primary human foreskin fibroblasts  (ATCC)

Journal: Nature Communications

Article Title: Selectively expressed RNA molecules as a versatile tool for functionalized cell targeting

doi: 10.1038/s41467-024-55547-6

Figure Lengend Snippet: Using keratin as a selectively expressed target for seRNA activation, keratin-negative human foreskin fibroblasts (HFF) and primary neurons, as well as keratin-positive breast cancer (MCF-7) and glioblastoma cell lines (exemplarily shown for SF188) were stained. Scale bar = 50 µm. a Non-selective and selectively expressed RNA constructs with indicated domain architecture were used for expression analysis in non-target and target cells after transfer as DNA-expression plasmid ( b , c ). Flow cytometry-based eGFP positive cells were either given as a percentage of all cells ( b , c )) or as relative value +/− s.d. using transfection efficiencies of non-selectively expressed RNA constructs as reference. P -values of 0.01 based on a one-way ANOVA test are indicated by two stars ( d ). n = at least 3 independent experiments for ( b – d ). Flow-cytometry analyses of primary cortical neurons (top) and U87 glioblastoma cells (bottom) expressing non-selective and seRNA constructs. The threshold (red line) indicates 99% of all cells in an untransfected control ( e ).

Article Snippet: Human foreskin fibroblasts (HFF) ((SCRC-1041, ATCC), 86HG39 glioblastoma cells (kindly provided by IBG-1, Research Center Juelich, Germany), HeLa cells (CCL-2, ATCC) and HEK293T cells (CRL-3216, ATCC) were cultivated in DMEM Glutamax medium (Gibco) supplemented with 10% FBS (Gibco) + 1x PenStrep (Thermo Fisher).

Techniques: Activation Assay, Staining, Construct, Expressing, Plasmid Preparation, Flow Cytometry, Transfection, Control

Journal: Nature Communications

Article Title: Selectively expressed RNA molecules as a versatile tool for functionalized cell targeting

doi: 10.1038/s41467-024-55547-6

Figure Lengend Snippet: seRNA Δ3-5 ( a ) and seRNA ( b ) constructs with either eGFP or ca-Caspase3 were expressed in indicated cell types. Target-specific expression of eGFP (green) and cell viability was analyzed by light microscopy and flow cytometry, given in %. For ( a ) and ( b ) n = 3 or more. c Western blot analysis of HFF and U87 cells 24 h after transfection with seRNA-ca-Caspase3. Tubulin was used as an internal marker. n = 3. d Indicated glioblastoma cell lines were transfected with seRNA-ca-Caspase3 and tested for survival after 24 h. Toxicity is indicated in phase contrast. Numbers show relative killing efficiencies based on flow cytometry corrected by cell line-specific transfection efficiencies received with seRNA Δ3-5 −eGFP. n = 10 or more independent experiments. e seRNA-HBx expression plasmids were co-transfected with control and HBx expression plasmids. Maximal killing efficiencies were determined by using a constitutive ca-Caspase3 expression plasmid. 24 h after transfection, killing efficiencies were determined. n = 4 or more independent experiments. A student’s t test based significance level of 0.001 is displayed with three stars. Only positive relative killing efficiencies are illustrated in the figure. f Switch from plasmid-based expression to IVT seRNA. Indicated in vitro transcribed seRNAs were transfected into non-target (primary neurons) and U87 glioblastoma cells and analyzed for GFP expression and cell death. As a control, an IVT GFP-mRNA was used. Absolute GFP-transfection efficiencies and killing efficiencies are indicated based on flow cytometry analysis. n = 3 independent experiments. g Stably red nuclear labeled MCF-7 breast cancer cells and cytoplasmic GFP labeled human fibroblasts (HFF) without seRNA treatment and treatment for indicated times with seRNA-ca-Caspase3. Nuclear shapes are magnified for indicated areas. All scale bars = 200 µm except for ( g ) with 50 µm. n = 3.

Article Snippet: Human foreskin fibroblasts (HFF) ((SCRC-1041, ATCC), 86HG39 glioblastoma cells (kindly provided by IBG-1, Research Center Juelich, Germany), HeLa cells (CCL-2, ATCC) and HEK293T cells (CRL-3216, ATCC) were cultivated in DMEM Glutamax medium (Gibco) supplemented with 10% FBS (Gibco) + 1x PenStrep (Thermo Fisher).

Techniques: Construct, Expressing, Light Microscopy, Flow Cytometry, Western Blot, Transfection, Marker, Control, Plasmid Preparation, In Vitro, Stable Transfection, Labeling