1. Volume 144, Issue 1, Pages 179-191.e4 (January 2013)
SPOCK1 Is Regulated by CHD1L and Blocks Apoptosis and Promotes HCC Cell Invasiveness and Metastasis in Mice 
Yan Li, Leilei Chen, Tim Hon Man Chan, Ming Liu, Kar–Lok Kong, Ji–Liang Qiu, Yan Li, Yun–Fei Yuan, Xin–Yuan Guan 
Gastroenterology 
Volume 144, Issue 1, Pages e4 (January 2013)
DOI: /j.gastro
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2. Figure 1
CHD1L activates SPOCK1 transcription by binding to the 5′ upstream region of SPOCK1. (A) QGY-7703 cells were transiently transfected with an ectopic CHD1L-expressing construct. Huh7 cells were treated with siCHD1L or scramble small interfering RNA. The relative expression of CHD1L and SPOCK1 was detected by qRT-PCR; 18S ribosomal RNA was used as an internal control. The bars represent the mean ± standard deviation of 3 independent experiments. (B) The correlation between CHD1L and SPOCK1 expression was detected by qRT-PCR in 135 pairs of HCCs and matched nontumor tissues with linear regression lines and Pearson correlation significance (P < .001, Pearson χ2 test). (C) The protein levels of CHD1L and SPOCK1 were detected by Western blot analysis in 5 representative primary HCC tissues (T) and their paired nontumor (N) tissues. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The Western blot data were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). The expression levels of CHD1L relative to those of SPOCK1 after normalization were plotted in the line chart. (D) Schematic diagram representing the distribution of the CHD1L-binding loci (ellipse) in the regulatory region of SPOCK1. The 5 individual fragments represent different 5′ upstream sequences of SPOCK1. (E) Electrophoretic mobility shift assay was used to detect the interaction between CHD1L and SPOCK1 double-stranded DNA probes. NE, nuclear extract. (F) Fragment E was subcloned into the pGL3-basic vector (pGL3-SPOCK1) and co-transfected with pcDNA3.1-CHD1L or empty vector for dual luciferase assays. The results were normalized to pGL3-basic activity, and the pGL3-control vector was used as a positive control.
Gastroenterology  , e4DOI: ( /j.gastro )
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3. Figure 2
Clinical significance of SPOCK1 in human HCC. (A) Scatterplots of the relative expression of SPOCK1 in normal liver, nontumor tissues, and their matched HCC counterparts. Black lines, mean ± standard deviation. (B) SPOCK1 protein expression levels in 8 representative primary HCC tissues (T) and their paired nontumor (N) tissues was evaluated via Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. SPOCK1 bands were quantified with ImageJ software (National Institutes of Health) and are shown in the bar chart after normalization. (C) Representative image of IHC staining with an anti-SPOCK1 antibody (from left to right: normal liver; nontumor tissue; paired tumor tissue; HCC tissue, 200× magnification; HCC tissue, 400× magnification). (D) Scatterplots of the relative expression of SPOCK1 in patients with metastasis or without metastasis. Black lines, mean ± standard deviation. (E) Kaplan–Meier overall survival curve (left panel) and disease-free survival curve (right panel) of HCC patients correlated with SPOCK1 expression. SPOCK1(+), patients with SPOCK1 overexpression; SPOCK1(−), patients without SPOCK1 overexpression.
Gastroenterology  , e4DOI: ( /j.gastro )
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4. Figure 3
SPOCK1 has strong tumorigenic ability. (A) Ectopic expression of SPOCK1 was detected in SPOCK1-transfected cells by Western blot. β-actin was used as a loading control. (B) The cell growth rates were determined with an XTT proliferation assay (*P < .05; **P < .001; independent Student t test). Representative images of (C) foci formation and (D) colony formation in soft agar induced by Vec-7703, SPOCK1-7703, Vec-8024, and SPOCK cells. The numbers of foci and colonies were calculated and are depicted in the bar chart. The values indicate the mean ± standard deviation of 3 independent experiments (*P < .05; **P < .001; independent Student t test). (E) Representative examples of tumors (indicated by arrows) formed in nude mice injected with the indicated cells. Tumor growth curves are summarized in the line chart. The average tumor volume was expressed as the mean ± standard deviation of 5 mice. (*P < .05; **P < .001; independent Student t test).
