Journal: Communications Biology

Article Title: In-depth analysis of obesity-associated changes in adipose tissue-derived mesenchymal stromal/stem cells and primary cilia function

doi: 10.1038/s42003-025-08986-w

Figure Lengend Snippet: A Subcutaneous ASCs (ASCsub) were stained for the ciliary markers 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. B Cilium lengths were measured and are presented as scatter dot plots (mean ± SD). The results are based on at least six individual samples per group (ASCsub25, n = 6 individual individual samples, 180 cilia; ASCsub35, n = 6, 178 cilia; and ASCsub40, n = 7, 208 cilia) and statistically analyzed. C , D The heatmaps depict significantly ( p < 0.05) differentially expressed genes from CiliaCarta in both ASC subgroups of donors with obesity, or in ASCsub35 vs. ASCsub 25, respectively. Significantly enriched genes have a red color code and reduced genes are encoded in blue. Red and blue colored genes are sorted alphabetically. E – H Truncated violin plots present selected ciliary genes, which are differentially expressed. Values reflect the fragments per kilobase per million mapped fragments (fpkm) of genes from the RNA‑seq data. Each violin plot displays the median (central dashed line) and quartiles (upper and lower dotted lines). I Relative gene levels of GLI1 (glioma-associated oncogene homolog 1), SNX10 (sorting nexin 10), ADCY3 (adenylate cyclase 3), and CLUAP1 (clusterin associated protein 1) were corroborated with RT-PCR. The results are obtained from five individual samples in each group and presented as relative quantification (RQ) with SEM. GAPDH was used as endogenous control. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test was used to assess statistical significance relative to ln25 ( I ). For non-Gaussian data ( B, I , GLI1 ), the Kruskal-Wallis test followed by Dunn’s post hoc test was applied. *p < 0.05, ** *p < 0.001.

Article Snippet: ASCs were transfected with 30 nM of control small interfering RNA (AllStars Negative Control siRNA, Qiagen, Hilden, #1027281) or siRNA targeting ADCY3 (Santa Cruz Biotechnology, Inc., sc-29600) using the 4D-Nucleofector TM Core Unit with X Unit (Amaxa TM P1 Primary Cell 4d- Nucleofector TM Kit, #V4XP-102) with the program DO-101 (Lonza, Maastricht).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative Proteomics, Control

Journal: Communications Biology

Article Title: In-depth analysis of obesity-associated changes in adipose tissue-derived mesenchymal stromal/stem cells and primary cilia function

doi: 10.1038/s42003-025-08986-w

Figure Lengend Snippet: 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.

Article Snippet: ASCs were transfected with 30 nM of control small interfering RNA (AllStars Negative Control siRNA, Qiagen, Hilden, #1027281) or siRNA targeting ADCY3 (Santa Cruz Biotechnology, Inc., sc-29600) using the 4D-Nucleofector TM Core Unit with X Unit (Amaxa TM P1 Primary Cell 4d- Nucleofector TM Kit, #V4XP-102) with the program DO-101 (Lonza, Maastricht).

Techniques: Staining, Fluorescence, Control, Marker, Quantitative Proteomics, MANN-WHITNEY

Journal: Communications Biology

Article Title: In-depth analysis of obesity-associated changes in adipose tissue-derived mesenchymal stromal/stem cells and primary cilia function

doi: 10.1038/s42003-025-08986-w

Figure Lengend Snippet: A Truncated violin plots of the members of the regulatory factor X (RFX) family ( RFX1 , RFX2 , and RFX3 ). Values reflect the fragments per kilobase per million mapped fragments (fpkm) of genes from the RNA‑seq data. Each violin plot displays the median (central dashed line) and quartiles (upper and lower dotted lines). B Relative gene levels of RFX2 were corroborated with RT-PCR. The results are obtained from five individual samples for the lean and four individual samples of the obesity group, and presented as relative quantification (RQ) with SEM. GAPDH was used as endogenous control. C – G Treatment of lean ASCsub with TGFβ for 48 h. C Primary cilia of lean ASCsub were stained for the ciliary markers 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. D Cilium lengths were measured and are presented as scatter dot plots (mean ± SD). The results are based of three individual samples per group. E – G Relative gene levels of RFX2 , ADCY3 (adenylate cyclase 3), IFT88 (intraflagellar transport), IFT172 , COL1A1 (collagen 1A1), and SERPINE1 (serpin family E member 1) 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. Statistical significance was analyzed with the Student’s t test. * p < 0.05, ** *p < 0.001.

