Journal: iScience

Article Title: Neuroactive steroids activate membrane progesterone receptors to induce sex specific effects on protein kinase activity

doi: 10.1016/j.isci.2025.112352

Figure Lengend Snippet: Examining the effects of ORG on PKA and PKC activity in cell lines expressing mPRs (A) Representative immunoblots for MDA231, mPRδ-GFP, and mPRε-GFP cells treated for 20 min with the pan-mPR agonist, ORG at 3, 10, 30, 100, and 300nM. Cells were lysed, the proteins resolved on SDS-PAGE and subject to immunoblotting. (B) Densitometry was carried out to quantify the difference between host and mPR expressing cells and between ORG treatment concentrations. The ratio of phosphorylated kinase to total kinase was determined and normalized to values in MDA231 cells (100%). two-Way ANOVA was used to compare treatment responses in each cell line. Post hoc comparisons of each ORG concentration were calculated using the Šidák multiple comparison test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 ( n = 4). Data are represented as mean ± SEM.

Article Snippet: MDA-MB-231 cells were transduced with lentivirus for mouse mPRδ and mouse mPRε with a c-terminal GFP tag (Origene, mPRδ: MR216535L4V, mPRε: MR215906L4V).

Techniques: Activity Assay, Expressing, Western Blot, SDS Page, Concentration Assay, Comparison

Journal: iScience

Article Title: Neuroactive steroids activate membrane progesterone receptors to induce sex specific effects on protein kinase activity

doi: 10.1016/j.isci.2025.112352

Figure Lengend Snippet: Examining the effects of ORG on mPR cell surface stability (A) Representative images of GFP signal in cells imaged using TIRF microscopy following 20-min treatment with aCSF or 100nM mPR agonist, ORG. (B) Quantified and normalized fluorescence comparing baseline to ORG treatment for 20 min in mPRδ-GFP and mPRε-GFP cells. Data were normalized to aCSF treatment within each cell line. ∗∗∗ p < 0.001, N = 3, n = 9–12. Data are represented as mean ± SEM.

Article Snippet: MDA-MB-231 cells were transduced with lentivirus for mouse mPRδ and mouse mPRε with a c-terminal GFP tag (Origene, mPRδ: MR216535L4V, mPRε: MR215906L4V).

Techniques: Microscopy, Fluorescence

Journal: iScience

Article Title: Neuroactive steroids activate membrane progesterone receptors to induce sex specific effects on protein kinase activity

doi: 10.1016/j.isci.2025.112352

Figure Lengend Snippet: Measuring the effects of ALLO on mPR signaling (A) Representative immunoblots for MDA231, mPRδ-GFP, and mPRε-GFP cells treated for 20 min with ALLO at 3, 10, 30, 100, and 300nM. Cells were lysed, the proteins resolved on SDS-PAGE and subject to immunoblotting. (B) Densitometry was carried out to quantify the difference between host and mPR expressing cells and between ALLO treatment concentrations. The ratio of phosphorylated kinase to total kinase was determined and normalized to control treatment (100%). two-Way ANOVA was used to compare treatment responses relative to control MDA231 cells. Post hoc comparisons of each ALLO concentration were calculated using the Šidák multiple comparison test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n = 4. Data are represented as mean ± SEM.

Article Snippet: MDA-MB-231 cells were transduced with lentivirus for mouse mPRδ and mouse mPRε with a c-terminal GFP tag (Origene, mPRδ: MR216535L4V, mPRε: MR215906L4V).

Techniques: Western Blot, SDS Page, Expressing, Control, Concentration Assay, Comparison

Journal: iScience

Article Title: Neuroactive steroids activate membrane progesterone receptors to induce sex specific effects on protein kinase activity

doi: 10.1016/j.isci.2025.112352

Figure Lengend Snippet: Measuring the effects of SGE516 on mPR signaling. Only cells expressing mPRδ demonstrate PKC activation in response to SGE-516 treatment (A) Representative immunoblots for MDA231, mPRδ-GFP, and mPRε-GFP cells treated for 20 min with either SGE-516 at 3, 10, 30, 100, and 300nM. Cells were lysed, the proteins resolved on SDS-PAGE and subject to immunoblotting. (B) Densitometry was carried out to quantify the difference between host and mPR expressing cells and between SGE-516 treatment concentrations. The ratio of phosphorylated kinase to total kinase was determined and normalized to control treatment (100%) for each cell type. mPR-GFP cell responses were then normalized to MDA231 means for each SGE-516 concentration. two-Way ANOVA was used to compare treatment responses in mPRδ-GFP or mPRε-GFP to MDA231 cells. Post hoc comparisons of each SGE-516 concentration were calculated using the Šidák multiple comparison test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n = 4. Data are represented as mean ± SEM.

Article Snippet: MDA-MB-231 cells were transduced with lentivirus for mouse mPRδ and mouse mPRε with a c-terminal GFP tag (Origene, mPRδ: MR216535L4V, mPRε: MR215906L4V).

Techniques: Expressing, Activation Assay, Western Blot, SDS Page, Control, Concentration Assay, Comparison

Journal: iScience

Article Title: Neuroactive steroids activate membrane progesterone receptors to induce sex specific effects on protein kinase activity

doi: 10.1016/j.isci.2025.112352

Figure Lengend Snippet: Analyzing the effects of NAS on G q and G s activation (A) Dose-response curves for ORG, ALLO, and SGE-516, using a G q reporter assay in mPRδ-GFP cells. Cells were exposed to 1-300nM ORG, ALLO, or SGE-516 for 6 h. Data were normalized to the minimum and maximum luminescent values curves fitted to calculate EC 50 values ( n = 4). (B) Dose-response curves for ORG, ALLO, and SGE-516, using a G s reporter assay in mPRε-GFP. Cells were exposed to 1-300nM ORG, ALLO, or SGE-516 for 6 h. Data were normalized to the minimum and maximum luminescent values curves fitted to calculate EC 50 values ( n = 4). (C) EC 50 values were determined for ORG, ALLO, and SGE-516 in both mPRδ-GFP and mPRε-GFP cells. Data are represented as mean ± SEM.

Article Snippet: MDA-MB-231 cells were transduced with lentivirus for mouse mPRδ and mouse mPRε with a c-terminal GFP tag (Origene, mPRδ: MR216535L4V, mPRε: MR215906L4V).

Techniques: Activation Assay, Reporter Assay