Journal: Cells

Article Title: Hypoxia Affects Stem Cell Fate in Patient-Derived Ileum Enteroids in a HIF-1α-Dependent Manner

doi: 10.3390/cells15010031

Figure Lengend Snippet: HIF-1α stabilization by roxadustat in normoxia reduces stemness and proliferation in human ileum-derived enteroids. Enteroids were treated with 100 μM roxadustat (Rox, purple) to stabilize HIF-1α protein expression in normoxia and compared to solvent (DMSO)-treated enteroids incubated in normoxia (N, red) or hypoxia (H, blue). ( a ) To confirm HIF-1α stabilization, enteroids were lysed 24 h post-treatment, and HIF-1α protein expression was assessed via Western blotting, and β-actin was used as a loading control. A representative Western blot image of enteroids derived from donor 1 is shown. ( b , c ) Gene expression of the HIF-1α target gene CA9 ( b ) and the stem cell-associated gene OLFM4 ( c ) from all three donors was assessed using qRT-PCR 48 h post-treatment. The graphs depict the mean ± SEM ( n = 3 independent experiments), and a 1-way ANOVA with multiple comparisons was applied. p < 0.05 = *, <0.01 = **, <0.001 = ***, <0.0001 = ****. ( d ) Enteroids from all three donors were seeded into untreated high-Wnt media. After two days, media were exchanged and enteroids were incubated with 100 μM roxadustat (purple) or a solvent control (DMSO, red) in normoxia. Brightfield images were acquired at the indicated time points post-media change using a ZEISS Celldiscoverer 7 microscope using a 5× 1× magnification. Enteroid growth was quantified by counting the number of enteroids per field of view. Means ± 95% confidence intervals are depicted from 8 fields of view. A 2-way ANOVA with multiple comparisons was applied. p ≥ 0.05 = ns (not significant), <0.05 = *, <0.01 = **. ( e , f ) Enteroids from donor 1 were seeded into normoxia (red) into untreated high-Wnt media. After two days, media were exchanged, and enteroids were incubated with 100 μM roxadustat (purple) or a solvent control (DMSO, red) for the indicated incubation spans. On day 5, enteroids were split into untreated high-Wnt medium, and imaging was performed on day 7. Brightfield images were acquired two days post-splitting using a ZEISS Celldiscoverer 7 microscope using a 5× 0.5× magnification. ( e ) Schematic depicting the experimental setup to assess enteroid formation efficiencies. ( f ) Enteroid formation efficiency was determined by quantifying the number of enteroids. Means ± 95% confidence intervals are depicted from ≥7 fields of view per independent experiment ( n = 3 independent experiments). A 2-way ANOVA with multiple comparisons was applied. p < 0.01 = **, <0.0001 = ****.

Article Snippet: Roxadustat (FG-4592, MCE #HY-13426) was dissolved in DMSO and applied at 100 μM.

Techniques: Derivative Assay, Expressing, Solvent, Incubation, Western Blot, Control, Gene Expression, Quantitative RT-PCR, Microscopy, Imaging