Journal: The Journal of Biological Chemistry
Article Title: ERAD deficiency promotes mitochondrial dysfunction and transcriptional rewiring in human hepatic cells
doi: 10.1074/jbc.RA120.013987
Figure Lengend Snippet: Genetic ablation of ERAD sensitizes HepG2 cells to TNFα– and IL-12–induced cell death. A, diagram illustrating CRISPR/Cas9-mediated editing of the Sel1L gene in HepG2 cells. F, R1, and R2 represent PCR primers used for genotyping of Sel1L-edited cells. Black bars represent exons. B , top , PCR analysis of Sel1L-normal (Sel1L +/+ ) and deleted (Sel1L −/− ) cells. Bottom, Western blotting analysis of SEL1L expression in Sel1L +/+ and Sel1L −/− cells; the upper band is a nonspecific band. C, average cell death level of Sel1L +/+ and Sel1L −/− cells as indicated by the mean PI fluorescence intensity. Sel1L −/− ( 1-3 ) represent three independent HepG2 cell clones. D–F, MTT assays of Sel1L +/+ and Sel1L −/− cells after treatment with increased concentrations of TNF-α ( D ), IL-12 ( E ), and TNFα + IL-12 ( F ). G and H, fluorescence reporter assay of ER stress in TNF-α ( G ) and IL-12 ( H ) treated Sel1L +/+ and Sel1L −/− cells. I, MTT assay of Sel1L +/+ and Sel1L −/− cells after treatment with TNFα or TNFα + Nec-1. J , FITC and PE dual channel FACS assay of Sel1L +/+ and Sel1L −/− cells after treatment with PBS (Mock), TNF-α, TNF-α + Ru360, or TNF-α + NAC. All data are mean ± S.E. ( n = 3). *, p < 0.05; **, p < 0.01 by Student's t test.
Article Snippet: For Cas9-mediated gene editing, sgRNAs flanking exon 6 were designed using the Optimized CRISPR Design tool ( https://www.synthego.com/products/bioinformatics/crispr-design-tool ) and cloned into the expression vector pGL3-U6-2sgRNA.
Techniques: CRISPR, Western Blot, Expressing, Fluorescence, Clone Assay, Reporter Assay, MTT Assay
