Figure 3.

Hierarchical clustering of phosphorylated proteins in hyperammonemic myotubes reveals distinct temporal differences

Hierarchical clustering, heatmap, and cluster analysis of differentially phosphorylated phosphosites (DPPS) and differentially expressed phosphoproteins (DEpP) in untreated (UnT) myotubes or those treated with 6h or 24h of 10mM ammonium acetate (AmAc).

(A) Heatmap of DPPS with hierarchical clustering into 5 groups based on the direction of temporal change: Persistent increase (DPPS that have increased phosphorylation at both 6hAmAc and 24hAmAc compared to UnT), Late increase (DPPS that have no change at 6hAmAc but have increased phosphorylation at 24hAmAc compared to UnT), Late decrease (DPPS that have no change at 6hAmAc but have decreased phosphorylation at 24hAmAc compared to UnT), Transient change (DPPS that have increased phosphorylation at 6hAmAc but have decreased phosphorylation at 24hAmAc compared to UnT), and Persistent decrease (DPPS that have decreased phosphorylation at both 6hAmAc and 24hAmAc compared to UnT).

(B–F) Pathway enrichment of DEpP in each of the 5 identified clusters: Persistent increase, Late increase, Late decrease, Transient change, and Persistent decrease. All experiments were done in n = 3 biological replicates (one 24hAmAc replicate was removed from downstream analyses because of outlier status). Statistical significance cutoff for DEpP/DPPS was padj<0.05 (Student’s t test with Benjamini-Hotchberg correction). Significance for canonical pathways was the default-log(p value) ≥1.3 using a right-sided Fisher exact test.