Article Snippet: ASCs were transfected with 30 nM of control small interfering RNA (AllStars Negative Control siRNA, Qiagen, Hilden, #1027281) or siRNA targeting ADCY3 (Santa Cruz Biotechnology, Inc., sc-29600) using the 4D-Nucleofector TM Core Unit with X Unit (Amaxa TM P1 Primary Cell 4d- Nucleofector TM Kit, #V4XP-102) with the program DO-101 (Lonza, Maastricht).

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative Proteomics, Control, Staining

Journal: Communications Biology

Article Title: In-depth analysis of obesity-associated changes in adipose tissue-derived mesenchymal stromal/stem cells and primary cilia function

doi: 10.1038/s42003-025-08986-w

Figure Lengend Snippet: A , B The effect of the ADCY inhibitor SQ22536 (SQ) or its activator NKH477 (NKH) was proven by the measurement of intracellular concentrations of acetylated cAMP via a direct cAMP ELISA. The results are presented as mean ± SD. Student’s t test was used. * p < 0.05. ASCs were treated with the ADCY inhibitor SQ22536 (SQ) ( C – E ) or its activator NKH477 (NKH) ( F – H ) for 24 or 48 h with indicated concentrations. Primary cilia of ASCsub were stained for the ciliary markers 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 ( C , F ). Cilium lengths were measured and are presented as scatter dot plots (mean ± SD) ( D , E and G , H ). The results are based on three individual samples per group (n = 3, 150 cilia) and statistical significance was analyzed with the Kruskal-Wallis test followed by Dunn’s post hoc test. ** *p < 0.001. Abbreviations: ADCY3 adenylate cyclase 3, cAMP cyclic adenosine monophosphate, con control, DMSO dimethyl sulfoxide, ELISA enzyme-linked immunosorbent assay, min minutes.

Article Snippet: ASCs were transfected with 30 nM of control small interfering RNA (AllStars Negative Control siRNA, Qiagen, Hilden, #1027281) or siRNA targeting ADCY3 (Santa Cruz Biotechnology, Inc., sc-29600) using the 4D-Nucleofector TM Core Unit with X Unit (Amaxa TM P1 Primary Cell 4d- Nucleofector TM Kit, #V4XP-102) with the program DO-101 (Lonza, Maastricht).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Communications Biology

Article Title: In-depth analysis of obesity-associated changes in adipose tissue-derived mesenchymal stromal/stem cells and primary cilia function

doi: 10.1038/s42003-025-08986-w

Figure Lengend Snippet: Analysis of cell motility. A Experimental schedule of treatment with the ADCY inhibitor SQ22536 (SQ) or its activator NKH477 (NKH). B Representative trajectories are depicted for individual cells (n = 1, 50 cells in each group). The velocity ( C ), the accumulated distance ( D ), and the directionality ( E ) of ASC motility are shown as mean ± SD (n = 3, 150 cells pooled from three experiments). Statistical significance was analyzed with the Kruskal-Wallis test followed by Dunn’s multiple comparison test. * p < 0.05; ** *p < 0.001; n.s., not significant. F Schematic illustration of the proposed working model. Obesity-associated factors, such as TGFβ, are able to shortened primary cilia in ASCs associated with deregulated pathways and genes/proteins, fueling inflammation and metabolic dysfunction and facilitating the progression of obesity. Treatment with NHK477 reactivates the ADCY3-cAMP signaling axis, prolongs the primary cilium, improves cell motility, and thus possibly prevents the progression of obesity. Abbreviations: ADCY3 adenylate cyclase 3, ADIPOQ adiponectin, cAMP cyclic adenosine monophosphate, CDH1 cadherin-1, COL collagen, CXCL12 CXC motif chemokine 12, DMSO dimethyl sulfoxide, FABP4 fatty acid-binding protein 4, FN1 fibronectin, HH hedgehog, ITGB5 integrin beta 5, ln lean, LPL lipoprotein lipase, ob obese, PGF placental growth factor, PPARG peroxisome proliferator-activated receptor gamma, TGFβ transforming growth factor-β, THBS1 thrombospondin 1.

Article Snippet: ASCs were transfected with 30 nM of control small interfering RNA (AllStars Negative Control siRNA, Qiagen, Hilden, #1027281) or siRNA targeting ADCY3 (Santa Cruz Biotechnology, Inc., sc-29600) using the 4D-Nucleofector TM Core Unit with X Unit (Amaxa TM P1 Primary Cell 4d- Nucleofector TM Kit, #V4XP-102) with the program DO-101 (Lonza, Maastricht).

Techniques: Comparison, Binding Assay