Journal: Molecular Oncology

Article Title: Cytomegalovirus infection is common in prostate cancer and antiviral therapies inhibit progression in disease models

doi: 10.1002/1878-0261.70073

Figure Lengend Snippet: CMV in prostate epithelial cells. (A) Illustration, made with Biorender.com , of prostate collection from postmortem donors. (B) Immunoblot of prostate tissue homogenates for the proteins CMV‐UL97 (with three bands as expected , marked with asterisks), CMV‐US28, and β‐actin from eight men. kDa is kilodalton. (C) Images of prostate sections with CMV antibody staining in brown in the epithelium. Nuclei are labeled in purple with hematoxylin. Scale bar 50 μm. (D) Quantification of CMV‐pp71 abundance in epithelial cells. All data points ( n = 41) are shown in the graph and summarized in the box plot (median, 25–75th percentiles, error bars show min‐max values). (E) Linear regression comparing CMV‐pp71 and CMV‐pp65 abundance in the prostate epithelium ( n = 41). (F) Co‐labelling of CMV‐pp71 (green) and CMV‐IE1 (red). Scale bar 25 μm. Asterisks label cell nuclei with partial CMV‐IE1 protein expression. (G) Co‐labelling of CMV DNA (green) and CMV‐pp71 (red). Scale bar 25 μm. (H) CMV qPCR heatmap. CMV qPCR assays detected CMV DNA in the positive control (purified CMV DNA) but not in the negative control (mouse prostate cancer cell line MyC‐CaP). Numbers denote how many prostates were positive and evaluated with qPCR with or without Bsr1 restriction enzyme pretreatment. Three prostate samples were evaluated in the screening of qPCR assays. Na is not analyzed. (I) Pearson correlation of the percentage of Ki‐67 + cells and CMV‐pp71 abundance ( n = 13). Labeling with Ki‐67 (green). Star points out a Ki‐67 + epithelial cell. Scale bar: 25 μm. (J) Pearson correlation between CMV‐pp71 epithelial abundance and age ( n = 41). In (F), (G) and (I), nuclei are labeled in blue with DAPI and dotted lines depict the basal lamina of the epithelium. CMV, Human cytomegalovirus.

Article Snippet: Primary antibodies used: Cytomegalovirus US28 (rabbit, 1:150, PA5‐39864, polyclonal, Thermo Fisher), Cytomegalovirus pp65 (mouse, 1:50, clone CH12, sc‐56 976, monoclonal, Santa Cruz Biotechnology), Cytomegalovirus pp71 (goat, 1:200, clone vC‐20, sc‐33 323, polyclonal, Santa Cruz Biotechnology), Cytomegalovirus IE1 (mouse, 1:10, pp72, clone 6E1, sc‐69 834, Santa Cruz Biotechnology), Cytomegalovirus IE1/2 (mouse, 1:150, MAB810R, monoclonal, Millipore), Chromogranin A (mouse, 1:100, LK2H10, MA5‐13096, Thermo Fisher), keratin 5 (rabbit, 1:200, EP1601Y, ab52635, Abcam), keratin 18 (rabbit, 1:100, H‐80, sc‐28 264, Santa Cruz Biotechnology), TP63 (mouse, 1:100, clone 4A4, CM163A, Biocare Medical, Pacheco, CA, USA), Ki‐67 (rabbit, 1:100–250, clone SP6, Thermo Fisher), wide spectrum cytokeratin (rabbit, 1:100, ab9377, abcam, Cambridge, UK), pan‐keratin (mouse, 1:200, clone C11, #4545, Cell Signaling, Danvers, MA, USA), and AR (rabbit, 1:50–100, clone SP107, Thermo Fisher).

Techniques: Western Blot, Staining, Labeling, Expressing, Positive Control, Purification, Negative Control