Journal: bioRxiv

Article Title: Antidepressants interact with sex steroid receptors and their intracellular signaling components

doi: 10.64898/2026.03.17.712321

Figure Lengend Snippet: Antidepressants selectively activate MAPK signaling and induce ERα-dependent gene expression in MCF-7 cells. (A) Quantification of MAPK phosphorylation (pMAPK/MAPK) by Western blot densitometry following treatment with vehicle, imipramine, S-ketamine, or estradiol (E2), in the presence or absence of the ERα degrader/antagonist fulvestrant and/or tunicamycin. Antidepressant treatment significantly increased MAPK phosphorylation, indicating enhanced MAPK pathway activation. (B) Total MAPK levels normalized to β-actin were unchanged across treatment conditions. Representative immunoblots for MAPK and pMAPK are shown above. (C) Quantification of Akt phosphorylation (pAkt/Akt) revealed no significant changes following antidepressant or E2 treatment, but there was a trend for a reduction by imipramine. (D) Total Akt levels normalized to β-actin were unaltered. Representative immunoblots for Akt and pAkt are shown above. One imipramine-treated sample was excluded from analysis due to an outlier pMAPK/MAPK ratio exceeding 1. Data from technical and biological replicates were pooled. (E–F) mRNA expression of the extranuclear-initiated ERα target gene LRRC54 (E) and the classical nuclear ERα target gene PgR (F) , quantified by qPCR following treatment with E2, imipramine, or S-ketamine. Antidepressants significantly increased LRRC54 and PgR expression, comparable to E2 and relative to vehicle control. Pretreatment with fulvestrant attenuated these effects, indicating ERα dependency. Data are presented as mean ± SEM from three independent biological replicates (n = 3). Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns as not significant.

Article Snippet: Membranes were then stripped for 10 minutes and reprobed with antibodies against ERα (mouse anti-ERα (1D5)) and β-actin (mouse or rabbit Licor 926–42212/Licor 926-42210).

Techniques: Gene Expression, Phospho-proteomics, Western Blot, Activation Assay, Expressing, Control

Journal: Antioxidants & Redox Signaling

Article Title: Mitochondrial Complex I Reversible S-Nitrosation Improves Bioenergetics and Is Protective in Parkinson's Disease

doi: 10.1089/ars.2017.6992

Figure Lengend Snippet: Nitrite pretreatment improves the recovery of mitochondrial respiration and induces Keap1 S-nitrosation. (A) Nitrite administration stimulates recovery of mitochondrial respiration after a transient 4 h exposure to MPP+. SH-SY5Y cells were seeded in a Seahorse Bioscience V7 tissue culture plate, incubated with nitrite for 48 h, and finally treated with 100 μM MPP+. After 4 h, MPP+ was removed and basal OCR was monitored for 8 h with a Seahorse XF24 Extracellular Flow Analyzer. Nitrite pretreated cells reached control OCR levels after 76 (10 μM, gray arrowhead) and 130 (1 μM, black arrowhead) minutes, whereas respiration in cells treated only with MPP+ remained consistently lower than controls. (B) Representative in-gel detection of S-nitrosated (SNO, left panel) and reduced (SH, central panel) cysteine thiols in immunocaptured mitochondrial complex I extracted from cells treated with nitrite and/or MPP+ reveals reduction of mitochondrial complex I protein cysteines in MPP+ specimen pretreated with nitrite (compare lane 7 with other lanes). Gel protein loading was assessed by silver staining (right panel). (C, D) Nitrate administration induces S-nitrosation of Keap1. SH-SY5Y cells received a 48 h treatment with nitrite (1 or 10 μM) or a 30′ treatment with H2O2 (0.5 mM), and the biotin switch reaction was performed in all samples in the presence of biotin-NEM, Cu++, and ascorbate. Biotinylated proteins were captured with streptavidin-conjugated beads, separated on a gel, and probed with an anti-Keap1 antibody (C, representative image, full gel in Supplementary Fig. S3). S-nitrosated-Keap1 levels were normalized on the precaptured SNO-modified total lysates labeled with NHS-ester (D). **p < 0.01; one-way ANOVA followed by Dunnett's multiple-comparison post doc test. Graphs represent mean ± SEM.

Article Snippet: The membrane was blocked for 1 h at RT in a PBS-T solution containing BSA 5%, and SNO-biotinylated proteins were detected for Keap1 (1:1000, ON at 4°C, 8046S; Cell Signaling, Danver, MA) and the goat anti-rabbit HRP-conjugated secondary antibody (1:5000, P0487; DAKO-Agilent Technologies).

