Fig 2: Immunohistochemical staining for CK18. (A) Extramammary Paget’s disease (EMPD) lesional skin. (B) EMPD lymph node metastasis. (C) Cutaneous metastasis from breast cancer. (D) Normal skin (sweat gland as positive control). (E) Squamous cell carcinoma. Scale bars=50 µm.

Fig 3: Characterization of immortalized human prostate cancer cell line ACRJ-PC28 growing as 3D organoids. A, ACRJ-PC28 cells were seeded on and embedded in Matrigel as described in the Materials and Methods and monitored over time by phase contrast microscopy. Organoids are shown at days 7 and 10. Lens 10×. Scale bar: 400 μm. Both ACRJ-PC28 and hPrEC-C1 cells form round organoids, but ACRJ-PC28 show lumens. PC-3 organoids form invasive protrusions (protrusions are marked with white arrows on day 7, and a green arrow marks the same organoid with many more protrusions). B and C, IF staining with CK5, CK18, and p63, and AR antibodies and imaged by confocal microscopy, 63×, demonstrates expression of basal markers CK5 and p63. However, expression of luminal marker CK18, and AR was negative. PC-3 and LNCaP organoids were used as positive controls for CK18 and AR, respectively. D, ACRJ-PC28 organoid vimentin and E-cadherin expression patterns suggest an opposite graded relative expression (designated H:high, I:intermediate, L:low). E, Expression of laminin β1 and integrin β1. B–E, Primary antibody incubation was followed by staining with fluorescence conjugated secondary antibody and phalloidin. Lens: 63×. Scale bar: 100 μm. Zoom factor: 0.75.

Fig 4: Characterization of immortalized human prostate cancer cell line ACRJ-PC28 in 2D. A, Expression analysis of AR, CK5, CK18, pRB, p53, p16, and p14 in ACRJ-PC28 and control cell lines (293T, PC3, DU145, LNCaP). ACRJ-PC28 cells express both CK5 and CK18. The absence of p16 and p14 expression confirming the successful knockout of CDKN2A. ACRJ-PC28 cells do not express detectable AR: B. LNCaP, DU145, and ACRJ-PC28 cells were seeded on coverslips and cultured with medium containing DHT for 24 hours before fixation. Cells were incubated with AR primary antibody followed by fluorescence conjugated secondary antibody and phalloidin. Coverslips were mounted with mounting buffer containing DAPI to the glass slides and sealed with nail polish before imaging with confocal microscope. Lens: 63×. Scale bar: 10 μm. Zoom factors are as indicated in the figure. Images confirm the absence of AR from ACRJ-PC28: C. The p63 basal marker is expressed in ACRJ-p28. cells. B and C, Protein expression levels were determined by Western blot analysis. D, Cells were counted at 1-day intervals over a 6-day period and indicates a doubling time of 45 hours.