Additional file 2: Table S1. of Maintenance of stemness is associated with the interation of LRP6 and heparin-binding protein CCN2 autocrined by hepatocellular carcinoma

Correlations between CCN2/LRP6 and clinicopathology feature in 374 patients with HCC. Table S2. Univariate analysis of factors associated with survival and recurrence in 374 patients with HCC. Table S3. Multivariate analysis of factors associated with survival and recurrence in 374 patients with HCC. Table S4. Primary antibodies used for western blot and immunohistochemistry. Table S5. Sequences of primers used for qRT-PCR. Table S6. Primers for vectors construction. Table S7. vshRNA target sequences for CCN2 and LRP6. (DOC 87 kb

rAd-p53 induced apoptosis was reverted by Fbxw7-specific siRNA.

<p>A) Fbxw7-siRNA treatment in Hep3B successfully down-regulated Fbxw7 protein but enhanced c-Myc and Cyclin expression as shown by immunostaining. Over-expression of rAd-p53 in the same cell line could enhance the levels of Fbxw7 and decrease c-Myc and Cyclin E expression. Fbxw7 knockdown in p53 over-expressing cells rescued partially the phenotype showing lower Fbxw7 levels with higher c-Myc and Cyclin E expression. β-actin served as loading control. B) The proliferation rate of rAd-p53 infected Hep3B cells was increased in the presence of Fbxw7-specific siRNA (MTT assay). C) The percentage of apoptotic Hep3B cells infected with rAd-p53 was decreased by Fbxw7-specific siRNA. Values are mean±standard error, n=3. * <i>P</i><0.05 <i>vs</i> control; ** <i>P</i><0.05 <i>vs</i> Fbxw7 siRNA; *** <i>P</i><0.05 <i>vs</i> rAd-p53.</p

Additional file 1: Figure S1. of BCORL1 is an independent prognostic marker and contributes to cell migration and invasion in human hepatocellular carcinoma

BCORL1 knockdown inhibits cell migration and invasion in HCCLM3 cells. A) HCCLM3 cells that were tranfected with scrambled siRNA or BCORL1 siRNA-1 were subjected to Western blot for BCORL1. n = 6; * P < 0.05 by t test. B) Transwell migration assays showed that BCORL1 knockdown inhibited cell migration in HCCLM3 cells. As assessed by Transwell invasion assays, HCCLM3 cell invasion was inhibited by BCORL1 knockdown. * P < 0.05 by t test; n = 3. (TIF 1951 kb

Effect of Fbxw7 knockdown on LO2 and Hep3B cell lines.

<p> A) qRT-PCR confirmed knockdown in LO2 and Hep3B cells treated with Fbxw7 siRNA. B) Expression of c-Myc and Cyclin E proteins were enhanced by Fbxw7 siRNA, as confirmed by Western blot analysis. β-actin served as loading control. C) Proliferation rates were assessed by MTT assay. Cell viability was normalized to time point 0 h. The proliferation rate of LO2 and Hep3B cells treated with Fbxw7 siRNA was significantly greater than in control siRNA cells. Values are depict as mean±standard error, n=3. *<i>P</i><0.05 <i>vs</i> control siRNA.</p

rAd-p53 induces apoptosis in HCC cell lines with increased Fbxw7 and decreased c-Myc and Cyclin E.

