Fig. 5: Reduced ADCY3 intensity at the cilium of ASCs from donors with obesity.

A Cells were stained for the ciliary markers clusterin associated protein 1 (CLUAP1, red) and acetylated α-tubulin (ace tubulin, green), and DNA (DAPI, blue). Representatives are shown for measurements of primary cilium staining of CLUAP1. Scale: 5 µm. B Line-scan analyses of axonemal CLUAP1 are shown for ASCs. Each point of the curve represents the mean fluorescence intensity ±SD and the results are based on three individual samples (n = 3, 73 cilia), normalized to the cilium length in percentage; a.u., arbitrary unit. C Cells were stained for the ciliary markers ADCY3 (adenylate cyclase 3, red) and acetylated α-tubulin (ace tubulin, green), and DNA (DAPI, blue). Representatives are shown for measurements of the primary cilium staining of ADCY3. Scale: 5 µm. D Line-scan analyses of axonemal ADCY3 are shown for ASCs. Each point of the curve represents the mean fluorescence intensity ±SD and the results are based on three individual samples (n = 3, 72 cilia), normalized to the cilium length (in percent); a.u., arbitrary unit. Statistical significance was assessed using the Kruskal-Wallis test followed by Dunn’s post hoc test to compare lean and obese groups at each 10% interval of relative ciliary length (0–100%). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. E–G Lean ASCs were treated with 30 nM of control siRNA (sicon) or with siRNA against ADCY3 for 48 h. E Primary cilia were stained for the ciliary marker acetylated α-tubulin (ace tubulin, green) and ARL13B (ADP-ribosylation factor-like GTPase 13B, red), and DNA (DAPI, blue). Representatives are shown. Scale: 5 µm. F The lengths of individual cilia were measured and are presented as scatter dot plots (mean ± SD). The results are based on three individual samples per group (n = 3, 135 cilia) and statistically analyzed. G Relative gene levels of ADCY3 are shown. The results were obtained from three individual samples in each group and presented as relative quantification (RQ) with SEM. GAPDH served as endogenous control. Unpaired Mann–Whitney U test (F) or Student’s t test was used (G). *p < 0.05, ***p < 0.001.