Ctcflos is a core regulator of alternative splicing in brite adipogenesis promoting the expression of thermogenic Prdm16 isoform.Figure 6(A) GO term analysis of genes upregulated in response to Ctcflos knockdown. In dark gray, negative log10 p-values of overrepresented GO terms 24 hours after KD and percentages of genes belonging to the respective GO terms among upregulated and among all genes. Small numbers next to the bars show rank positions of GO terms. In light grey, corresponding values for the same GO terms after 72 hours, hypergeometric test. (B-D) Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis, (B) expressed as Ucp1 relative to Fabp4 mRNA levels, (C) Ucp1 relative to Gtf2b and (D) Fabp4 relative to Gtf2b. Mean values ± SD, n=3 (biological replicates), one-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Effect of Ctcflos KD on Serine/Arginine rich Splicing factor 2 (SC35/SRSF2) and nuclear speckle abundance in Ctcflos tr1 (ASO 1) KD and control cells (KD day 1 of differentiation, analysis after 48h). (E) Immunocytochemistry of SC35. SC35 signal (blue) alone (middle images) and SC35 (blue), DNA staining by DAPI (green) and phase contrast (PC) (gray) combined (outer images). Effect of Ctcflos KD on Serine/Arginine rich Splicing factor 2 (SC35/SRSF2) and nuclear speckle abundance in Ctcflos tr1 (ASO 1) KD and control cells (KD day 1 of differentiation, analysis after 48h). (F) Quantification of SC35 fluorescence signal as whole image fluorescence intensity subtracted by background and normalized to total cell number. Mean and individual values, one representative replicate of three experiments with comparative results (see Fig EV4B and C), unpaired t test, **p< 0.01. (G) Quantification of nuclear speckle number as mean number of SC35 signals per nucleus. Mean and individual values, one representative replicate of three experiments with comparative results (see Fig EV4D and E), unpaired t test, **p< 0.01. Effect of Ctcflos KD on Serine/Arginine rich Splicing factor 2 (SC35/SRSF2) and nuclear speckle abundance in Ctcflos tr1 (ASO 1) KD and control cells (KD day 1 of differentiation, analysis after 48h). (H, I) Correlation of SC35 and UCP1 fluorescence signals across individual cells in (H) control and (I) Ctcflos tr1 (ASO 1) KD cells. Extreme values were excluded to avoid distortion of correlation analysis. One representative replicate of four experiments with comparable results (see Fig S6 F-Q). Pearson correlation, ****p<0.0001. Influence of Ctcflos KD on Prdm16 alternative splicing. (J) Splice graph of Prdm16 and resulting Prdm16 long and short isoforms. Primers used to amplify both Prdm16 isoforms at once by PCR are displayed in orange, primers used to amplify specifically Prdm16 short and Prdm16 long, respectively, are displayed in blue. Influence of Ctcflos KD on Prdm16 alternative splicing. (K) Gel picture of PCR products using exon 16 spanning primers to detect Prdm16 long (upper band (348 bp)) and Prdm16 short (lower band (173 bp)) comparing Ctcflos tr1 (ASO 1) KD and control samples. Influence of Ctcflos KD on Prdm16 alternative splicing. (L) Time course of relative expression of Prdm16 total, long and short isoforms during brite adipogenesis, assessed by qPCR using isoform specific primers. Mean values ± SD, one representative replicate of two experiments with comparable results, Two-way ANOVA (Šídák-test), ***p< 0.001, ****p< 0.0001. Influence of Ctcflos KD on Prdm16 alternative splicing. (M) Levels of Prdm16 long and short isoforms in Ctcflos tr1 (ASO1) KD and control cells. Signal intensities quantified from PCR gel picture subtracted by background and normalized to overall mean signal intensity of the respective biological replicate. Mean and individual values. Two-way ANOVA (Šídák-test), *p<0.05, ****p<0.0001. Influence of Ctcflos KD on Prdm16 alternative splicing. (N) Correlation of Prdm16 short and Ucp1 expression levels across Ctcflos tr1 (ASO1) KD and control samples and within both groups. Pearson correlation, n.s. p>0.05, **p< 0.01, ****p< 0.0001. Influence of Ctcflos KD on Prdm16 alternative splicing. (O) Relative Prdm16 total, (P) Relative Prdm16 long transcript levels 24 hours after Ctcflos KD, comparing Ctcflos tr1 (ASO1) and tr3, 4 (ASO2) KD and control samples, assessed by qPCR, Mean values ± SD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, * p<0.05, **p<0.01, ***p<0.001. Influence of Ctcflos KD on Prdm16 alternative splicing. (Q) Relative Prdm16 short transcript levels 24 hours after Ctcflos KD, comparing Ctcflos tr1 (ASO1) and tr3, 4 (ASO2) KD and control samples, assessed by qPCR, Mean values ± SD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, * p<0.05, **p<0.01, ***p<0.001. Influence of Ctcflos KD on Prdm16 alternative splicing. (R, S) Fold change of Prdm16 short and Prdm16 long (R) ASO1 to NC and (S) ASO2 and NC 24 hours after Ctcflos KD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, ** p<0.01. Influence of Ctcflos KD on Prdm16 alternative splicing. (T) Relative Prdm16 total, (U) Relative Prdm16 long and (V) Relative Prdm16 short transcript levels 72 hours after Ctcflos KD, comparing Ctcflos tr1 (ASO 1) KD and control samples, assessed by qPCR, Mean values ± SD, n=3-4 (biological replicates), unpaired t tests, *p<0.05, ***p<0.001. Influence of Ctcflos KD on Prdm16 alternative splicing. (W, X) Fold change of Prdm16 short and Prdm16 long (W) ASO1 to NC and (X) ASO2 and NC 72 hours after Ctcflos KD, n=3-4 (biological replicates), unpaired t tests, * p<0.05. Global transcriptome analysis of Ctcflos KD impact on alternative splicing (KD day 1 of differentiation, analysis after 24h). (Y) MA-plot of differential alternative splicing analysis using SGSeq algorithm comparing Ctcflos tr1 (ASO 1) KD with controls. Each data point depicts a splice event. Splice events of differential abundance between Ctcflos tr1 (ASO 1) KD and controls (p-adj. <0.05) are colored in red. Global transcriptome analysis of Ctcflos KD impact on alternative splicing (KD day 1 of differentiation, analysis after 24h). (Z) Overview of selected genes involved in adipogenesis, lipid metabolism, mitochondria or splicing, with differential splicing profiles comparing Ctcflos tr1 (ASO 1) KD and control samples. Genes previously described to be spliced into alternative isoforms of varying function in brown adipogenesis are marked in red.