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HEP_24479_sm_SuppInfoFigures.doc6415KSupporting Fig. 1. Hypoxia induces ANGPTL4 expression through HIF-1α but not HIF-2α. (A) Western blot analysis of HIF-1α and ANGPTL4 of SMMC-7721 cells in HIF-1α siRNA experiments under hypoxic conditions (Hyp). (B) Western blots of HIF-2α and ANGPTL4 in SMMC-7721 cells in HIF-2α siRNA experiments under hypoxic conditions (Hyp). (C) The effect of 2ME2 treatment on HIF-1α and ANGPTL4 protein levels in SMMC-7721 cells under hypoxic conditions (Hyp). (D) ChIP experiments were performed in MHCC-97L and SMMC-7721 cells grown under normoxia (Nor) or hypoxia (Hyp) for 24 h, and they demonstrated HIF-1α did not bind at HRE2, 3, 4 and 5. The genomic DNA inputs were 5%. Supporting Fig. 2. The expression of ANGPTL4 in HCC cell lines. (A) The mRNA levels of ANGPTL4 were determined by qRT-PCR analysis in 11 HCC cell lines. (B) Secreted ANGPTL4 in the cell culture supernatant was quantified by ELISA in 11 HCC cell lines. (C) The protein levels of ANGPTL4 were determined by western blotting in 11 HCC cell lines. Supporting Fig. 3. The expression levels of ANGPTL4 in the stable cell lines infected with ANGPTL4-expressing lentivirus. (A) Western blot analysis of ANGPTL4 from whole cell lysates and secreted ANGPTL4 from the conditioned medium (CM) of SMMC-7721 and Huh7 cells infected with either ANGPTL4-expressing lentivirus or control lentivirus. (B) Relative mRNA expression of ANGPTL4 was determined by qRT-PCR analysis in SMMC-7721 and Huh7 cells infected with either ANGPTL4-expressing lentivirus or control lentivirus. (C) Secreted ANGPTL4 in the culture supernatant was quantified by an ELISA in two stable ANGPTL4-overexpressing cell lines and control cell lines. (D) Validation of HUVECs used in the trans-endothelial migration assay by immunostaining with the vascular biomarkers CD31, CD34 and von Willebrand factor (vWF). The negative staining control (Ctrl) was only incubated with secondary antibody and not with primary antibodies. Original magnification: 400×. Supporting Fig. 4. (A) Serum ANGPTL4 protein levels were determined by ELISA in HCC patients with macrovascular invasion (n=29) and without macrovascular invasion (n=133). (B) Serum ANGPTL4 protein levels were determined by ELISA in HCC patients with hepatitis B infection (n=213) and without hepatitis B infection (n=33). (C) Serum ANGPTL4 protein levels were determined by ELISA in HCC patients with cirrhosis (n=130) and without cirrhosis (n=14). (D) Serum ANGPTL4 protein levels were determined by ELISA in HCC patients with extra-hepatic metastasis (n=15) and without extra-hepatic metastasis (n=395). (E) Depiction of ROC curves of serum ANGPTL4. The area under the ROC curves was 0.709 ± 0.026 (with a 95% confidence interval for the area being between 0.659 and 0.759). A cut-off point of 93.5 ng/ml discriminated between normal controls and HCC patients with a sensitivity of 44.7% and a specificity of 87.4%. The arrows indicate the cut-off point for serum ANGPTL4 at 93.5 ng/ml. Supporting Fig. 5. (A) Serum ANGPTL4 levels in tumor-bearing mice with SMMC-7721-lenti-control or MMC-7721-lenti-ANGPTL4 were determined by an ELISA. (B) Western blots of ANGPTL4 from the whole cell lysate and secreted ANGPTL4 from the CM of COS7 cells expressing ANGPTL4 or vector control. (C) The protein levels of integrin α4 and integrin β1 were determined by western blotting in SMMC-7721 and Huh7 cells. Supporting Fig. 6. Secreted ANGPTL4 enhances HCC cell trans-endothelial migration in vitro through the VCAM-1/integrin β1 pathway. (A) Trans-endothelial migration assays of SMMC-7721 cells treated with CM-control, CM-ANGPTL4, anti-integrin β1 antibody or anti-ANGPTL4 neutralizing antibody. Original magnification: 200×. (B) The quantification of six randomly selected fields is shown. (C) The protein levels of the members of NF-κB signal pathway were determined by western blotting in HUVECs treated with CM containing ANGPTL4 or control. Supporting Fig. 7. The effect of hypoxia and Inflammatory cytokines on induction of ANGPTL4 in MHCC-97L cells. (A) Secreted ANGPTL4 protein in the culture supernatant was quantified by an ELISA in MHCC-97L cells treated with interleukin 1β (0.1 ng/ml and 1 ng/ml), interleukin 6 (5 ng/ml and 50 ng/ml) or TNF α (1 ng/ml and 10 ng/ml) under normoxic (21% O2) or hypoxic (2% O2) conditions. (B) Western blot analysis of ANGPTL4 from whole cell lysates of MHCC-97L cells treated with 1 ng/ml interleukin 1β, 50 ng/ml interleukin 6 or 10 ng/ml TNF α under normoxic (21% O2) or hypoxic (2% O2) conditions.
HEP_24479_sm_SuppInfoTables.doc95KSupporting Table 1. Clinicopathologic characteristics of HCC specimens with ANGPTL4 expression and HIF-1α expression Supporting Table 2. Antibodies used in this study Supporting Table 3. The sequences of siRNA duplexes Supporting Table 4. The sequences of gene-specific primers used for qRT-PCR Supporting Table 5. Sequences of primers used for ChIP assay
HEP_24479_sm_SuppInfo.doc55KSupporting Informaion

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