Fig 2: Knockdown of COUP-TFII in THRA-PV myoblasts improves myoblast differentiation. (a) Phase contrast images of myogenic differentiation of WT and THRA-PV myoblasts transfected with a scramble (siSCRAMBLE) siRNA or with a siRNA for COUP-TFII (siCOUP-TFII) before (d0) and after 1 (d1), 2 (d2) and 3 (d3) days of induction. Scale bar = 1000 µm (b) Immunostaining for Myosin heavy chain (MyHC) three days after induction of differentiation of WT and THRA-PV myoblasts transfected with a scramble (siSCRAMBLE) siRNA or an siRNA for COUP-TFII (siCOUP-TFII). MyHC in red, DAPI in blue. Scale bar = 200 µm (c) Fusion index, (d) number of nuclei per myotube and (e) number of myotube per field for control (siSCRAMBLE) and COUP-TFII silenced (siCOUP-TFII) in primary myoblasts from WT and THRA-PV mice three days after induction of differentiation. Data are shown as mean ± SEM, n = 3. One-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Student’s t Test ## p < 0.01.

Fig 3: COUP-TFII interacts with THRA and modulates its binding to TREs. (a) Co-immunoprecipitation of COUP-TFII and THRA in nuclear proteins from skeletal muscle of WT and THRA-PV mice; COUP-TFII was used as bait. (b) Co-immunoprecipitation of COUP-TFII and THRA in nuclear proteins isolated from C2C12 myoblast (CTRL) and C2C12 myoblasts after COUP-TFII silencing (siCOUP-TFII); COUP-TFII was used as bait. ‘INPUT’ is the total nuclear fraction, ‘1st wash’ is the negative fraction after precipitation, ‘3rd wash’ is the last wash, ‘CoIP’ is the positive fraction after 3 washes. (c) ChIP- qPCR analysis of the recruitment of THRA to the TRE in the MyoD1, Ucp3 and Ppargc1a promoter regions. Bar graph showed the enrichment of DNA fragments pulled down by THRA antibody. Data are shown as mean ± SEM. Two-tailed Student’s t test; **p < 0.01, ****p < 0.0001. Fold Change is indicated.

Fig 4: RNA-sequencing profiling of THRA-PV and WT myoblast after COUP-TFII silencing. (a) 3D Principal Component Analysis plot (PCA) for WT and THRA-PV myoblasts before (WT and THRA-PV) and after (siCOUP-TFII WT and siCOUP-TFII THRA-PV) COUP-TFII silencing. (b) Heatmap and hierarchical clustering analysis of the 3072 differentially expressed genes among WT, WT siCOUP-TFII, THRA-PV, and THRA-PV siCOUP-TFII. Downregulated genes are represented in green, upregulated genes are represented in red. (c) Number of total, upregulated and downregulated differentially expressed genes in THRA-PV myoblasts (THRA-PV vs WT) and after COUP-TFII silencing in WT (siCOUP-WT vs WT) or THRA-PV (siCOUP-TFII THRA-PV vs THRA-PV) myoblasts and siCOUP-TFII THRA-PV vs WT (d) Volcano plot representation of differential expression analysis of genes after COUP-TFII silencing in WT and THRA-PV myoblasts. The x-axis shows log10fold-changes in expression and the y-axis the log10 odds of FDR. (e) Venn diagrams representing summary of the total differentially expressed, upregulated and downregulated genes after COUP-TFII silencing in WT and THRA-PV myoblasts.

Fig 5: GSEA profiling of COUP-TFII silenced WT and THRA-PV myoblasts. (a) Heatmap representation of overall gene expression for thyroid hormone signaling, myogenic markers and ECM markers for differentially expressed genes between siCOUP-TFII THRA-PV and THRA-PV (p < 0.05, and |FC|> 2.0). Black dots indicate THRA-PV vs WT DEGs, white dots indicate DEGs between siCOUP-TFII WT and WT, grey dots indicate DEGs between siCOUP-TFII THRA-PV and WT. (b) Disease and functional analysis using the IPA knowledgebase of the THRA-PV vs WT and THRA-PV siCOUP-TFII vs THRA-PV groups focusing on the ‘Skeletal and Muscular System Development and Function’ category. Diseases and functions with z-scores > 2 or z-scores = 2, and p-value < 0.01 were selected. (c) Enriched gene sets from siCOUP-TFII THRA-PV vs THRA-PV and THRA-PV vs WT comparisons. The number in brackets represents the ranking of the gene sets after GSEA of siCOUP-TFII THRA-PV vs THRA-PV. NES: normalized enrichment score. Only gene sets with FDR > 0.25 were considered. (d) Heatmap representation of genes related to ‘Muscle Cell Migration’ and ‘Muscle Cell Proliferation’ GO terms after leading edge analysis of DEGs of siCOUP-TFII THRA-PV cells. Black dots indicate THRA-PV vs WT DEGs, white dots indicate DEGs between WT siCOUP-TFII and WT, grey dots indicate DEGs between siCOUP-TFII THRA-PV and WT.