Fig 1: 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. β-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 2: 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. β-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. β-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 3: Induction of ARNTL2 by differentiation medium (DM) and single compounds.Confluent ASCs were serum-starved (SS) followed by treatment with serum-free ASC1 medium (A), differentiation medium (DM), insulin (I), dexamethasone (D), FCS (F), transferrin (T) and IBMX (X) for 4 h. Left panel: a representative Western blot of n = 3 independent experiments (i.e., donors) is shown. PI3K/Akt/mTOR and MAPK signaling was analyzed to confirm activity of each compound used. β-Actin served as loading control. Right panel: densitometric analysis of the Western blot shown on the left. Values are presented as mean ± SEM of three measurements. Statistical comparison was done using One-way ANOVA and Dunnett´s Multiple Comparison Test (vs. SS; *) and the two-tailed paired/unpaired t test (#).
Fig 4: 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 5: Impact of short-term stimulation of ASCs with adipogenic differentiation medium on ARNTL1 and ARNTL2 expression.A ARNTL2 mRNA expression. Values are given as mean ± SEM of n = 3 individual donors. Statisitcal analysis between groups was achieved using One-way ANOVA with Dunnett´s multiple comparison test. B A representative ARNTL2 Western blot of n = 3 different donors is shown. β-Actin served as loading control. C Densitometric quantification of ARNTL2 Western blots of n = 3 individual donors. Values are shown as mean ± SEM. Statistical comparison was done with the paired t test. D ARNTL1 mRNA expression. Values are given as mean ± SEM of n = 3 individual donors. Statisitcal analysis between groups was achieved using One-way ANOVA with Dunnett´s multiple comparison test. E A representative ARNTL1 Western blot of n = 3 different donors is shown. β-Actin served as loading control. F Densitometric quantification of ARNTL1 Western blots of n = 3 individual donors. Values are shown as mean ± SEM. Note: scaling of y-axis is different in (C) and (F). DM differentiation medium.
Supplier Page from DNASU for ARNTL2 (Homo sapiens) in pENTR223 (Gateway donor/master vector)