Journal: bioRxiv

Article Title: Estradiol propionate reduction of nuclear size in prostate cancer lines lacking DHRS7 suggests DHRS7 absence as a biomarker for the effectiveness of estrogen therapy

doi: 10.1101/2023.09.18.558190

Figure Lengend Snippet: DHRS7 expression, subcellular localization, and orientation. (A) Plotted as x-fold over the median value across 84 human tissues, expression data extracted from BioGPS reveals DHRS7 to be highly preferentially expressed in normal prostate (14 extracted tissues shown). (B) Levels of DHRS7 protein in LNCaP and PC3 cells determined by Western blot. Numbers on the left indicate molecular weights. (C) Antibody staining for DHRS7 in LNCaP cells reveals a strong nuclear envelope accumulation along with diffuse staining in the ER. Notably, levels are very low in the bottom cell indicating expression variability in the population. Scale bar, 10 µm. (D) A model for DHRS7 transmembrane helices was generated using TMHMM v2.0. (E) Determination of topology using the digitonin assay. Cells expressing a DHRS7-mRFP fusion were permeabilized with either Triton X-100 which removes all membranes or digitonin which preferentially pokes holes in the plasma membrane where cholesterol is abundant. Cells were then fixed and stained with an antibody against mRFP. If the epitope is in the lumen it is masked and so will be inaccessible to the antibody so that there would be less signal from the antibody than from the mRFP. The similar staining pattern indicates the epitope on the C-terminal region of the protein is accessible to the cytoplasm/ nucleoplasm. Scale bar 10 μm.

Article Snippet: PC3 (late-stage prostate cancer line, ATCC CRL-1435), PC3-H2B-RFP (stable plasmid integration in PC3), and HT1080 fibrosarcoma cell lines were grown in DMEM, 10% fetal bovine serum (FBS).

Techniques: Expressing, Western Blot, Staining, Generated, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Estradiol propionate reduction of nuclear size in prostate cancer lines lacking DHRS7 suggests DHRS7 absence as a biomarker for the effectiveness of estrogen therapy

doi: 10.1101/2023.09.18.558190

Figure Lengend Snippet: DHRS7 can direct nuclear size changes in prostate cancer cells. (A) Protein levels of DHRS7 by Western before and after siRNA knockdown (25 nM) in LNCaP cells. Numbers on the left indicate molecular weights. (B) Whisker-scatterplot chart showing quantification of volume reconstructions for ∼100 LNCaP cells knocked down for DHRS7 shows its loss resulted in increased nuclear size. (C) Whisker-scatterplot chart showing the quantification of volume reconstructions for ∼100 PC3 and HT1080 cells with or without exogeneous DHRS7 expression as indicated. Exogenous expression of DHRS7 in late-stage PCa PC3 cells yields decreased nuclear size. All statistical analyses were performed with unpaired t-test with Welch’s correction: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: PC3 (late-stage prostate cancer line, ATCC CRL-1435), PC3-H2B-RFP (stable plasmid integration in PC3), and HT1080 fibrosarcoma cell lines were grown in DMEM, 10% fetal bovine serum (FBS).

Techniques: Western Blot, Knockdown, Whisker Assay, Expressing

Journal: bioRxiv

Article Title: Estradiol propionate reduction of nuclear size in prostate cancer lines lacking DHRS7 suggests DHRS7 absence as a biomarker for the effectiveness of estrogen therapy

doi: 10.1101/2023.09.18.558190

Figure Lengend Snippet: Medium-throughput screen for compounds decreasing nuclear size in PC3 cells. (A) Screening approach. Drug-treated PC3 cells stably expressing H2B-RFP to determine nuclear size were stained after fixation with CellMask Deep Red dye to determine the total cell area. (B) Replicate screens of plate 6 from the Microsource Spectrum library (compounds listed on Table 1, concentration: 10 μM). Nuclear size (vertical axis) was averaged over a total of 350-4,000 cells per well (horizontal axis). A1/A12, B1/B12 … G1/G12: DMSO controls. Estradiol propionate (EP) is indicated (red circles). Nuclear size reduction by at least 20% was statistically significant (p<0.001, Wilcoxson rank test). (C) Molecular structures of estradiol (top) and EP (bottom). (D) Characteristic images of EP (left) and estradiol benzoate (right)-treated cells (blue: H2B-RFP nuclear signal; red: CellMask Deep Red cytosolic signal).

Article Snippet: PC3 (late-stage prostate cancer line, ATCC CRL-1435), PC3-H2B-RFP (stable plasmid integration in PC3), and HT1080 fibrosarcoma cell lines were grown in DMEM, 10% fetal bovine serum (FBS).

Techniques: Stable Transfection, Expressing, Staining, Concentration Assay

Journal: bioRxiv

Article Title: Estradiol propionate reduction of nuclear size in prostate cancer lines lacking DHRS7 suggests DHRS7 absence as a biomarker for the effectiveness of estrogen therapy

doi: 10.1101/2023.09.18.558190

Figure Lengend Snippet: Estradiol propionate only corrects the cancer nuclear size defect in the absence of DHRS7. (A-D) Whisker-scatterplot charts showing the quantification of nuclear volume in ∼100 cells in presence of the indicated concentrations of estradiol propionate (EP). (A) Wild-type LNCaP cells. (B) LNCaP cells with siRNA-mediated DHRS7 knockdown. (C) Wild-type PC3 cells. (D) PC3 cells with exogenously restored DHRS7 expression. All statistical analyses were performed with unpaired t-test with Welch’s correction: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: PC3 (late-stage prostate cancer line, ATCC CRL-1435), PC3-H2B-RFP (stable plasmid integration in PC3), and HT1080 fibrosarcoma cell lines were grown in DMEM, 10% fetal bovine serum (FBS).

Techniques: Whisker Assay, Knockdown, Expressing

Journal: bioRxiv

Article Title: Estradiol propionate reduction of nuclear size in prostate cancer lines lacking DHRS7 suggests DHRS7 absence as a biomarker for the effectiveness of estrogen therapy

doi: 10.1101/2023.09.18.558190

Figure Lengend Snippet: DHRS7 dehydrogenase activity is likely required for its effects on nuclear size. (A) Sequence alignment of DHRS7 against 11β-HDS-B1 and 17-β-HDS-1. The mutated arginine (R82 in DHRS7) is highlighted in blue while residues conserved are in green and conservation of amino acid functionality is indicated in yellow. (B) Structural prediction for DHRS7 generated with the Phyre2 web server, www.sbg.bio.ic.ac.uk/phyre2 /, using human 11β-HDSB1 - PDB 2ILT 1BHS. (C-D) Whisker-scatterplot charts showing the quantification of nuclear volume in ∽100 PC3 cells expressing DHRS7 either wild-type or R82E (C) or PC3 cells expressing DHRS7 R82E mutant in presence of the indicated concentrations of estradiol propionate (EP) (D). All statistical analyses were performed using unpaired t-test with Welch’s correction: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: PC3 (late-stage prostate cancer line, ATCC CRL-1435), PC3-H2B-RFP (stable plasmid integration in PC3), and HT1080 fibrosarcoma cell lines were grown in DMEM, 10% fetal bovine serum (FBS).

Techniques: Activity Assay, Sequencing, Structural Proteomics, Generated, Whisker Assay, Expressing, Mutagenesis