Fig. 8
From: Evidence of renal angiomyolipoma neoplastic stem cells arising from renal epithelial cells
Everolimus induces cell cycle arrest in AML tumour allografts. a Sulforhodamine B cell number assay of total populations of Cdkn2a∆/∆ primary kidney cells infected with empty shRNA (shRNA-Ctrl/Cdkn2a∆/∆) or with shRNA targeting Tsc2 (shRNA-Tsc2/Cdkn2a∆/∆)and two single cell-derived sphere clones (shRNA-Tsc2/Cdkn2a∆/∆ SC3 and SC6) treated with everolimus for 7 days. Cells were grown under adherent conditions and values represent A540 ratios of treated to untreated cells. b Phase contrast images of shRNA-Tsc2/Cdkn2a∆/∆ spheres formed in suspension culture conditions in the presence or absence of everolimus (1.6 nM, 48 h). Scale bars: 50 μm. c–f Number c, diameter d, cellular viability e and cell cycle phase flow cytometry analysis f (x-axis propidium iodide (PI) staining, y-axis α-BrdU staining) of shRNA-Tsc2/Cdkn2a∆/∆ sphere cells after 48 h everolimus (1.6 nM) treatment. Cells were incubated with BrdU (30 μM) for 12 h prior to harvesting. g mRNA abundance of the indicated genes in shRNA-Tsc2/Cdkn2a spheres differentiated in the presence or absence of everolimus (1.6 nM, 48 h). h, i Long term h and short term i growth of shRNA-Tsc2/Cdkn2a∆/∆ AML allograft tumours treated with saline (control) or everolimus (10 mg/kg i.p. daily for 2 weeks). The timepoints of starting and stopping therapy are indicated. Images of tumours from each group at the end points of the two experiments are shown. Scale bar: 1 cm. j Representative histological images (H&E staining) and PMEL(HMB45) immunofluorescence stainings of shRNA-Tsc2/Cdkn2a∆/∆ AML-like allograft tumours from the experiment shown in i. k Percentage of PMEL positive cells in shRNA-Tsc2/Cdkn2a∆/∆ AML-like allograft tumours from the experiment shown in i. l Immunofluorescence staining using α-BrdU antibody (red) and DAPI staining (blue), arrowheads highlight positive nuclei. Proliferating cells were labelled by injection of mice with BrdU (80 μg/g body weight i.p.) 32, 24 and 8 h prior to harvesting tumours. m Quantification of percentage of nuclei in tumours from i that were labelled with BrdU. n Quantification of number of cells per ×40 microscopy field staining positively for cleaved caspase 3 in tumours from i. All graphs depict mean ± s.d. Student’s t test, n = 3. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001
