Supplemental Figure legends

Supplemental Figure 1. Anti-cancer drugs induce CEBPD expression in liver cancer cells. Two liver cancer cell lines (Huh7 and HepG2) were cultured in the presence of 30 μM cisplatin (CDDP), 1 mM paclitaxel (TAX), 10 nM velcade® (Vel; bortezomib), 10 μM 5-AzadC, 20 μM actinomycin D (ActD), 10 μg/mL 5-fluorouracil (5-FU), 1 μM dexamethasone (Dex), or 10 μM retinoic acid (Ra), respectively. HepG2 and Huh7 cells were cultured in the indicated anti-cancer chemicals for 2 h and 6 h, respectively. Subsequently, total lysates were harvested for Western blotting analysis with CEBPD antibody.

Supplemental Figure 2. Scheme of depression of epigenetic effects to enhance HMDB-induced CEBPD expression and apoptosis.

Supplemental Figure 3. The p38/CREB pathway mediates HMDB-induced CEBPD transcriptional activation and CEBPD participates in HMDB-induced apoptosis in liver cancer cells. (A) A luciferase assay was performed with lysates harvested from liver cancer cells transfected with CEBPD reporter vector and the dominant negative form of p38 (DN-p38) or CREB (DN-CREB) expression vectors. (B) Huh7 or HepG2 cells were infected with lentiviruses encoding shLacZ or shCEBPD for 3 days. Flow cytometry analysis was performed on lentivirus-infected cells after 18 h of HMDB treatment.

Supplemental Figure 4. Combinatorial treatment of HMDB and 5-AzadC can additively affect cell death. Typical cell cycle histograms were analyzed using flow cytometry after exposure in the presence or absence of 5-AzadC for 48 h, followed by HMDB at the indicated time points.

Supplemental Figure 5. Enhanced cytotoxic effects of HMDB combined with 5-AzadC on Huh7 and HepG2 cells. (A) Huh7 and HepG2 cells were treated with 5-AzadC for 48 h and then incubated with HMDB at the indicated concentration. Total lysates were harvested for Western blotting analyses. (B) Co-treatment with 5-AzadC and HMDB enhanced the apoptosis of liver cancer cells. An apoptosis assay (TUNEL assay) was performed as previously described in Supplementary Methods.

Supplemental Figure 6. Loss of CEBPD attenuated dual treatment-induced tumor killing in liver cancer cells. (A) Huh7 and HepG2 cells were infected with lentiviruses encoding shLacZ or shCEBPD (shD) and subsequently treated with or without HMDB and 5-AzadC; the cells were then harvested for TUNEL assay. (B) Quantification of the data was analyzed by ImageJ.

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