Figure 6.

Inhibition of CAT-1 expression suppresses HG3 tumor growth in vivo. (A) HG3 cells, that had been transduced with a doxycycline-inducible shRNA CAT-1 knockdown construct (HG3_pLKO_tet_on_shCAT-1, shRNA: TRCN0000042967, termed iCAT-1 k.o.) were cultured in the absence (–) or the presence (+) of doxycycline (DOX) for 4, 6, 12 or 21 days. To allow arginine-independent expansion, cell medium was supplemented with 1 mM citrulline and 67 μM lysine tripeptide. Protein expression of CAT-1 and GAPDH was analyzed by Western Blot. (B–D) HG3_pLKO_tet_on_shCAT-1 (iCAT-1 k.o.) or HG3 cells transduced with an inducible non-target shRNA construct (HG3_pLKO_tet_on_non_target, iNT) were cultured for 6 days in the absence (–) or presence (+) of 1 μg/ml doxycycline and then subjected to the following analyses: (B) Cell proliferation was determined by the incorporation of [³H]thymidine for 16 h. Proliferation in HG3_pLKO_tet_on_non_target cells in the absence of doxycycline (mean: 165,043 ± 36,096 cpm) was set as 100% (n = 9). (C,D) Cells were stained with Annexin V and PI and were then analyzed by flow cytometry (n = 3). Values are shown as mean ± SD. (B–D) Statistical calculations were performed by one way ANOVA with Tukey post-test. (E–G) 2.5 × 10⁶ iCAT-1 k.o. (red lines) or iNT (blue lines) cells were injected s.c. in the flank of NSG mice. Mice received drinking water with (continous line) or without (dashed line) doxycycline (1 mg/ml) ad libitum (5 mice/group), depicted is one representative of two set of groups. (E) Tumor size was calculated based on caliper measurements (Volume = height × width²) and mice were sacrificed when tumor volume exceeded 3 cm³. (F) Kaplan Meier survival curves were generated. (G) CAT-1, ASS, and GAPDH protein expression was detected by Western Blot in explanted tumors, derived from iCAT-1 k.o. with (+) or without (−) oral administration of 1 mg/ml doxycycline via drinking water ad libitum (n = 5).