Ectopic expression of TSC-22 in malignant cells inhibits cell proliferation and delays tumor formation in vivo. (A) Growth of YAC-1 cells transfected with a pMSCV vector containing TSC-22 exhibited significant inhibition of proliferation in vitro compared with YAC-1 cells transfected with the empty vector alone (P < .05). This experiment was repeated 3 times with similar results. (B) In vivo tumorigenesis resulting from subcutaneous injection of YAC-1 cells ectopically expressing TSC-22 or the pMSCV vector alone into NOD-SCID mice. Tumor size was measured daily. Overexpression of TSC-22 in YAC-1 cells delays the onset of tumor formation in vivo by an average of 2 days (P < .05). (C) The weights of tumors collected from mice killed at the end of the in vivo experiment (day 17). The average weight of tumors formed from YAC-1 cells ectopically expressing TSC-22 was less than that of tumors formed from the YAC-1 cells transfected with the vector alone (P < .05). (D) Photograph of the representative tumor pairs excised from killed mice that had been injected with YAC-1 cells ectopically expressing the pMSCV vector alone (top) or TSC-22 (bottom). (E) Western blot detection of the TSC-22 protein levels in tumors derived from the injection of YAC-1 cells ectopically expressing the pMSCV vector alone or TSC-22 into NOD-SCID mice. Presence of MYC-tag indicates ectopic TSC-22 expression. The tumorigenicity assay in panels B-E was performed twice with a total of 16 mice. Injected transfected YAC-1 tumor cells were first purified to 99% or more purity by cell sorting for GFP. Error bars in panels A-C indicate SDs (n ≥ 3) in one representative experiment.