.. The p18INK4c CKI is Ixazomib proteasome specific for complexes between D type Cyclins and either CDK4 or CDK6 (ref. 23). We measured the CKI activity of recombinant HM103p18 and Hp18 proteins with an in vitro kinase assay against Rb substrate. Both proteins had similar IC50s against CDK6, which improved (from 4 ��mol/l to <250 nmol/l) after the refolding step in their preparation. However, the intracellular activity of the cell-permeable HM103p18 protein was considerably greater than that of the control Hp18 protein as assessed either by the inhibition of Rb phosphorylation or by secondary effects on p53 phosphorylation, p21 expression and ATM phosphorylation (Figure 3a).

This experiment also examined five other recombinant proteins: HM101p18 is identical to HM103p18 but utilized the MTD101 sequence (Supplementary Table S1); Hp18M101 and Hp18M103 have MTD sequences positioned on the carboxyl- rather than amino terminal ends of the protein; and HM103p18M103 and HM101p18M101 each contain an additional MTD sequence at the carboxyl-terminal end of the protein (Supplementary Table S3 and Supplementary Figure S5). HM103p18 was a considerably more efficient inducer of apoptosis than control proteins in HCT116 cancer cells, as assessed either by annexin V staining or by Caspase-3 activation (Figure 3b and Supplementary Figure S6). In addition, no changes in annexin V staining or Caspase-3 activity were observed in untransformed RAW264.7 and NIH3T3 cells treated with CP-p18INK4c. We conclude that differences in the biological activities of HM103p18 or HM101p18 as compared to Hp18 are due to differences in protein uptake mediated by the MTD sequences.

Systemic delivery of cell-permeable p18INK4c High levels of HM103p18 were observed in ~60% of peripheral white blood cells after 1 hour of intraperitoneal injection of FITC-labeled HM103p18 (Figure 4a). Similar levels of protein uptake were observed in both leukocytes and lymphocytes; however, neutrophils contained relatively low levels of the protein, suggesting phagocytic cells are not intrinsically more active at internalizing recombinant p18INK4c proteins (Figure 4b). HM103p18 was also delivered to the spleen and liver with similar kinetics, as assessed by either immunostaining (Figure 4c) or western blot analysis (Figure 4d) and persisted for several hours, declining with a half-life of ~1 hour (Figure 4d).

By contrast, proteins lacking the MTD sequence failed to accumulate in any the blood cells or tissues analyzed (Figure 4). In addition, Intraperitoneally administrated Q-dot-labeled recombinant HM103p18 was delivered to major organs such as liver and lung by fusing to only MTD (MTD103), but not Batimastat PTD (HIV Tat) (Supplementary Figure S7, left panel). Intravenously injected HM103p18 was sufficiently delivered in vivo, distributed to solid tumors, persisted from 6 to 24 hours (Supplementary Figure S7, left panel).