1. Volume 139, Issue 2, Pages 632-643.e4 (August 2010)
C/EBPα Is Up-regulated in a Subset of Hepatocellular Carcinomas and Plays a Role in Cell Growth and Proliferation 
Guo–Dong Lu, Carol Ho–Wing Leung, Benedict Yan, Colyn Mui–Yeong Tan, Sie Yieh Low, Myat Oo Aung, Manuel Salto–Tellez, Seng Gee Lim, Shing Chuan Hooi 
Gastroenterology 
Volume 139, Issue 2, Pages e4 (August 2010)
DOI: /j.gastro
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2. Figure 1
cebpa mRNA is up-regulated in HCC tumors. (A) The cebpa mRNA expression in HCC and adjacent nontumor tissues was analyzed by qRT-PCR and normalized against several housekeeping genes. The histogram shows the mean cebpa expression ± SD. (B) HCC tumors were classified into different categories according to their cebpa expression ratio (tumor against matched nontumor) normalized against sfrs4: up-regulated (≥2.0-fold), no change (0.50- to ∼2.0-fold), or down-regulated (≤0.5-fold). **P < .01, *P < .05 comparing the mRNA expression in tumor and nontumor.
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3. Figure 2
C/EBPα protein expression in HCC tumors and HCC cell lines. (A) Representative Western blot showing 7 pairs of HCC (Tu) and adjacent nontumor tissues (NT). Protein markers (50, 37, and 25 kDa) are shown on the left. (B) Immunohistochemistry of C/EBPα expression in normal liver (top), adjacent nontumor (middle), and HCC (bottom). The “normal” liver sample was obtained from a patient with liver metastasis secondary to colon cancer. Magnification: ×40. Bars: 50 μm. (C) The cebpa mRNA expression in HCC cell lines was quantified by qRT-PCR and normalized against sfrs4. (D) Western blot showing C/EBPα protein expression in HCC cell lines. (C and D) Lane 1 indicates HepG2; 2, Hep3B; 3, HCC-M; 4, Huh7; 5, PLC/PRF/5; 6, THLE-3 cells. (E) Western blot showing the distribution of C/EBPα in the cytosol and nuclear subfractions. Lamin A/C and α-tubulin were used as nuclear and cytosol markers, respectively. Lane 1 indicates HepG2; 2, Hep3B; 3, PLC/PRF/5; 4, Huh7 cells.
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4. Figure 3
C/EBPα is transcriptionally active in Hep3B and Huh7 cells. (A) The C/EBPα transcriptional activity was measured indirectly by luciferase reporter assays and normalized against TK-renilla. (B) The alb and (C) afp mRNA expression was quantified by qRT-PCR. (D) The cebpa, alb, and afp expression in Hep3B cells knocked down for cebpa (sh4 and sh7) were quantified by qRT-PCR. (A–C) Lane 1 indicates HepG2; 2, Hep3B; 3, HCC-M; 4, Huh7; 5, PLC/PRF/5; 6, THLE-3 cells.
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5. Figure 4
Transient knock-down of C/EBPα inhibited colony formation in the HCC cells expressing high levels of C/EBPα. C/EBPα was transiently knocked-down with the use of either specific siRNA (A and B) or shRNA sequences targeting C/EBPα (C and D). (A) The mean percentage of colonies in each of the HCC cell lines after C/EBPα siRNA knock-down (R1, R2, and R3, and scramble control siRNA NC) 7–10 days after transfection. (B) Western blot verifying the efficacy of R1, R2, and R3 in knocking down C/EBPα expression in Hep3B cells. (C) Relative cebpa expression (expressed against that in respective shNC groups) by qRT-PCR after knocking down C/EBPα with the use of shRNA (sh4, sh5, and sh7). shNC is a universal shRNA-negative control. (D) Colony formation assay with the use of shRNA targeting C/EBPα or negative control shNC. Representative plates are shown. The bottom panel is a summary of the data (percentage of shNC ± SD). *P < .05 comparing cells knocked-down for cebpa and cells with scrambled control siRNA (NC) or shRNA (shNC).
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6. Figure 5
HCC cells stably knocked down for C/EBPα has decreased cell proliferation. (A) The cell growth rate of Hep3B stably knocked down for cebpa (sh4 and sh7) was monitored with the use of methylthiotetrazole (MTT) assay. (B) Flow cytometry results showing bromodeoxyuridine incorporation of Hep3B cells stably knocked down for cebpa. FITC(-) is a negative control without incubation of fluorescein isothiocyanate (FITC)–tagged antibody. (C) Western blot showing expression of key cell cycle proteins in nontransfected (Ctrl), control (shNC), and stable cebpa knock-down cells (sh4 and sh7). (D) The E2F- and pRb-responsive transcriptional activity was measured indirectly by luciferase reporter assays. (E) The association of Rb with E2F1 was investigated by coimmunoprecipitation and compared between C/EBPα-expressing cells (shNC) and knocked-down cells (sh4 and sh7) and between Hep3B cells treated with vector control and with 20 μmol/L LY (PI3K inhibitor) or 10 nmol/L OA (PP2A inhibitor) for 24 hours. Immunoglobulin G (IgG) indicates addition of rabbit/goat IgG in coimmunoprecipitation. (F) The Hep3B stable cells with decreased C/EBPα expression (sh4 and sh7) or control cells (shNC) were treated with LY or OA as indicated. Cell growth was determined by MTT assay and normalized to the untreated cells.
