Figure 5: GILZ knockdown delayed tumour formation and increased tumourigenicity in syngeneic mice.

Twenty C57BL6/rj mice were injected (s.c.) with 5 × 10³ B16F1-GFP-M or B16F1-GFP-D cells after transient transfection with control siRNA (siCTR) or GILZ-specific siRNA (siGILZ). (a) Delayed tumour appearance (mean ± SEM) was observed under the indicated conditions (n = 5). ***p < 0.001 vs. B16F1-GFP-M siCTR cells; ^§§§p < 0.001 vs. B16F1-GFP-D siCTR cells. (b) Graph showing the tumour sizes at each time point after transient transfection with control (●) or GILZ-specific (■) siRNA in B16F1-GFP-M (black) and B16F1-GFP-D (red) cells. The numbers in the upper left corner of the graph indicate the number of mice with tumours/the total number of mice; C = control siRNA, and G = GILZ-specific siRNA. Tumour size was evaluated as described in the Methods. (c) qRT-PCR assay of Tsc22d3 expression in B16F1-GFP-M (white) and B16F1-GFP-D cells (grey) transfected with control (siCTR) or GILZ-specific (siGILZ, stripes) siRNA. **p < 0.01; *p < 0.05 (n = 5). (d) The percentage of mice bearing human HBL melanoma tumours and the delay in tumour appearance. CB17-SCID mice were injected (s.c.) with 5 × 10⁶ HBL H2B-GFP cells transfected with either control or GILZ-specific siRNA (5 mice per condition).