1. Self-sufficiency in growth signals Sustained angiogenesis Insensitivity to anti-growth signals Evading apoptosis Tissue invasion & metastasis Limitless replicative potential Cancer The development of human cancer cells

2. Cdc6 is essential in the initiation of DNA replication: A). Assembling pre-replication complex (pre-RC) with ORC, and loading MCMs to replication origins; B). Licensing DNA replication to ensure one round DNA replication per cell cycle.

3. LA-N-2 MCF7 CHLA255 Cdc6 shRNA THTD6Ai THTD6Ai THTD6Ai THTP Mock Mock THTP THTP Cdc6 Cdc6 knockdown -actin 1 2 3 4 5 6 7 8 The novel function of GLS101/TSUKUSI FL1 is the silencing of human Cdc6, the specific genes for cell proliferation: Specific Cdc6 shRNAs produced from the viral vector Inhibit Cdc6 and block Cancer cell proliferation, but not normal cells.

4. Apoptosis Differentiation M G1 Recombinant Lentivirus GLS101 G2 STOP S Senescence The Cell Cycle

5. ——   THTD  ———IP/mouse IgG —    IP/β-p16INK4a Phosph-Rb-C Ser780 Phosph-Rb-C Ser807/811 IgG, light chain 1 2 3 4 ——— THTD 1:10 1:25 1:5  IP/β-p16INK4a Phosph Rb-C Ser 807/811 IgG, light chain 1 2 3 4 Reactivation of p16INK4a CDK inhibitory activity by Cdc6 knockdown

6. MCF7 MCF7/THTP MCF7/THTD6Ai Senescence-associated β-glactosidase (SA-β-Gla) positive staining caused by Cdc6 knockdown

7. Anti-H3K9me3/me2 indirect fluorescent staining Mock THTD6Ai Confocal fluorescent microscopy Mock THTD6Ai The formation of senescence associated heterochromatic foci via Cdc6 knockdown