Journal: PLoS ONE
Article Title: Engineering human cell spheroids to model embryonic tissue fusion in vitro
doi: 10.1371/journal.pone.0184155
Figure Lengend Snippet: A-C: Quantification of the relative extent of HPEKp in the seam between spheroids (a metric established to quantify the progression of fusion) with HWJSCs (mean ± SEM for 6 replicates aggregated from 2 independent experiments), normalized to the day 0 time point, upon treatment with either DMSO (0.1%), CH5183284 (20 μM) (A.i), Erlotinib (6.5 μM) (B.i), or SB431542 (10 μM) (C.i). The DMSO control is shown on all three graphs for comparison. Asterisks denote statistical significance relative to DMSO at each time point with two-way ANOVA and Dunnett post-hoc test (α = 0.05). Fluorescent micrographs represent a single representative z-plane (~55–60μm from the bottom of the amalgam of fusing spheroids) after 4 days of fusion in the presence of CH5183284 (A.ii), Erlotinib (B.ii), or SB431542 (C.ii). White asterisks denote HPEKp in the seam between adjacent spheroids. D: Relative cellular ATP content of HPEKp/HWJSC spheroids after 4 days of fusion as quantified using CellTiter Glo 3D assay following the manufacturer’s instructions, normalized to the DMSO control and presented as mean ± SEM for at least 12 replicates aggregated from 2 at least independent experiments. E: Quantification of the relative extent of HPEKp in the seam between spheroids, normalized to the day 0 time point, upon treatment with either vehicle control (white) or human recombinant EGF at 2 ng/mL (green), 10 ng/mL (blue), or 50 ng/mL (black) and presented as mean ± SEM for 14 replicates aggregated from 4 independent experiments. Asterisk denotes statistical significance relative to control at each time point with two-way ANOVA and Dunnett post-hoc test (α = 0.05). F: Area under the curve analysis of EGF-treated spheroids throughout fusion, tabulated using the trapezoid method, presented as mean ± SEM for 14 replicates aggregated from 4 independent experiments. The vehicle control (0 ng/mL EGF) is displayed as 0.1 ng/mL in the figure. Asterisk denotes statistical significance relative to control with one-way ANOVA and Fisher’s least significant difference test (α = 0.05).
Article Snippet: Alternatively, spheroids were treated at day 0 with various concentrations of recombinant human EGF (BioLegend) that was reconstituted at 10 μg/mL in sterile 1% BSA in DPBS, diluted with 1% BSA in DPBS, and added to spheroids at either 2, 10, or 50 ng/mL final concentration in CnT-PR-CC with 1% antibiotic-antimycotic, with the vehicle control containing the same dilution of 1% BSA in DPBS.
Techniques: Control, Comparison, Recombinant
