Fig. 4.

Ectopic N1^IC rescues USP9x knockdown in KBP allografts. (A) Estimated weight of KBP shUSP9x; hN1^IC allografts given regular (n = 6 tumors) or Dox diet (n = 7 tumors). (B) Immunoblot of USP9x, JAG1, and N1^IC in KBP shUSP9x; hN1^IC tumors 3 wk after tumor onset. The expression of β-actin is included as the loading control. Molecular weight markers are shown in kilodaltons. (C) Expression of USP9x, JAG1, and N1^IC from B, determined by densitometry and presented, relative to β-actin. (D) Enzyme-linked immunosorbent assay analyses of secreted IL-1β in conditioned media from overnight cultures of tumor cells from KBP shUSP9x; hN1^IC tumors (−Dox, n = 4 tumors and +Dox, n = 7 tumors). (E) Quantification (by flow cytometry) of TAMs, M2 TAMs, CD4⁺ T cells, CD8⁺ T cells, and cytotoxic T cells in KBP shUSP9x; hN1^IC tumors 3 wk after tumor onset (n = 5 tumors per group). All values are expressed as a percentage of CD45⁺ cells. Error bars represent SEM. *P < 0.05. P values are determined by the two-tailed Student’s t test with Welch’s correction for unequal variances. a.u = arbitrary units.