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7. Figure 6
Epigenetic regulation of the CEBPA gene in HCC cell lines. (A) The methylation status on the CEBPA upstream (left panel; −1374 to −1051) and core promoters (right panel; −142 to −12) were determined by methylation-specific PCR (MSP). H2O served as a negative control. DNA from HCC-M was treated with SssI methyltransferase (New England Biolabs) and served as the positive control (HCC-M + SssI). (B) Bisulfite sequencing of the CEBPA upstream promoter region in HepG2 and Hep3B cells. Each circle represents a cytosine phosphate guanine dinucleotide (open circles indicate unmethylated; filled circles, methylated) in the primary sequence, and each line of circles represents the analysis of a single cloned allele. The locations of the MSP primers for the CEBPA upstream promoter are shown by dark lines. (C) The methylation status on the CEBPA upstream promoters in HCC tumors with down-regulated (left) or up-regulated (right) C/EBPα expression was determined by MSP. (D) The HCC cell lines were treated with 5-aza dC (5-aza; 5 μmol/L) for 3 days with/without TSA (75 ng/mL) for the final day. The cebpa expression was normalized to that of control treated (solvent) cells. (E) Hep3B cells were treated with increasing doses of TSA for 24 hours. (F) The Hep3B cells were knocked-down for HDAC1 or HDAC2 or both by specific siRNAs and then subjected to Western blotting.
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8. Figure 7
Different chromatin configurations in Hep3B and HepG2 cells. (A) Hep3B cells were treated with 75 ng/mL TSA for 3 hours. The chromatin bound with acetylated histone H3 or trimethyl histone H3 were isolated and amplified by PCR (left). The mRNA levels of cebpa were analyzed by RT-PCR (right). (B) HepG2 cells were treated as in Figure 6D, and ChIP was performed as in panel A. A+T indicates treatment with 5-azadC and TSA. (C) ChIP was performed with anti-HDAC1– or anti-HDAC2–specific antibodies in Hep3B cells treated with/without TSA and in HepG2 cells. NC indicates no addition of antibody; C, control cells treated with respective solvents.
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9. Supplementary Figure 1
Validation of anti-C/EBPα antibody in Western blot and immunofluorescence. (A) Hep3B cells were seated on coverslips for immunofluorescent detection of C/EBPα. Two antibodies against C/EBPα, no (from Cell Signaling; left), and C-18 (from Santa Cruz Biotechnology; right) were used. The confocal images obtained with the antibody no (left) showed clear nuclear localization of C/EBPα. In contrast, the confocal images obtained with the antibody C-18 (right) showed nonspecific cytoplasmic staining in Hep3B cells. Although the blocking peptide prescribed by Santa Cruz Biotechnology (sc-9314p) could block nuclear C/EBPα staining, it failed to block the nonspecific cytoplasmic staining. Green or red indicates C/EBPα; blue, DAPI. Negative Ctrl indicates no addition of primary antibody. (B) HCC cells were collected for Western blot analysis of C/EBPα. Three antibodies against C/EBPalpha, including no (from Cell Signaling; left), 14AA (from Santa Cruz Biotechnology; middle), and C-18 (from Santa Cruz Biotechnology; right) were used. The 14AA antibody (against rat-origin epitope) detects molecular weight–similar but nonspecific bands, whereas no and C-18 (both of which against human-origin epitope) had similar results. Lane 1 indicates HepG2; 2, Hep3B; 3, PLC/PRF/5; 4, Huh7.
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10. Supplementary Figure 2
Knocking-down of C/EBPβ did not affect cell colony growth. (A) HepG2 and HCC-M cells, both of which express C/EBPβ: but undetectable levels of C/EBPα, were knocked down for C/EBPβ by specific siRNAs. The efficacy of knocking-down was shown by Western blotting. (B) After C/EBPβ silencing as in panel A, HepG2 and HCC-M cells were grown in 6 plates for colony formation assay. Ctrl indicates cells without transfection; NC, cells transfected with nonspecific siRNAs.
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11. Supplementary Figure 3
Decreased C/EBPα expression after HDAC inhibition by TSA and PXD101. Hep3B cells were treated with 75 ng/mL TSA for different exposure time (A) or increased concentration of PXD101 for 24 hours (B). The cebpa expression was determined by qRT-PCR.
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