8908-SH expressed in HEK293 cells (15 nM) by rhWIF1 (0–1000 nM); EC 50 = 3.78 ± 0.13 nM. Panel B: Inhibition of rhShh_1314-SH expressed in E. coli (15 nM) by rhWIF1 (0–1200 nM); EC 50 = 6.83 ± 0.89 nM. Panel C: Inhibition of rhShh_1845-SH expressed in E. coli (15 nM) by rhWIF1 (0–1200 nM); EC 50 = 15.71 ± 1.75 nM. In all assays, Gli Reporter-NIH3T3 cells were grown to confluency, culture medium was removed, and cells were treated with 50 μL aliquots of rhShh preincubated for 5 min with rhWIF1. Data represent representative experiments performed in triplicate (B, C) or quadruplicate (A) and are expressed as fraction of luminescence relative to 0 nM rhWIF1.

Article Snippet: The ONE-StepTM Luciferase Assay System and the Gli Reporter-NIH3T3 cell line were purchased from BPS Bioscience (Inc., San Diego, CA, USA).

Techniques: Inhibition, Reporter Assay