![]() ![]() ( F–H) L Cells were transfected or not with mCherry-tagged TFAP2 family members for 48 hr before fixation, labeling and imaging as in Figure 1A. Representative images are shown in E (nuclei in magenta lipid droplets in green). Arrow indicate position of 50 kDa marker. Inset: TFAP2A protein levels of each clone determined by Western blot. In D), the number of lipid droplets was quantified as in Figure 1A and is expressed as fold induction relative to the control cells in five independent experiments ± SEM. The corresponding knock-out clones as well as control cells were treated with Wnt3a-conditioned media for 24 hr. ( D–E) HeLa-MZ cells were transfected with targeted CRISPR/Cas9 plasmids against TFAP2A. RNA was isolated and analyzed by qPCR using primers to the indicated genes, and that data are expressed relative to the non-target control and are presented as the mean mRNA amounts of two to five independent experiments ± SEM. ( C) L cells were treated with siRNAs against both TFAP2A and TFAP2C or with non-targeting controls as in ( A), before the addition of Wnt3a- or control-conditioned media for an additional 24 hr. 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. ( 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. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |