Supplementary Fig. 1. RT-PCR showed that tumor tissues have elevated Mst1 mRNA levels in most of the HCC patients tested. GAPDH RT-PCR products were shown as the endogenous loading controls. N: the mRNA sample from the adjacent normal tissue. T: the mRNA sample from the tumor tissue.

Normal LiverHep3BPLC/PRF/5 Huh * * * Normalized Mst1 mRNA levels (a.u.) Supplementary Fig. 2. Elevated Mst1 mRNA expression was detected in HCC cell lines with RT-PCR. RT- PCR generated from the GAPDH mRNA was used as the endogenous loading control.

Supplementary Fig. 3. Mst1 antibody detects both the 56kD full-length (FL) and the 36kD N-terminal cleaved form (NT) in adjacent normal liver tissues of 2 HCC patients. The FL form is predominant form in HCC tissues. Western blot using the same Mst1 antibody on the HEK293T lysates transfected with Mst1 was included to show the position of the NT form induced by Mst1 overexpression. FL: full-length form. NT: amino terminal cleaved form

Supplementary Fig. 4. Quantitation of the changes in Mst1 full length protein levels in the 25 HCC tumors relative to their adjacent normal tissues (normalized with endogenous actin levels).

Supplementary Fig. 5. Mst2 was undetectable in Hep3B and Huh-7 cells, but was present in normal human liver tissues as detected by Western Blotting. Normal Liver Mst2 Actin Hep3B HuH7

Supplementary Fig. 6. Representative fluorescent immunocytochemical images of the expressed HA-Mst1 (red: anti-HA tag) in Hep3B cells were shown. The morphology of the nuclei (blue: DAPI stained nuclei) in the HA-Mst1 expressing cells did not show any sign of chromatin condensation. n = 3 experiments. Overnight treatment of 10mM cisplatin in Hep3B was done as the positive control for chromatin condensation for the experiments.

Supplementary Fig. 7. More densely grown areas were seen in Mst1 overexpressing stable cells than in the control stable cells after 3-day culturing in high initial plating density (2×10 5 cells per well). Dense cell growth areas were indicated by black arrows. No obvious difference in cell number was seen between the Mst1 overexpressing and the control stable cells when started with low plating density (8×10 4 per well).

Supplementary Fig. 8. Quantitation of the changes in NORE1B expression between tumors and their adjacent normal tissues in the HCC patient samples.

Supplementary Table 1. Characteristics of the HCC patient cohort.

Supplementary Table 2. Nucleotide sequences of the PCR primers

PatientAdjacent NormalTumor SEEDEMDSGTM 330 … 342 RVASTMTDGAN SEEDEMDSGTM 330 … 342 RVASTMTDGAN 352 *Caspase-3 recognition sites in wildtype Mst1: …DEMD 326 S…TMTD 349 G… Supplementary Table 3. Translated amino acid sequences around the caspase-3 recognition sites from the Mst1 mRNA nucleotide sequences in selected HCC patient tumor- adjacent normal tissue pairs.