Techniques: Incubation, Control, Silver Staining, Modification, Labeling, Comparison

Journal: Journal of Extracellular Vesicles

Article Title: Potential Role of Menstrual Fluid‐Derived Small Extracellular Vesicle Proteins in Endometriosis Pathogenesiss

doi: 10.1002/jev2.70048

Figure Lengend Snippet: Antibodies used.

Article Snippet: Rabbit anti‐human β‐actin HRP (mAb) , # 12620S/ Cell Signaling Technology , 1:1000 , 45 KDa.

Techniques: Recombinant

Journal: Bone

Article Title: Postnatal expression and possible function of RANK and RANKL in the murine inner ear.

doi: 10.1016/j.bone.2020.115837

Figure Lengend Snippet: Fig. 2. Expression of OPG, RANK and RANKL in the cochlear soft tissue and the otic capsule at 5 days (N=4 mice) and 7 weeks (N=3 mice) of age. (A) Representative western blot with each lane showing protein lysate from one mouse (2 cochleae). β-Actin serves as a protein loading control. (B) The ratio of protein expression at 7 weeks relative to 5 days of age for the cochlear soft tissues and the otic capsule. The measurements are based on quantification of western blots shown in (A). Bars depict mean ± standard error of the mean. Individual dots represent individual animals. *, p<0.05, **, p<0.01, ***, p<0.005, as compared to OPG.

Article Snippet: The anti-OPG (#sc-8468), anti-RANK (#sc-9072), and anti-RANKL (#sc-7628) antibodies were from Santa Cruz Biotechnology and the anti-β-actin (#9470) antibodies were from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Control

Journal: Nature Communications

Article Title: Multiplexed and reproducible high content screening of live and fixed cells using Dye Drop

doi: 10.1038/s41467-022-34536-7

Figure Lengend Snippet: a Relative cell viability in OptiPrep™ fixed cells as compared to live cell microscopy following 24 h treatments with increasing concentrations of dinaciclib, paclitaxel, staurosporine, and vincristine in MCF 10A-H2B-mCherry cells. Error bars represent the standard error of the mean of eight technical replicates from one representative biological replicate. b Deep Dye Drop protocol steps: EdU and LDR dye are added in 10% OptiPrep™ followed by fixation with 4% formaldehyde in 20% Optiprep™. Cells are then permeabilized with 0.5% Triton X-100 in 10% OptiPrep, and the EdU is labeled with a fluorescent dye azide via Click chemistry in 20% OptiPrep™. The contents of the well are aspirated, cells are blocked, and then stained with a conjugated antibody against phospho-histone H3 (pH3) in 10% OptiPrep™. One well of a multi-well plate is depicted. c Schematic and representative image of cells stained with the Deep Dye Drop protocol. The image shown is an example of typical of Deep Dye Drop staining as performed in 58 breast cancer cell lines in this work. Nuclei are stained with Hoechst (gray-scale), dead cells are stained with LIVE/DEAD red (blue), S-phase cells are labeled with EdU (red) and M-phase cells are stained with phospho-histone H3 (green). Scale bar is 100 µm. d Thresholds set to classify dead cells shown on a distribution of LDR intensity values and e to identify cells in M-phase shown on a distribution of pH3 intensity values. f Scatter plot of EdU intensity versus DNA content. The red dotted lines represent gating applied to assign cells to the sub G1, G1, G2, beyond G2, and S-phases of the cell cycle (see online “Methods”). g DNA content in BT20 cells treated for 72 h with inhibitors targeting CHK1 (1 µM LY2606368), CDK1/2 (3.16 µM BMS-265246) and PLK4 (0.316 µM CFI-400495) and untreated controls. All cells from a single well in a 384-well plate are shown per condition. h The difference in GR values calculated from Deep Dye Drop and conventional assays with respect to the GR value from the Deep Dye Drop assay. The gray bars represent the 90% confidence intervals for GR values from Deep Dye Drop experiments performed in biological triplicate. Source data are provided as a Source Data file.

Article Snippet: Alternate antibodies were used in the place of pH3: actin (1:500, catalog number 8844, clone 13E5, lot number 4, Cell Signaling Technologies, Danvers, MA), 53BP1 (1:500, catalog number 612523, clone 19, lot number 9017821, BD Biosciences, Franklin Lakes, NJ), cytochrome C (1:200, catalog number MA338200, clone 6H2.B4, lot number RK242765, Invitrogen, Carlsbad, CA), pRb (1:500, catalog number 4277, clone D20B12, lot number 4, Cell Signaling Technologies, Danvers, MA).