<p>A) MTT activity was measured after infecting Hep3B cells with rAd-p53 in an MOI gradient to determine their IC₅₀ after 48 h. Mean OD values were used to calculate the IC₅₀ via the modified Kou-type method: lgIC₅₀ = Xm-I (P-(3-Pm-Pn)/4), in which Xm: lg maximum dose; I: lg (maximum dose/ adjacent dose); P: sum of positive response rate; Pm: the largest positive response rate; Pn: the smallest positive response rate. B) Over-expression of <i>p53</i> 24 h after infection as confirmed by RT-PCR. Quantification of RT-PCR for p53 and Fbxw7 indicated elevated mRNA levels for rAd-p53 infected Hep3B cells, whereas c-Myc and Cyclin E levels were not significantly decreased. C) Protein expression of p53 and Fbxw7 was enhanced, while c-Myc and Cyclin E expression was suppressed by rAd-p53 in Hep3B, HepG2 and Huh7 cells, as confirmed by Western blot analysis. β-actin served as loading control. D) Hep3B proliferation rate assessed by MTT assay was significantly lower in rAd-p53 infected cells compared to control cells. E) Quantification of apoptotic cell population (AnnexinV positive/ PI negative) by flow cytometry. rAd-P53 infected Hep3B cells were composed of a larger subset of apoptotic cells after 72 hours of infection compared to control. Values are depicted as mean±standard error, n=3. *<i>P</i><0.05 <i>vs</i> control.</p

Additional file 3: Figure S3. of Long non-coding RNA CASC2 suppresses epithelial-mesenchymal transition of hepatocellular carcinoma cells through CASC2/miR-367/FBXW7 axis

miR-367 reversed the anti-metastatic effects of CASC2 on HCC cells. (A) FBXW7 level was reversed by miR-367 in CASC2-overexpressing MHCC-97H cells. (B) miR-367 expression was rescued by miR-367 mimics in CASC2-overexpressing MHCC-97H cells. (C) miR-367 abolished the inhibitory effects of CASC2 on migration and invasion of MHCC-97H cells. (D) miR-367 abrogated the inhibitory effects of CASC2 on EMT progression of MHCC-97H cells. *P < 0.05, **P < 0.01, ***P < 0.001. (TIFF 1008 kb

rAd-p53 inhibits tumor growth <i>in vivo</i>.

<p>Multicenter intratumoral administration of rAd-P53 in Hep3B subcutaneous tumor in nude mice was monitored over time. Tumor size and weight was plotted in A), and B) respectively. C) Exemplarily, tumor bearing mice and explanted tumors are depicted. Values are mean±standard error. * <i>P</i><0.05 <i>vs</i> rAd-p53.</p

Additional file 4: Figure S4. of Long non-coding RNA CASC2 suppresses epithelial-mesenchymal transition of hepatocellular carcinoma cells through CASC2/miR-367/FBXW7 axis

FBXW7 rescued the pro-metastatic effects of miR-367 on HCC cells. (A) FBXW7 overexpression increased the expression of E-cadherin and decreased the expression of Vimentin in Hep-3B cells. (B) FBXW7 abolished the promoting effects of miR-367 on EMT progression of Hep-3B cells. (C) FBXW7 abrogated the pro-metastatic effects of miR-367 on migration and invasion of Hep-3B cells. **P < 0.01. (TIFF 878 kb

Additional file 1: of MicroRNA-1468 promotes tumor progression by activating PPAR-γ-mediated AKT signaling in human hepatocellular carcinoma

Figure S1. Modulation of CITED2 or UPF1 mimics miR-1468-induced cellular processes in HCC. (A) Hep3B-miR-control cells that were transfected with empty vector (EV) or CITED2, UPF1 overexpression plasmid were subjected to western blot for CITED2 and UPF1. MHCC-97 L-anti-miR-NC cells that were transfected with scrambled siRNA or CITED2, UPF1 siRNA were subjected to western blot for CITED2, UPF1. CITED2 or UPF1 restoration mimics the effects of miR-1468 knockdown on cell proliferation (B, C), colony formation (D), cell cycle progression (E) and apoptosis (F) of Hep3B cells. CITED2 or UPF1 knockdown mimics effects of miR-1468 overexpression in MHCC-97 L cells (B-F). (G) Alteration of CITED2 or UPF1 mimics the effects of miR-1468 on cell cycle and apoptosis associated factors. *P < 0.05. (TIFF 704 kb

Additional file 3: of MicroRNA-1468 promotes tumor progression by activating PPAR-γ-mediated AKT signaling in human hepatocellular carcinoma

Figure S3. Overview of miR-1468-induced tumor progression and activates PPAR-γ-mediated AKT signaling in human hepatocellular carcinoma. (TIFF 77 kb