Journal: Frontiers in Endocrinology

Article Title: Upregulation of an estrogen receptor-regulated gene by first generation progestins requires both the progesterone receptor and estrogen receptor alpha

doi: 10.3389/fendo.2022.959396

Figure Lengend Snippet: The progestogens display similar efficacies, but not potencies, to each other and E 2 for increased proliferation of the MCF-7 BUS breast cancer cell line, while enhancing anchorage-independent growth of the MCF-7 BUS cells to differential extents to each other and E 2 . The MCF-7 BUS cell line was incubated for 120 hours with (A) increasing concentrations of E 2 (■), P 4 (●), MPA (▲), NET (♦) or DRSP (×) or (D) 1 nM E 2 in the absence and presence of 1 nM progestogens. Cell proliferation was quantified using the MTT cell viability assay, and the vehicle response was set as one, with all other responses calculated relative to this. (B) Plots of the maximal response and (C) log EC 50 values of the test compounds for proliferation from (A) are shown. (E, F) MCF-7 BUS cells were treated for 21 days with (E) 1 nM E 2 , P 4 , MPA, NET or DRSP, or (F) 1 nM progestogen in the presence of 1 nM E 2 . Anchorage-independent growth was quantified using the soft agar assay. The colonies formed were quantified using ImageJ software (Version 1.49). Results are shown as relative colony formation with the response obtained with the vehicle control set as one, and all other responses calculated relative to this. One-way ANOVA with Tukey’s multiple comparison or two-way ANOVA with Bonferroni’s multiple comparison post-tests were used for statistical analysis. Statistically significant differences are indicated by the letters a, b, c or d, where the values that differ significantly from others are assigned a different letter, or ** or *** to indicate p<0.01 or p<0.001. No statistical significance (p>0.05) is indicated by ns. One-way ANOVA with a Dunnett’s multiple comparison post-test was performed to compare responses of estrogen-progestogen combinations relative to E 2 alone, and no statistical differences were obtained between the E 2 response and E 2 in combination with any progestogen.

Article Snippet: Proteins were visualized using enhanced chemiluminescence (Biorad, RSA) and a MyECL imager and quantified using ImageJ software (version 1.49).

Techniques: Incubation, Viability Assay, Soft Agar Assay, Software, Control, Comparison

Journal: Frontiers in Endocrinology

Article Title: Upregulation of an estrogen receptor-regulated gene by first generation progestins requires both the progesterone receptor and estrogen receptor alpha

doi: 10.3389/fendo.2022.959396

Figure Lengend Snippet: Both the PR and ERα are required for the upregulation of the ER-regulated gene CTSD by MPA and NET. The MCF-7 BUS cell line transfected with (A-C) 10 nM NSC or PR-A/B siRNA or (A, B, D) 25 nM NSC or ERα siRNA were treated with 100 nM E 2 , MPA or NET for 24 hours. (A) For verification of PR-A/B or ERα knockdown, total protein from the MCF-7 BUS cells transfected as described above was harvested, and western blotting performed using antibodies specific for ERα, PR-A/B and GAPDH. A representative blot is shown and (B) PR-A, PR-B and ERα expression levels were quantified relative to the GAPDH loading control using ImageJ software (Version 1.49). Western blots of three independent experiments were quantified to determine the percentage protein knocked down. (E) MCF-7 BUS cells were treated with 100 nM E 2 , MPA or NET in the absence and presence of 10 µM ICI for 24 hours. (C–E) Total RNA was isolated, reverse transcribed and real-time qPCR was conducted to determine the relative expression of CTSD mRNA levels relative to GAPDH (reference gene). The vehicle control of each condition was set as one and the relative mRNA expression in the treated samples set relative to this. Two-way ANOVA with Bonferroni’s multiple comparison post-test was used for statistical analysis. Statistically significant differences are indicated by *, ** or *** to indicate p<0.05, p<0.01 or p<0.001. No statistical significance (p>0.05) is indicated by ns.

Article Snippet: Proteins were visualized using enhanced chemiluminescence (Biorad, RSA) and a MyECL imager and quantified using ImageJ software (version 1.49).

Techniques: Transfection, Knockdown, Western Blot, Expressing, Control, Software, Isolation, Reverse Transcription, Comparison