lentiviral vector plko 1 shrna control shctrl  (Addgene inc)

Journal: Frontiers in Oncology

Article Title: An inflammation-related gene landscape predicts prognosis and response to immunotherapy in virus-associated hepatocellular carcinoma

doi: 10.3389/fonc.2023.1118152

Figure Lengend Snippet: Validation of expression and tumorigenicity of hub genes. (A) qRT-PCR validation of MEP1A, CCL20, ADORA2B, TNFSF9, ICAM4, and SLC7A2 in HCC and normal plasmas. (B, C) The mRNA expression level of CCL20 and SLC7A2 in HCC cell lines (SMMC7721, Huh7, HepG2, and HCCLM3) and the normal liver cell lines (WLR68, and LO2) was indicated by qRT-PCR assays. (D) The protein and (E) mRNA expression of CCL20 and SLC7A2 was analyzed by western blotting and RT-PCR in stable SMMC-7721 cells expressing-shRNA against luciferase or CCL20 and over SLC7A2. (F) Morphology of HCC cells after knockdown of CCL20 and overexpression of SLC7A2. (G) Tumorigenicity of SMMC7721-shCtrl cells and SMMC7721-shCCL20/overSLC7A2 cells in nude mice. (H) Tumor volume was measured every 3 days after tumor formation in nude mice injected with SMMC7721 cells transfected with shCtrl or shCCL20/overSLC7A2. (I, J) Tumor weight was measured in nude mice injected with SMMC7721 cells transfected with shCtrl or shCCL20/overSLC7A2 after 30 days. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.0001, ns, not significant.

Article Snippet: Full-length SLC7A2 cDNA was synthesized and cloned into the pCS-CG vector (Addgene, Cambridge, MA, USA). shRNA sequences specifically against CCL20 (shCCL20) and control-shRNA against luciferase (shCtrl) were expressed from pLKO.1-puro (Addgene, Cambridge, MA, USA).

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction, shRNA, Luciferase, Knockdown, Over Expression, Injection, Transfection