Journal: Oncotarget

Article Title: Targeting endogenous DLK1 exerts antitumor effect on hepatocellular carcinoma through initiating cell differentiation

doi: 10.18632/oncotarget.12214

Figure Lengend Snippet: ( A ) Quantitative RT-PCR was used to evaluate the efficacy of the inducible shRNA against endogenous DLK1 through examining DLK1 mRNA in Huh-7 and Hep3B cells with stably ectopic plasmids as Dox treatment. The Dox-induced DLK1 knockdown may inhibit colony formation ( B ), anchorage-independent growth, as shown by in vitro soft-agar assay ( C ), and spheroid colony formation ( D ) derived from Huh-7 and Hep3B cells. Plasmid shLUC encoding shRNA against luciferase was used as control. The representative images are shown and statistical analyses are performed (right). The colony formation data were analyzed by one-way ANOVA. For comparison with control, Student's t -test was performed. Data of soft-agar assay were analyzed by unpaired, two-tailed Student's t -test was performed for shDLK-1-Dox versus control ( n = 3, for both two cell lines). The spheroid formation data were also first analyzed for both two cell lines by one-way ANOVA, followed by comparison with control by Student's t -test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Plasmid Tet-pLKO-neo (Addgene plasmid #21916) was used to construct recombinant vector generating small hairpin RNA (shRNA) against human DLK1 , the stuffer DNA was removed from plasmid Tet-pLKO-neo by AgeI/EcoRI enzyme digest and then replaced with double-stranded oligonucleotides encoding the desired shRNA and AgeI/EcoRI sites.

Techniques: Quantitative RT-PCR, shRNA, Stable Transfection, Knockdown, In Vitro, Soft Agar Assay, Derivative Assay, Plasmid Preparation, Luciferase, Control, Comparison, Two Tailed Test

Journal: Oncotarget

Article Title: Targeting endogenous DLK1 exerts antitumor effect on hepatocellular carcinoma through initiating cell differentiation

doi: 10.18632/oncotarget.12214

Figure Lengend Snippet: ( A ) The growth curves of xenograft tumors derived from Huh-7 and Hep3B cells with inducible DLK1 knockdown; ( B ) The images of xenograft tumors with inducible DLK1 knockdown; ( C ) Weight of xenograft tumors with inducible DLK1 knockdown was significantly reduced. In this study, nude mice were given 1 mg/ml of Dox for 15 days as xenograft tumors reached 4 mm in diameter, and shLUC encoding shRNA against ectopic luciferase was used as control. * p < 0.05; ** p < 0.01.

Article Snippet: Plasmid Tet-pLKO-neo (Addgene plasmid #21916) was used to construct recombinant vector generating small hairpin RNA (shRNA) against human DLK1 , the stuffer DNA was removed from plasmid Tet-pLKO-neo by AgeI/EcoRI enzyme digest and then replaced with double-stranded oligonucleotides encoding the desired shRNA and AgeI/EcoRI sites.

Techniques: Derivative Assay, Knockdown, shRNA, Luciferase, Control

Journal: Oncotarget

Article Title: Targeting endogenous DLK1 exerts antitumor effect on hepatocellular carcinoma through initiating cell differentiation

doi: 10.18632/oncotarget.12214

Figure Lengend Snippet: ( A ) Scheme and representative pseudocolor images of orthotopic xenograft tumors within nude mice at 3 and 8 weeks after inoculation. 2 × 10 6 Huh-7 cells expressing luciferase were injected into the left liver lobe of nude mice. Luciferase imaging of these mice was performed once a week until tumor luminescence was observed, and then recombinant adenoviruses were administered at week 3 post cell implantation by tail vein injection. Luminescence imaging of these mice was performed at week 8 after inoculation. ( B ) Tumor luminescence was analyzed. Data represent the mean of 4 mice for each group ± sem (error bars). ( C ) Liver/body weight ratio was determined when these nude mice were sacrificed. ( D ) Western blotting assay was used for evaluating the efficiency of adenovirus-mediated DLK1 knockdown in these orthotopic xenograft tumors. Adenoviral vector containing scrambled shRNA was used as negative control. * p < 0.05.

Article Snippet: Plasmid Tet-pLKO-neo (Addgene plasmid #21916) was used to construct recombinant vector generating small hairpin RNA (shRNA) against human DLK1 , the stuffer DNA was removed from plasmid Tet-pLKO-neo by AgeI/EcoRI enzyme digest and then replaced with double-stranded oligonucleotides encoding the desired shRNA and AgeI/EcoRI sites.

Techniques: Expressing, Luciferase, Injection, Imaging, Recombinant, Western Blot, Knockdown, Plasmid Preparation, shRNA, Negative Control

Journal: Oncotarget

Article Title: Targeting endogenous DLK1 exerts antitumor effect on hepatocellular carcinoma through initiating cell differentiation

doi: 10.18632/oncotarget.12214

Figure Lengend Snippet: ( A – C ) The effect of early adenovirus-mediated DLK1 knockdown on DEN-induced mouse HCC tumors. (A) Scheme of adenovirus-mediated mDLK1 knockdown in DEN-induced mouse HCC model. BL/6 mice were treated with DEN plus eight injections of TCPOBOP, where adenovirus administration for four times in total was performed via tail vein injection at an interval of 3 weeks. ( B ) Representative images of livers and H&E-stained sections from the mice were shown. And (C) numbers of tumors (> 2 mm in diameter), size of largest tumors, and liver:body weight ratio were statistically analyzed. ( D – F ) The effect of later adenovirus-mediated Dlk1 knockdown on DEN-induced mouse HCC tumors. (D) Scheme of adenovirus-mediated mDLK1 knockdown in the mouse HCC model. BL/6 mice were treated with DEN plus eight injections of TCPOBOP and either received Dlk1 shRNA containing adenovirus or control injection starting at week 18 for a total of five times at an interval of 3 weeks. Mice were sacrificed at week 34. (E) Representative images of livers and H&E-stained sections from the mice were shown. (F) Numbers of tumors, size of largest tumors and liver:body weight ratio were statistically analyzed. Data are represented as means ± SD. * p < 0.05; ** p < 0.01.

Article Snippet: Plasmid Tet-pLKO-neo (Addgene plasmid #21916) was used to construct recombinant vector generating small hairpin RNA (shRNA) against human DLK1 , the stuffer DNA was removed from plasmid Tet-pLKO-neo by AgeI/EcoRI enzyme digest and then replaced with double-stranded oligonucleotides encoding the desired shRNA and AgeI/EcoRI sites.

Techniques: Knockdown, Injection, Staining, shRNA, Control