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5. Figure 4
shRNA-mediated SPOCK1 silencing abolishes the tumorigenic effect of SPOCK1. (A) BEL-7402 and QGY-7701 cells were treated with scrambled shRNA or shRNA against SPOCK1. Western blot analysis and qRT-PCR were performed to detect SPOCK1 expression; β-actin and 18S were used as loading controls, respectively. (B) The cell growth rates were determined with an XTT proliferation assay (*P < .05; **P < .001; independent Student t test). Representative images of (C) foci formation and (D) colony formation in soft agar induced by shCTL-7402, shSPOCK1-7402, shCTL-7701, and shSPOCK cells. The numbers of foci and colonies were calculated and are depicted in the bar chart. The values indicate the mean ± standard deviation of 3 independent experiments (*P < .05; **P < .001; independent Student t test). (E) Representative examples of tumors (indicated by arrows) formed in nude mice injected with the indicated cells. The tumor incidence rate during the 4-week observation period is shown in the table section of panel E.
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6. Figure 5
SPOCK1 exerts anti-apoptotic function via the Akt–cyt c–caspase-9–caspase-3 pathway. (A) After treatment with STS, apoptosis was determined by TUNEL assay. Apoptotic cells were stained with fluorescein isothiocyanate–labeled DNA strand breaks (green signal), and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue signal). The apoptotic index, defined as the percentage of apoptotic cells, was calculated and is summarized in the bar chart. The values represent the mean ± standard deviation of 3 independent experiments (*P < .05; **P < .001; independent Student t test). (B) After treatment with STS for the indicated time, the levels of phosphorylated Akt (P-Akt), total Akt, caspase-9, caspase-3, and PARP were detected in Vec-7703, SPOCK1-7703, shCTL-7402, and shSPOCK cells by Western blot analysis. β-actin was used as a loading control. (C) Representative images of JC-1 dye staining. JC-1 dye aggregates at high ΔΨm mitochondria (red) or forms monomers at low ΔΨm mitochondria (green). (D) SPOCK cells were incubated for 24 hours with different concentrations of Akt1 inhibitor varying from 0 to 80 μmol/L. The levels of phosphorylated Akt (ser473) and BAD (ser136) were detected by Western blot analysis. β-actin was used as a loading control. (E) Vec-7703 and SPOCK cells were treated with STS (1 μmol/L) alone or in combination with Akt1 inhibitor (80 μmol/L). Apoptosis in Vec-7703 and SPOCK cells was compared at the indicated times by flow cytometry. Cells stained with Annexin-V–fluorescein isothiocyanate were counted as apoptotic, and the apoptotic index was defined as the percentage of apoptotic cells. (F) Six hours after STS treatment, the apoptotic index of Vec-7703 and SPOCK cells was calculated and is summarized in the bar chart. The values represent the mean ± standard deviation of 3 independent experiments (*P < .05; independent Student t test).
Gastroenterology  , e4DOI: ( /j.gastro )
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7. Figure 6
SPOCK1 promotes tumor invasion and metastasis. (A) A Matrigel invasion assay was performed to study the invasion ability of Vec-7703, SPOCK1-7703, shCTL-7402, and shSPOCK cells. The number of invaded cells was calculated and is depicted in the bar chart. The values indicate the mean ± standard deviation of 3 independent experiments (*P < .05; **P < .001; independent Student t test). (B) An in vivo experimental metastasis assay was performed to evaluate the effect of Vec-7703 and SPOCK cells on tumor metastasis. Representative images of livers derived from severe combined immunodeficient mice after tail vein injection of Vec-7703 or SPOCK cells. The right panel summarizes the numbers of metastatic nodules. The values for the individual mice are shown above the bars; the values by group are also denoted (*P < .05, **P < .001; independent Student t test). (C) Representative images of H&E staining of lung tissues from severe combined immunodeficient mice injected with Vec-7703 or SPOCK cells (200× magnification, upper panel). Representative images of IHC staining of liver tissues from severe combined immunodeficient mice (200× magnification, lower panel). (D) The relative expression of MMP-9 and SPOCK1 was detected in empty vector– and SPOCK1-transfected cells by qRT-PCR; 18S ribosomal RNA was used as an internal control. (E) Gelatin zymography was performed to quantify MMP-9 activity in Vec-7703 and SPOCK cells. The gelatin digested by MMP-9 was visualized as a clear band on a dark background. MMP-9 activity was quantified by ImageJ software (National Institutes of Health) and is shown in the bar chart. The value is expressed as the average of 2 independent experiments. (F) Representative images of cells invading through the Matrigel in the presence of MMP-9 inhibitor at concentrations of 0–20 μmol/L. The number of invaded cells was calculated and is depicted in the bar chart. The values indicate the mean ± standard deviation of 3 independent experiments (*P < .05, **P < .001; independent Student t test).
Gastroenterology  , e4DOI: ( /j.gastro )
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8. Supplementary Figure 1
A ChIP-PCR assay was performed to detect the existence of indicated DNA fragments pulled down by GFP antibodies (B-2) against GFP tagged CHD1L protein. RNA polymerase antibody was used as a positive control and IgG was used as a negative control.
Gastroenterology  , e4DOI: ( /j.gastro )
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