Figure 2. Incorporation of SILAC isotopes in organoid cultures and effect of the class I HDAC inhibitor CI994 on organoid growth and development.

(a) Organoids were grown continuously in SILAC medium containing arginine and lysine with light isotopes of carbon, hydrogen and nitrogen (i.e. ¹²C¹⁴N) (light), or with medium containing L-arginine-¹³C₆-¹⁵N₄ and L-lysine-¹³C₆-¹⁵N₂ (heavy). Intestinal organoids were amplified and split every five days. From days 10 to 30, levels of isotope incorporation were evaluated by mass spectrometry analysis by combining equal amounts of organoid lysates prior to sample processing, protein identification and quantification. (b) Micrograph of intestinal organoids before and after 5-day treatment with either DMSO or CI994 at 5 μM. Scale bars represent 100 μm. (c) Evaluation of reproducibility between the different assays: ratios from one experimental repeat (N1) CI994 5 μM/DMSO (x axis) versus the second repeat (N2) CI994 5 μM/DMSO (y axis) for identified proteins. A correlation between N1 and N2 CI994 5 μM/DMSO experiments was observed with Pearson correlation (R = 0.848).(d) The average CI994/DMSO ratios of proteins over the average peptide intensities, to identify proteins with an increase or decrease in expression following treatment with the HDAC inhibitor CI994.