Global transcriptome profiling reveals Ctcflos-dependent thermogenic regulatory program.Figure 5(A) Experimental setup of two-time point transcriptome analysis. Ctcflos tr1 (ASO 1), tr 3 and 4 (ASO 2) or control treatments were performed at the first day after induction (d1 diff) and cells were harvested for transcriptome analysis 24 hours and 72 hours later. (B), (C) Heat maps of differentially expressed genes between Ctcflos tr1 (ASO 1) KD and control cells (B) 24 hours after the KD (780 up- and 657 down-regulated genes) and (C) 72 hours after the KD (692 up- and 715 down-regulated genes). Row-wise centered log2 normalized counts are displayed. ( (D) Relative expression levels of brite adipocyte marker genes uncoupling protein 1 (Ucp1), cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea), cytochrome c oxidase subunit 7a1 (Cox7a1), peroxisome proliferator activated receptor γ coactivator 1α (Pgc1a) and elongation of very long chain fatty acids-like 3 (Elovl3), comparing Ctcflos tr1 (ASO 1) and tr 3 and 4 (ASO 2) KD with their respective controls. Mean values ± SD, n=6 (biological replicates), unpaired t tests or in grey parenthesis two-way ANOVA (Šídák-test) n.s. p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. (E) GO term analysis of genes downregulated 72 hours after the knockdown. Negative log10 p-values of overrepresented GO terms, hypergeometric test. (F) Transcriptomic effect of Ctcflos tr1 (ASO 1) KD on brown and brite core regulatory gene expression network comparing Ctcflos tr1 (ASO 1) KD with controls. Visualized by Path Visio software. Color code visualizes log2 fold change of genes 24 hours (left part of box) and 72 hours (right part of box) after the KD. Red t-bars represent inhibitory effects, greed arrows represent activating effects. Impact of Ctcflos KD on Ucp1 expression is largely mediated via reduced Prdm16 transcription. (G) Relative transcript levels of PR domain containing 16 (Prdm16) 24 and 72 hours after the knockdown of Ctcflos tr1 (ASO1) compared to control cells, assessed by qPCR, Mean values ± SD, n=3 (biological replicates), two-way ANOVA (Šídák-test), ****p< 0.0001, *p< 0.05. Impact of Ctcflos KD on Ucp1 expression is largely mediated via reduced Prdm16 transcription. (H) Relative Ucp1, (I) Ctcflos tr1 and (J) Prdm16 transcript levels in single and double KD (day1 diff) of Ctcflos tr1 (ASO 1) and Prdm16 (siPrdm16), nontargeting LNA Gapmer (light grey) and siRNA (dark grey) controls, assessed by qPCR, mean and individual values, one-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001. (K-M) Rescue of Ctcflos KD impact on Ucp1 gene transcription by Prdm16 overexpression. Primary iWAT cells infected with Prdm16- or turbo green fluorescent protein (GFP)-expressing viral particles at the second day of proliferation, followed by reverse transfection at the first day of differentiation using nontargeting control (NC) or Ctcflos-targeting ASO1. Gene expression analyzed 72 hours later by qPCR (K) Transcript levels of Ctcflos tr1 relative to Fabp4 (L) Transcript levels of Prdm16 relative to Fabp4 (M) Transcript levels of Ucp1 relative to Fabp4. Mean and individual values. Each graph presents pooled data from two experiments slightly varying virus titers. RM One-way ANOVA (Tukey-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001.