Cells treated with non-target siRNAs or with siRNAs to both TFAP2A and TFAB2C are shown in panel B (nuclei in magenta lipid droplets in green). In ( A), data are presented as the normalized mean number of lipid droplets per cell of 5 independent experiments ± SEM. Cells were then fixed, labeled, imaged and analyzed by automated microscopy as in Figure 1A. ( A–B) L Cells were treated with siRNAs against the indicated targets for 48 hr before the addition of Wnt3a-conditioned medium for an additional 24 hr. In this figure, pValues are indicate as: *,<0.05 **, <0.005, and n.s., not significant. Green bars, control-conditioned media (control CM) Red bars, Wnt3a-conditioned media (Wnt3a CM). Nuclei are in magenta, and lipid droplets in green. Panel I illustrates the effects of compounds that induce droplet formation (left column) or that do (Trichostatin A) or do not (niclosamide, hexachlorophene) inhibit droplet formation (right column) in Wnt3a-treated cells. The number of droplets per cell was counted and the zscores established, in order to quantify the ability of each compounds to induce lipid droplets in untreated cells (H, left panel), or to inhibit lipid droplet formation in Wnt3a-treated cells (H, right panel). Cells were incubated for 24 hr with Wnt-3a- or control-conditioned media for 24 hr in the presence of the compounds at 1 µM and 10 µM, fixed, labeled with BODIPY (lipid droplets) and Hoechst 33342 (nuclei) and imaged by automated microscopy. ( H–I) High-content image-based screen of a library of compounds that affect the Wnt pathway in HeLa-MZ cells. The data are color-coded from a high (light) to a low (dark) number of lipid droplets induced by each Wnt ligand. Data are normalized to the empty vector control and were tested for significance and are presented as the mean number of lipid droplets per cell of two independent replicates of the screen ± SEM, normalized to the control condition. ( G) L Cells were transfected with plasmids containing each of Wnt ligand for 48 hr, imaged and analyzed as in ( A). Color indicates ability to induce lipid droplets as detailed in ( G). ( F) Evolutionary relationship of the 19 Wnt ligands. ( E) L cells were incubated with the indicated compounds together with Wnt3a for 24 hr, processed and analyzed as in ( A) and the data are presented as the mean number of lipid droplets per cell of five independent experiments ± SEM, normalized to the control condition. Efficient silencing was confirmed by qPCR ( Figure 1-figure supplement 1B) and the data are presented as the mean number of lipid droplets per cell of 3 independent experiments ± SEM, normalized to the control condition. ( C–D) As in ( A), except that HeLa-MZ cells ( C) or L Cells ( D) were transfected with siRNAs against the indicated targets for 48 hr, before the addition of Wnt3a. In ( B) the number of lipid droplets was quantified by automated microscopy (bar graph), and the data are presented as the mean number of lipid droplets per cell of five independent experiments ± SEM, normalized to the control condition. Cells were fixed, labeled with BODIPY (lipid droplets, green) and Hoechst 33342 (nuclei, magenta), and imaged by light microscopy.
#Microsynth primer iso#
Microsynth is ISO 9001:2015-, ISO 13485:2016- (oligonucleotide synthesis, kit production), ISO/IEC 17025:2017 (STS 0429) - (NGS, Sanger sequencing, fragment length analysis) and GMP certified (Sanger sequencing).( A–B) HeLa-MZ cells were transfected with plasmids encoding wild-type or a S9A mutant of GSK3B 24 hr before the addition of Wnt-3a- or control-conditioned media (CM) for a further 24 hr. Microsynth employs >100 people and has branch offices in Germany (Microsynth Seqlab), France (Microsynth France), Austria (Microsynth Austria) and Switzerland (ecogenics). CRISPR on/off target GxP analyses, rAAV characterization) › DNA/RNA isolations (all sources, including high-throughput format) › Genotyping (fragment length analysis, cell line characterization, STR marker development, GBS)
› Next generation sequencing (de novo & re-sequencing at the genome level, RNA-Seq, metagenome and amplicon sequencing, bioinformatics, etc.) › DNA Sanger sequencing of all types (incl. › antisense oligonucleotides as well as siRNAs for drug discovery OEM kit production) for molecular diagnostics › DNA/RNA oligonucleotides (unmodified, modified, various purity grades) for various R&D applications The main activities are divided into the following three business areas: Microsynth (founded in 1989) is a leading company in Europe in the field of DNA/RNA syntheses & analyses.
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