Journal: Stem Cell Research & Therapy
Article Title: Shrimp miR-965 induced the human melanoma stem-like cell apoptosis and inhibited their stemness by disrupting the MCL-1-ER stress-XBP1 feedback loop in a cross-species manner
doi: 10.1186/s13287-020-01734-3
Figure Lengend Snippet: Transcriptional regulation of MCL-1 by XBP1 in a feedback manner in MSLC. a MCL-1 promoter was activated by tunicamycin in MSLC. MCL-1 promoter (− 1000 to + 100 bp) was inserted into the PGL3 vector to generate PGL3-MCL-1 (− 1000) and then the recombinant vector was co-transfected with the internal control pRL-TK into MSLC pre-treated with 3 μg/mL of tunicamycin followed by evaluating the fluorescence in cells. b Prediction of transcriptional factors that activated MCL-1 promoter during ER stress. As predicted, XBP1s was a candidate transcriptional factor that induced MCL-1 promoter activation during ER stress. c MSLC were transfected with pcDNA-XBP1s (or pcDNA3.1-control) followed by assessing the MCL-1 expression. d , e Identifying the XBP1s binding sites in MCL-1 promoter. MSLC were transfected with the MCL-1 wild-type promoter pGL3-MCL-1 (− 1000) (or PGL3 vectors containing truncated promoters or mutant promoter) and pcDNA3.1-XBP1s (or pcDNA3.1-control) as well as pRL-TK. Twenty-four hours later, the fluorescence of cells was evaluated. f Binding of XBP1s to the MCL-1 promoter was determined by chromatin immunoprecipitation (ChIP) in MSLC. ChIP assays were performed using XBP1s antibody and non-immune IgG. − 390 to − 212 bp in MCL-1 promoter was amplified during PCR in ChIP assay. g MCL-1 expression was feedback regulated by XBP1s through ER stress. MSLC were treated with XBP1s-siRNA (or XBPS-siRNA-scrambled) and tunicamycin. Twenty-four hours later, MCL-1 expression was detected
Article Snippet: Cells were transfected with 200 ng of recombinant pGL3 plasmid containing truncated human MCL-1 promoters (or mutant MCL-1 promoter) and 20 ng of Renilla luciferase (pRL-TK) vector (Promega, USA) as well as pcDNA3.1-XBP1s or pcDNA3.1-control.
Techniques: Plasmid Preparation, Recombinant, Transfection, Control, Fluorescence, Activation Assay, Expressing, Binding Assay, Mutagenesis, Chromatin Immunoprecipitation, Amplification
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