Fig. 1: Cachexia reduces PKA activity and impairs CREB1-mediated transcription of mitochondria-related genes in skeletal muscle.
a, IPA of DEGs (filtered for P adjusted value <0.05) in gastrocnemius from C26 13 days (C26 13d) versus control (ctr). P adjusted value-based ranking top 10 terms are shown. n = 7 ctr, n = 7 C26 13d. b, Heatmap generated with bidirectional hierarchical clustering (gene- and sample-based) of normalized reads per kilobase per million mapped reads (RPKM) of cAMP signalling-related selected DEGs from RNA-seq in gastrocnemius from C26 13d versus ctr mice (for all genes listed: P adjusted value <0.01 and base mean >600). n = 7 ctr, n = 7 C26 13d. c, Serine/threonine kinase activity measured through PamGene peptide array in gastrocnemius muscle lysates from C26 tumour-bearing mice (day 13) versus non-tumour-bearing mice (ctr). Kinases are ordered by their kinase score, and the bubble colours represent the median kinase statistic score, calculated as the sum of the significance and specificity scores. Top 20 inhibited kinases (that is, with negative median kinase statistics) are shown. n = 6 mice for each experimental group. Red arrows highlight the PKA family members. d,e, Representative blot (d) and densitometric analysis of phospho(serine/threonine)-PKA substrates over GAPDH (e) of whole gastrocnemius lysate from non-tumour-bearing mice (ctr) or C26 13d. n = 5 for each experimental group. Two-tailed t-test. f, Volcano plot showing overall results of differential binding analysis of p-CREB1S133 ChIP-seq in C26 10d versus ctr. Each dot is a peak and is coloured according to differential binding status (based on P value ≤0.05 and log2FC lower or greater than 0): lower in C26, higher in C26, common (not changed). Differential binding analysis was performed using DiffBind (Methods). g, Heatmap of peak intensity from p-CREB1S133 ChIP-seq analysis in C26 10d versus ctr. Differential analysis defines two groups: lower or higher in C26. Signal is visualized within a ±2-kb window centred on the peak. Both quadriceps for each mouse were pooled together for the ChIP assay. n = 2 mice per experimental group. Two independent experiments with matched control and C26 samples were performed. h, Integrative Genomics Viewer (IGV) images showing representative p-CREB1S133 ChIP signals among the ‘lower in C26’ peaks (red bar) (Supplementary Table 3) aligned across the indicated gene. n = 2 mice per experimental group. i, Normalized RPKM of selected p-CREB1S133 target genes differentially expressed in RNA-seq of gastrocnemius from C26 13d versus ctr mice (Supplementary Table 1). n = 7 ctr, n = 7 C26 13d. Numbers indicate adjusted P values from DESeq2 analysis. Data are presented as mean values ± s.e.m. in e and i, and only significant P values (<0.05) are annotated in the graphs.