Techniques: Microscopy, Labeling, Staining

Journal: Nature Communications

Article Title: Multiplexed and reproducible high content screening of live and fixed cells using Dye Drop

doi: 10.1038/s41467-022-34536-7

Figure Lengend Snippet: a MCF7 cells stained with phospho-pRb and b actin untreated and after 72 h in 1 µM palbociclib; effects of increasing concentrations of palbociclib on the fraction of phospho-pRb positive MCF7 cells, or b on cell size as detected with actin staining after 72 h. c MCF 10 A cells stained with 53BP1 untreated and after 72 h in 1 µM etoposide; induction of DNA damage by increasing concentrations of etoposide in MCF 10 A cells as detected with 53BP1 staining after 72 h. d MCF7 cells stained with cytochrome C untreated and after 72 h in 0.1 µM actinomycin D; effect of increasing concentrations of actinomycin D on release of cytochrome C from the mitochondria in MCF7 cells after 72 h, performed in duplicate. Nuclei are stained with Hoechst (gray-scale), and EdU (red) in all images. Error bars represent the standard deviation of the mean of four replicates. Scale bars are 50 µm. e Schematic and representative image from three cell lines in a 384-well plate stained with the addition of a fifth channel to the Deep Dye Drop assay. Cells are stained with Hoechst (gray-scale), LDR (blue, 1), EdU (red, 2), pH3 (purple, 3) and 53BP1 (green, 4). Scale bar is 50 µm. Source data are provided as a Source Data file.

Article Snippet: Alternate antibodies were used in the place of pH3: actin (1:500, catalog number 8844, clone 13E5, lot number 4, Cell Signaling Technologies, Danvers, MA), 53BP1 (1:500, catalog number 612523, clone 19, lot number 9017821, BD Biosciences, Franklin Lakes, NJ), cytochrome C (1:200, catalog number MA338200, clone 6H2.B4, lot number RK242765, Invitrogen, Carlsbad, CA), pRb (1:500, catalog number 4277, clone D20B12, lot number 4, Cell Signaling Technologies, Danvers, MA).

Techniques: Staining, Standard Deviation

Journal: Nature Communications

Article Title: Multiplexed and reproducible high content screening of live and fixed cells using Dye Drop

doi: 10.1038/s41467-022-34536-7

Figure Lengend Snippet: a Schematic and representative image from technical triplicates of multiplexing Deep Dye Drop assays with cyclic immunofluorescence: the Hoechst (gray-scale), EdU (red), pH3 (green), beta-catenin (cyan), phospho-pRb (blue), and p21 (yellow) signals are displayed, and contrast was adjusted for visualization purposes only. Scale bar is 100 µm. b UMAP representation of MCF7 and MCF 10 A cells treated with BMS-265246 (1 µM, 10 µM), ribociclib (10 µM) or DMSO stained with Deep Dye Drop and cyclic immunofluorescence. c The number of MCF7 cells in S-phase following treatment with increasing concentrations of ribociclib, d palbociclib, and e abemaciclib after 6, 24, 48, and 72 h. Data are presented as mean +/− standard deviation of four technical replicates. EdU versus DNA content scatter plots show the single cell cell-cycle distributions from one well at 1 µM doses of each drug at the time points indicated, the percentage of cells in S-phase (average from four technical replicates) is indicated on each plot. The scatter plots show all cells in a single, representative well of a 384-well plate for each condition. f Illustration of possible patterns of the emergence of resistant cells. A, clonal, genetic resistance or B, non-clonal, non-genetic adaptation followed by representative images from biological duplicates performed in technical quadruplicate of MCF7 cells treated with 1 µM palbociclib for 24 h or 72 h. Nuclei are visualized with Hoechst in white and EdU positive cells are shown in red, scale bars are 100 µm. Source data are provided as a Source Data file and on Synapse.

Article Snippet: Alternate antibodies were used in the place of pH3: actin (1:500, catalog number 8844, clone 13E5, lot number 4, Cell Signaling Technologies, Danvers, MA), 53BP1 (1:500, catalog number 612523, clone 19, lot number 9017821, BD Biosciences, Franklin Lakes, NJ), cytochrome C (1:200, catalog number MA338200, clone 6H2.B4, lot number RK242765, Invitrogen, Carlsbad, CA), pRb (1:500, catalog number 4277, clone D20B12, lot number 4, Cell Signaling Technologies, Danvers, MA).

Techniques: Multiplexing, Immunofluorescence, Staining, Standard Deviation