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: International Journal of Molecular Sciences

Article Title: Efficient Redirection of NK Cells by Genetic Modification with Chemokine Receptors CCR4 and CCR2B

doi: 10.3390/ijms24043129

Figure Lengend Snippet: Efficient overexpression of CCR4 and CCR2B on NK-92 cells. ( A , D ) CCR4 or CCR2B expression was analyzed by flow cytometry on NK-92 cells transduced with CCR4- or CCR2B-encoding lentiviral particles and selected for 14 days using 2 µg/mL puromycin. T regs and monocytes that naturally express CCR4 and CCR2, respectively, were used for comparison. Histogram overlay shows the intensity of the APC signal of the CCR4/CCR2-specific antibody. Histogram shows one representative out of n = 3 independent experiments; bar chart shows CCR4-/CCR2-positive fractions and their MFIs. Mean values with SD are shown from n = 3 independent experiments. Ctrl NK-92: Untransduced NK-92 cells stained with CCR4 or CCR2 antibodies. ( B , E ) qPCR analysis of CCR4 or CCR2B transduced NK-92 cells versus T regs or monocytes, respectively. Ctrl NK-92: Untransduced NK-92 cells. Mean values with SD are shown from n = 3 independent experiments with three technical replicates each ( C , F ) Immunofluorescence microscopy of CCR4/CCR2B transduced NK-92 cells. Upper panel: bright field, lower panel: fluorescence staining of the respective antibodies directed against CCR4 and CCR2 chemokine receptors. Scalebar represents 10 µm. **: p ≤ 0.01. ****: p ≤ 0.0001.

Article Snippet: γ -Retroviral vector particles were generated by transient transfection of HEK293T cells using TransIT-VirusGEN (MirusBio, Madison, WI, USA) and third generation lentiviral plasmids (hEF1 α -H2B-mVenus-IRES-mCherry PGK-Puromycin vector (Addgene plasmid #99278 [ ]) containing DNA sequences for CCR4, CCR2, CCL22 or CCL2; BaEVRless envelope [ ] protein in pTwist CMV BetaGlobin WPRE Neo vector (Twist Bioscience, South San Francisco, CA, USA), pMDLg/pRRE-gagpol and pRSV-Rev (Addgene #12251 [ ] and #12253 [ ], kindly provided by Didier Trono)).

Techniques: Over Expression, Expressing, Flow Cytometry, Transduction, Staining, Immunofluorescence, Microscopy, Fluorescence