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. Author manuscript; available in PMC: 2015 Feb 9.
Published in final edited form as: Stem Cells. 2014 Jun;32(6):1437–1450. doi: 10.1002/stem.1707

Figure 5. Synergism between YB-1, RSK2, and hTERT conferred complete transformation.

Figure 5

(a) Quantification of HTRZ, HTRY, and HTRY-LT cell growth under anchorage-independent conditions. MDA-MB-231 and SUM149 cells acted as a positive control. (b) Immunoblot assessing RSK expression and activation. (c) Telomerase activity in HTRZ, HTRY, and HTRY-LT cell lysate. 184hTERT cells were used as a positive control. (d) Soft agar colony growth of HTRY cells expressing YB-1, RSK2, or YB-1 and RSK2. Uninduced HTRY cells served as the control. Immunoblotting confirmed transgene expression at 96 hours post-transfection. (e) Quantification of soft agar colony growth following treatment of HTRY-LT cells with scrambled (scr) or YB-1, RSK1, and RSK2 siRNA. Immunoblotting confirmed gene silencing. (f) The ability of HTRZ, HTRY-LT #1, and HTRY-LT #2 cells to form palpable tumors when transplanted into the mammary fat pad of NSG mice (6 mice/group). (g) qRT-PCR analysis of human YB-1 and BMI1 in tumors isolated from NSG mice inoculated with HTRZ and HTRY-LT #2 cells. Gene expression was normalized using eukaryotic 18S rRNA. (h) Hematoxylin-eosin (H&E) and immunoperoxidase staining with p300, BMI1, and YB-1 antibody in tumor tissue explanted 86 days and 142 days after implantation of HTRY-LT #1 cells into the mammary fat pad of NSG mice. The contralateral naïve gland served as a control. Scale bar represents 500 μm. Data represented as mean ± SEM. P values were determined using t test. **, P < 0.01.