Journal: The Journal of Clinical Investigation

Article Title: CDCP1 overexpression drives prostate cancer progression and can be targeted in vivo

doi: 10.1172/JCI131133

Figure Lengend Snippet: (A) Representative images of p-HP1γ. Senescence associated-β-galactosidase (SA-β-Gal) and Cyclin D1 staining in the anterior prostates of WT, CDCP1, Ptenpc–/–, and CDCP1 Ptenpc–/– mice. Scale bars: 125 μm. (B) Western blot analysis of p21, Cyclin D1, COUP-TFII, Smad4, and p53 in anterior prostate glands from the indicated genotypes. (C) qRT-PCR analysis of c-Myc, Cyclin D1, COUP-TFII, p21, p27, and p16 expression in prostates from 12- to 16-week-old Ptenpc–/– and CDCP1 Ptenpc–/– mice (n = 3). (D) Western blot analysis of Pten–/– and CDCP1 Pten–/– MEFs treated with saracatinib (100 nM) for 12 hours. (E) Representative images of SA-β-Gal staining in Pte–/– and CDCP1 Pten–/– MEFs treated with saracatinib (100 nM) and DMSO for 12 hours. Scale bars: 125 μm. Bar graph shows the fold change in growth by crystal violet in Pten–/– and CDCP1 Pten–/– MEFs treated with saracatinib (100 nM) or DMSO as control (n = 3). (F) Western blot analysis of Pten–/– and CDCP1 Pten–/– MEFs transfected with si-c-Myc and control si-scramble (si-Ctrl) after 48 hours. (G) Representative images of SA-β-Gal staining in Pten–/– and CDCP1 Pten–/– MEFs transfected with si-c-Myc and si-Ctrl after 48 hours. Scale bars: 125 μm. Bar graph shows the fold change in growth by crystal violet in Pten–/– and CDCP1 Pten–/– MEFs transfected with si-c-Myc and si-Ctrl (n = 3). (H) Schemes of Cyclin D1 and COUP-TFII promoters. qRT-PCR of ChIP-analysis showing the binding of c-Myc to COUP-TFII promoter and c-Myc and Smad4 to Cyclin D1 promoters in Pten–/– and CDCP1 Pten–/– MEFs. Normal mouse IgG serves as negative control (n = 2). Error bars indicate SD. *P < 0.05; **P < 0.01. Statistical test: 2-tailed t test.

Article Snippet: For IHC the following antibodies were used: Ki-67 (Thermo Fisher Scientific, clone SP6, catalog RM-9106-R7; rabbit polyclonal; unmasked water bath 98°C, pH 6, 20 minutes; Lab Vision dilution ready to use); CDCP1 (Cell Signaling Technology, catalog 4115, rabbit polyclonal; unmasked water bath 98°C, pH 6, 20 minutes; 1:50); p-HP1γ-Ser83 (Cell Signaling Technology, catalog 2600, unmasked water bath 98°C, pH 6, 20 minutes; 1:50); Cyclin D1 (Cell Signaling Technology, catalog 2978S); AR (N-20) (Santa Cruz Biotechnology, catalog SC-816, rabbit polyclonal; unmasked water bath 98°C, pH 6, 20 minutes; 1:300); wide spectrum cytokeratin (pankeratin) (DAKO, catalog Z0622; rabbit polyclonal; unmasked water bath 98°C, pH 9, 20 minutes; 1:2000).

Techniques: Staining, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Binding Assay, Negative Control

Journal: Scientific Reports

Article Title: Evidence for a non-stochastic two-field hypothesis for persistent skin cancer risk

doi: 10.1038/s41598-020-75864-2

Figure Lengend Snippet: Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following immunolabeling with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) HP1γ + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear γH2AX are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).

Article Snippet: Immunofluorescent staining of formalin-fixed paraffin-embedded mouse skin following heat-induced antigen retrieval was performed using the following antibodies: HP1γ (Cbx3) immunolabeling was done using rabbit anti-Cbx3 (1:250, Cat#IHC-00204, Bethyl Laboratories). γH2AX immunolabeling was done using monoclonal rabbit anti-γH2AX (1:100, Bethyl Laboratories, Cat#A700-053).

Techniques: Staining, Control, Immunolabeling, Labeling