Figure 6.

Dox-induced 3HA-V12Ras expression diminishes GH4 cell xenograft tumor growth and promotes a lactotrope phenotype. A, GH4 clone number 10 xenograft tumor growth in nude mice. Female nude mice were supplemented with estrogen pellets for 1 week. Mice were provided with water containing 5% sucrose with or without 2 mg/mL doxycycline for 3 days prior to cell injection. GH4 clone number 10 cells were injected bilaterally into flanks of nude mice using 2 × 10⁶ cells suspended in 200 μL DMEM for each injection. Animals were monitored for tumor formation every other day, and tumor size was measured with a digital caliper. Tumor volume was calculated using the following formula: (0.52 × length × width²). Tumor volume on day 30 after cell injection was plotted (n = 10). *, P < .05. B, Quantification of pH3-positive cells in GH4 clone number 10 xenograft tumors by immunohistochemical analysis. Sections of paraffin-embedded xenograft tumors from panel A were incubated with an anti-pH3 primary antibody and then stained with a biotin-conjugated secondary antibody. Cells that were positively stained for pH3 were manually counted from 10 fields per slide. The average number of positive cells per field is plotted for each tumor section (n = 7). *, P < .05. C, Immunohistochemical (IHC) analysis and scoring of PRL and GH staining in GH4 clone number 10 xenograft tumors. Sections of paraffin-embedded xenograft tumors from panel A were incubated with an anti-PRL primary antibody or an anti-GH primary antibody and then stained with a biotin-conjugated secondary antibody. Cells with weak to strong PRL or GH DAB staining were scored as positive, reported as percentage positive of total cells scored, and are graphed as a percentage of the total positive cells (PRL+ GH) (n = 3). D, IHC analysis of HA-tagged V12Ras expression in GH4 clone number 10 xenograft tumors. Sections of paraffin-embedded xenograft tumors from panel A were incubated with an anti-HA primary antibody, as detailed in Materials and Methods (n = 3).