Optimal cutoff value for low SIRT3 expression, based on the results of IHC evaluation. Results showed that ROC curve analysis for survival status has the shortest distance from the curve to the point (0.0, 1.0) (Fig. 2). Thus, we selected the cutoff value for survival status. Tumors with scores below the obtained cutoff value were considered 
to be with low SIRT3 expression, leading to the greatest number of tumors correctly classified as ML-240 having (i.e., case group) or not having (i.e.,SIRT3 as a Prognostic Biomarker in HCCTo determine the clinical significance of SIRT3 expression in HCC, relationship between SIRT3 expression and clinicopathological features was analyzed. Significant correlations were found between SIRT3 expression and variables including Eliglustat biological activity differentiation (P = 0.013), clinical stage (P = 0.005), serum AFP level (P,0.01), tumor multiplicity (P = 0.026) and relapse (P = 0.028). HCC patients with low SIRT3 expression had a higher tendency to be 22948146 with poor differentiation, advanced stage, high level of serum AFP and multiple tumor numbers. There were no statistical connections between SIRT3 expression and the other clinicopathological parameters, such as age, gender, HBsAg, cirrhosis, tumor size and vascular invasion (P.0.05, Table 1).Interrelation of SIRT3 Expression and HCC DifferentiationAs indicated in Table 1, expression of SIRT3 was related to HCC differentiation. We next further confirmed the reverse connection of SIRT3 expression in HCC and tumor differentiation. Another 30 HCC cases (10 cases in each group of well, moderate and poor differentiation) diagnosed from Mar 2011 to Oct 2011 were collected to determine the SIRT3 expression patterns. Results showed that SIRT3 expression in noncancerous tissue was not significantly changed in cases with different tumor differentiation (Fig. S1). However, SIRT3 was gradually decreased from well- to poor-differentiated HCC (Fig. 4A). Percentage of cases with high SIRT3 expression was 24.4 in poor-differentiated HCC, noticeably lower than 43.5 in well-differentiated HCC (Fig. 4B).Correlation of SIRT3 Expression with Survival of Postoperative HCC PatientsTo determine whether SIRT3 expression was related to survival of HCC patients after surgical resection, Kaplan-Meier survival analyses were performed. Survival data were available for 248 patients. The average survival time was 40.9 months for the patients with low SIRT3 expression, while it was 65.0 months for patients expressed high level of SIRT3. Patients with low SIRT3 expression were likely to be with significantly shorter overall survival (P,0.01) (Fig. 5A) and recurrence-free survival (P = 0.004) (Fig. 5B). The impact of SIRT3 on prognosis was further evident in HCC patients subclassified by the factors attributed to worse outcome. The 8 subgroups of HCC patients were identified as `tumor multiplicity (multiple)’, `tumor size ( 5 cm)’, `HBsAg (positive)’, `serum AFP ( 20 ng/ml)’, `grade (III V)’, `cirrhosis (yes)’, `stage (III V)’, and `relapse (yes)’. Results revealed that patients with low SIRT3 expression in all subgroups survived shorter than those with high SIRT3 expression (Fig. 6). Unfortunately, it seemed that SIRT3 expression was not of significance in predicting the recurrence-free survival for these subgroups of HCC cases (Fig. S2). Our further study indicated that low SIRT3 expression in HCC patients with tumor size (,5 cm), grade (I I), or stage (I I) associated with poorer overall survival but not recurrence.Optimal cutoff value for low SIRT3 expression, based on the results of IHC evaluation. Results showed that ROC curve analysis for survival status has the shortest distance from the curve to the point (0.0, 1.0) (Fig. 2). Thus, we selected the cutoff value for survival status. Tumors with scores below the obtained cutoff value were considered to be with low SIRT3 expression, leading to the greatest number of tumors correctly classified as having (i.e., case group) or not having (i.e.,SIRT3 as a Prognostic Biomarker in HCCTo determine the clinical significance of SIRT3 expression in HCC, relationship between SIRT3 expression and clinicopathological features was analyzed. Significant correlations were found between SIRT3 expression and variables including differentiation (P = 0.013), clinical stage (P = 0.005), serum AFP level (P,0.01), tumor multiplicity (P = 0.026) and relapse (P = 0.028). HCC patients with low SIRT3 expression had a higher tendency to be 22948146 with poor differentiation, advanced stage, high level of serum AFP and multiple tumor numbers. There were no statistical connections between SIRT3 expression and the other clinicopathological parameters, such as age, gender, HBsAg, cirrhosis, tumor size and vascular invasion (P.0.05, Table 1).Interrelation of SIRT3 Expression and HCC DifferentiationAs indicated in Table 1, expression of SIRT3 was related to HCC differentiation. We next further confirmed the reverse connection of SIRT3 expression in HCC and tumor differentiation. Another 30 HCC cases 
(10 cases in each group of well, moderate and poor differentiation) diagnosed from Mar 2011 to Oct 2011 were collected to determine the SIRT3 expression patterns. Results showed that SIRT3 expression in noncancerous tissue was not significantly changed in cases with different tumor differentiation (Fig. S1). However, SIRT3 was gradually decreased from well- to poor-differentiated HCC (Fig. 4A). Percentage of cases with high SIRT3 expression was 24.4 in poor-differentiated HCC, noticeably lower than 43.5 in well-differentiated HCC (Fig. 4B).Correlation of SIRT3 Expression with Survival of Postoperative HCC PatientsTo determine whether SIRT3 expression was related to survival of HCC patients after surgical resection, Kaplan-Meier survival analyses were performed. Survival data were available for 248 patients. The average survival time was 40.9 months for the patients with low SIRT3 expression, while it was 65.0 months for patients expressed high level of SIRT3. Patients with low SIRT3 expression were likely to be with significantly shorter overall survival (P,0.01) (Fig. 5A) and recurrence-free survival (P = 0.004) (Fig. 5B). The impact of SIRT3 on prognosis was further evident in HCC patients subclassified by the factors attributed to worse outcome. The 8 subgroups of HCC patients were identified as `tumor multiplicity (multiple)’, `tumor size ( 5 cm)’, `HBsAg (positive)’, `serum AFP ( 20 ng/ml)’, `grade (III V)’, `cirrhosis (yes)’, `stage (III V)’, and `relapse (yes)’. Results revealed that patients with low SIRT3 expression in all subgroups survived shorter than those with high SIRT3 expression (Fig. 6). Unfortunately, it seemed that SIRT3 expression was not of significance in predicting the recurrence-free survival for these subgroups of HCC cases (Fig. S2). Our further study indicated that low SIRT3 expression in HCC patients with tumor size (,5 cm), grade (I I), or stage (I I) associated with poorer overall survival but not recurrence.