Ctcflos is essential for brite adipocyte differentiation and thermogenic function.Figure 4(A) Time course of relative Ctcflos expression across proliferation, induction and differentiation of primary brite adipocytes, assessed by qPCR. One representative curve of three experiments with comparative results, mean values ± SD of 3 technical replicates, one-way ANOVA, *p< 0.05, ****p<0.0001. (B) Design of Ctcflos knockdown (KD) experiments. Ctcflos KD was performed at the first day after induction and cells were harvested and analyzed 72 hours later. (C, D) Efficiency of Ctcflos KD by (C) LNA Gapmer ASO1, ASO3 and ASO4 targeting Ctcflos transcript 1 and (D) LNA Gapmer ASO2, ASO5 and ASO6 targeting Ctcflos transcript 3 and 4 compared to nontargeting controls, assessed by qPCR. Mean values ± SD, n=3 (biological replicates), one-way ANOVA (Šídák-test ), ***p<0.001, ****p<0.0001. (E) Relative Ucp1 transcript levels in response to Ctcflos tr1, 3 and 4 KD by ASO 1, 3, 4 and 2, 5 6, respectively, compared to nontargeting controls, assessed by qPCR. Mean values ± SD, n=3-4 (biological replicates), one-way ANOVA (Šídák-test), ***p<0.001, ****p<0.0001. (F) Relative expression levels of cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea) and cytochrome c oxidase subunit 7a1 (Cox7a1) in Ctcflos tr1, 3 and 4 KD compared to control samples, assessed by qPCR. Mean values ± SD, n=3 (biological replicates)-4, unpaired t tests or in grey parenthesis two-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001. (G) Western blot analysis of UCP1 protein abundance in response to Ctcflos tr1, 3 and 4 KD by ASO 1 and 2, respectively compared to nontargeting controls. Actin-β as loading control. Arrow heads mark UCP1 specific bands. (H) Immunocytochemistry of UCP1 protein in response to Ctcflos tr1 KD by ASO 1 (UCP1 (red); DNA staining by DAPI (green); phase contrast (PC) (gray)). (I) Quantification of UCP1 fluorescent signal as whole image fluorescence intensity subtracted by background and normalized to cell number. Mean and individual values, n=3 (biological replicates) (with 7-8 analyzed images for each biological replicate), paired t test **p< 0.01. Ctcflos tr1 KD (ASO 1) and Ctcflos tr 3 and 4 KD (ASO 2) with their respective controls and (M-O) Ctcflos tr1 KD (ASO 1) and its control in UCP1 wildtype (WT) and UCP1 knockout (KO) cells, measured by microplate-based respirometry (Seahorse XF96 Analyzer). Oxygen consumption is recorded under basal conditions and in response to successive injection of oligomycin (5µM), isoproterenol (1.5µM), FCCP (1µM) and antimycin A (5µM) to determine basal leak, UCP1 dependent uncoupled, maximal and non-mitochondrial respiration, respectively. Data are expressed after deduction of non-mitochondrial respiration. Mean values ± SD, n=4-5 (biological replicates). Quantification of isoproterenol-stimulated UCP1-mediated uncoupled respiration, expressed as fold of basal leak respiration. Mean and single values, (K n=4 (biological replicates), paired t test *p<0.05, (L) n=5 (biological replicates), paired t test *p<0.05, (N, O) n=7 (technical replicates), unpaired t test, n.s. p>0.05, ****p<0.0001. Effect of Ctcflos KD on brite adipocyte respiratory capacity. Time course of oxygen consumption rates in primary brite adipocytes 72 hours after treatment (day 1 diff), comparing Ctcflos tr1 KD (ASO 1) and its control in UCP1 wildtype (WT) and UCP1 knockout (KO) cells, measured by microplate-based respirometry (Seahorse XF96 Analyzer). Oxygen consumption is recorded under basal conditions and in response to successive injection of oligomycin (5µM), isoproterenol (1.5µM), FCCP (1µM) and antimycin A (5µM) to determine basal leak, UCP1 dependent uncoupled, maximal and non-mitochondrial respiration, respectively. Data are expressed after deduction of non-mitochondrial respiration. Mean value ± SD, n=7 (technical replicates). Quantification of isoproterenol-stimulated UCP1-mediated uncoupled respiration, expressed as fold of basal leak respiration. Mean and single values, (K n=4 (biological replicates), paired t test *p<0.05, (L) n=5 (biological replicates), paired t test *p<0.05, (N, O) n=7 (technical replicates), unpaired t test, n.s. p>0.05, ****p<0.0001. (P) Effect of Ctcflos KD on brite adipocyte lipolysis. Basal and isoproterenol (iso) (0.5µM) stimulated glycerol release comparing Ctcflos tr1 (ASO 1), tr 3 and 4 (ASO 2) KD and their controls. Mean values ± SD, n=4 (biological replicates), two-way ANOVA (Šídák-test), n.s. p>0.05. Impact of Ctcflos KD on mitochondrial biogenesis. (Q) Western Blot analysis of Cytochrome C Oxidase subunit 4 (COX4) protein level in Ctcflos tr1 (ASO 1), tr 3 and 4 (ASO 2) KD and controls. Actin-β as loading control. Impact of Ctcflos KD on mitochondrial biogenesis. (R) Microscopic images of Mito Tracker stained brite adipocytes (Mito tracker (blue); DNA staining by DAPI (green); phase contrast (PC) (gray)) Impact of Ctcflos KD on mitochondrial biogenesis. (S) Quantification of Mito Tracker staining as whole image fluorescence intensity subtracted by background and normalized to cell number comparing Ctcflos tr1 (ASO 1) KD and control. Mean and individual values, n=10 (technical replicates) the experiment was repeated a second time, for a total of two biological replicates, for the second replication see (Fig EV1P) (with 10 analyzed images), unpaired t test **p< 0.01.