Fig 1: Induction of rhythmic gene expression by serum-shock in ASCs.A ARNTL2 mRNA expression. Values are presented as mean ± SEM from i = 3 different donors. Statistical analysis was done using One-way ANOVA and Bonferroni´s multiple comparison test. B ARNTL2 Western blot analysis. A representative result of n = 3 different donors is shown. ß-Actin served as loading control. C Densitometric analysis of ARNTL2 protein level. Values are presented as mean ± SEM from n = 3 different donors. Statistical comparison was achieved using the two-tailed paired t test (vs. t = 0; *) and the two-tailed unpaired t test (#). D ARNTL1 mRNA expression. Values are presented as mean ± SEM from n = 3 different donors. Statistical analysis was done using One-way ANOVA and Bonferroni´s multiple comparison test (*) in addition to the two-tailed unpaired t test (#). E ARNTL1 Western blot analysis. A representative result of n = 3 different donors is shown. ß-Actin served as loading control. F Densitometric analysis of ARNTL1 protein level. Values are presented as mean ± SEM from n = 3 different donors. Statistical comparison was done using One-way ANOVA and Bonferroni´s multiple comparison test. G C/EBPB mRNA expression. Values are presented as mean ± SEM from n = 3 different donors. Statistical analysis was achieved using One-way ANOVA and Bonferroni´s multiple comparison test. H C/EBP ß Western blot. A representative result of n = 3 different donors is shown. Short: short exposure; long: long exposure (~1 h). ß-Actin served as loading control. I Densitometric analysis of C/EBP ß protein level. Values are presented as mean ± SEM from n = 3 different donors. J Response of KLF4 KLF4 mRNA expression after circadian synchronization by serum-shock. Values are presented as mean ± SEM from n = 3 different donors. Statistical comparison was done with One-way ANOVA with Bonferroni´s multiple comparison test. K Representative KLF4 Western blot of n = 3 individual donors after serum-shock. ß-Actin served as loading control. L Densitometric analysis of KLF4 protein level. Values are presented as mean ± SEM from n = 3 different donors. Statistical comparison was done with One-way ANOVA and Bonferroni´s multiple comparison test.
Fig 2: Effects of ARNTL2 overexpression on adipogenesis.A Western blot analysis of control ASCs (Ctrl., -), harboring the empty vector, and ARNTL2 overexpressing (OE, + ) ASCs subjected to adipogenic differentiation. Left panel: representative Western blots of ASCs from n = 3 donors are shown. ß-Actin served as loading control. Right panel: Densitometric quantitation of the Western blots is shown. Values are presented as mean ± SEM of three measurements. Statistical comparison was achieved using the Paired t test (vs. normalized group) and the Unpaired t test. B RT-qPCR analysis of adipogenic marker genes. Representative result of n = 3 donors. Values are presented as mean ± SEM of three technical replicates. Statistical comparison within one group was done with One-way ANOVA and Dunnett´s multiple comparison test. Statistical comparison between groups was achieved using the paired t test (vs. normalized control group) and the unpaired t test. C Oil Red O staining of control ASCs (Ctrl.) harboring the empty vector and ARNTL2 overexpressing (OE) ASCs on d14 of adipogenic differentiation. A representative result of n = 3 different donors is shown. Microphotographs were taken at ×50 magnification. Scale bar: 200 µm D Quantification of Oil Red O staining. Values are presented as mean ± SEM of n = 3 different donors. Statistical comparison between groups was achieved using the paired t test (vs. normalized control group) and the unpaired t test.
Fig 3: ARNTL2 expression is downregulated upon weight-loss (WL) in human ASCs.Microarray analysis of freshly isolated ASCs was done as described in Ejaz et al. [34]. Tissue samples were acquired from age-matched normal-weight donors (NWDs; n = 3), obese donors (ODs; n = 3) and weight-loss donors (WLDs; n = 4).
Fig 4: ARNTL2-dependent feedback mechanism.A Pharmacological inhibition of signaling pathways. Serum-starved ASCs were pre-treated with the indicated compounds or vehicle (DMSO) for 30 min followed by DM stimulation for 30 min (left panel) and 4 h (right panel), respectively. A representative Western blot and densitometry of n = 3 independent experiments (i.e., donors) is shown. ß-Actin served as loading control. Values are presented as mean ± SEM of three measurements. Statistical comparison was done using One-way ANOVA and Dunnett´s Multiple Comparison test (vs. stimulated cells pre-treated with DMSO) and the unpaired two-tailed t test (between groups as indicated). B Stimulation of control (Ctrl.; -) and ARNTL2 overexpressing (OE; +) ASCs with DM for indicated time points. Left panel: A representative Western blot of n = 3 independent experiments (i.e., donors) is shown. ß-Actin served as loading control. Right panel: Densitometry corresponding to the Western blot shown on the left. Values are presented as mean ± SEM of three measurements. Statistical comparison was done with One-way ANOVA with Dunnett´s Multiple Comparison test (vs. t = 0) or the two-tailed paired and unpaired t test between groups as indicated.
Fig 5: PI3K/Akt/mTOR and MAPK signaling during adipogenesis in ARNTL2 OE ASCs.Corresponding result to Fig. 6A. See legend of Fig. 6 for details.
Supplier Page from DNASU for ARNTL2 (Homo sapiens) in pENTR223 (Gateway donor/master vector)