Journal: The Biochemical journal

Article Title: FoxA2 and RNA Pol II Mediate Human Islet Amyloid Polypeptide Turnover in ER-stressed Pancreatic β-cells

doi: 10.1042/BCJ20200984

Figure Lengend Snippet: RIN-m5f cells were transduced with empty vector (EV), hIAPP or rIAPP encoding lentivirus particles for 48h. (A) Immuno-confocal microscopy optical section (1μm-Z plane) of RIN-m5f cells transduced with empty vector (EV) and hIAPP encoding lentivirus. Cells were co-stained with hIAPP-specific IAPP monoclonal antibody (red) and DAPI (blue). (B) Representative single (1μm-Z plane) fluorescence confocal sections of hIAPP intracellular accumulation sites in hIAPP lentivirus-transduced cells. Arrows denote hIAPP’s nuclear locations in micrographs. Bars, 5μm.

Article Snippet: Lentiviral particle production and lentivirus-mediated transduction Flag-tagged ORF sequences for pre-pro-hIAPP and rIAPP, flanked with restriction enzyme sites (5’ SgfI and 3’ Mlu1) were synthesized using gBlock gene synthesis (Integrated DNA Technology) and cloned into a third-generation lentivirus destination vector (pLenti-C-Myc-DDK-IRES-Puro, Origene, cat# PS100069).

Techniques: Transduction, Plasmid Preparation, Confocal Microscopy, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Transcriptional repression of PTEN in neural cells using CRISPR/dCas9 epigenetic editing

doi: 10.1038/s41598-020-68257-y

Figure Lengend Snippet: The dCas9-KRAB system represses PTEN in the HEK 293T cell line and human mesenchymal precursor cells (hMPCs). dCas9-KRAB was stably expressed with no gRNA, with individual gRNAs targeting the PTEN proximal promoter and 5′ untranslated region (UTR), with a mix of gRNAs C and D, or with a mix of all four gRNAs. Empty vector and the combination of four shRNAs targeting the PTEN transcript were also stably expressed by lentiviral transduction. ( A , C ) Fold change in PTEN mRNA expression in qRT-PCR relative to empty vector in the HEK 293T cell line ( A ) and hMPCs ( C ). *p < 0.05, **p < 0.01 (Kruskall–Wallis test with Dunn’s multiple comparisons test to compare each condition to empty vector control), n = 3, error bars show standard error of the mean (SEM). ( B , D ) Western blot of PTEN and GAPDH in HEK 293T ( B ) and hMPCs ( D ). Conditions correspond to qRT-PCR data labeled above.

Article Snippet: The third-generation lentiviral packaging plasmids pMDLg/pRRE (Addgene plasmid #12251) and pRSV-Rev (Addgene plasmid #12253), and envelope plasmid pMD2.G (Addgene plasmid #12259; all gifts from Didier Trono), were used for lentiviral production.

Techniques: Stable Transfection, Plasmid Preparation, Transduction, Expressing, Quantitative RT-PCR, Control, Western Blot, Labeling