Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Machine learning-based biomarker selection: ( A ) LASSO regression curve for feature selection in the training cohort GSE71220 . The x-axis shows the log of the regularization parameter λ, and the y-axis shows the partial likelihood deviance. Two vertical dashed lines indicate the optimal λ values: the left dashed line corresponds to λ.min (the value that minimizes the cross-validated error), and the right dashed line corresponds to λ.1se (the largest λ within one standard error of the minimum). The optimal penalty parameter λ = 0.013 (λ.min) was selected, yielding 21 non-zero coefficient genes including HK3 , GPR15 , and RORC . ( B ) LASSO coefficient paths. Each colored line represents the trajectory of a gene coefficient as the regularization penalty increases (decreasing log λ). The vertical dashed line indicates the selected λ.min at log λ = −4.3182, corresponding to the optimal model complexity. ( C ) Boruta feature importance assessment. Boxplots display the importance scores of each feature across iterative random forest runs. The y-axis represents the importance score; the vertical position (median) of each boxplot reflects the feature’s importance, while the box length represents the interquartile range, indicating variability across runs. Core predictors (green) are distinguished from tentative (blue) and rejected (red) features. ( D ) Venn diagram showing the overlap between genes selected by LASSO and Boruta. Six hub genes ( GPR15 , HK3 , RORC , CLEC4D , TCF7 , and SLC4A10 ) were identified by both algorithms and retained for further analysis. ( E ) Validation of biomarker expression in the training and validation cohorts. Violin plots display the expression levels of the six hub genes. The upper panels show expression in the training cohort GSE71220 , and the lower panels show expression in the internal validation cohort GSE42057 significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The human RORC ORF clone (ROR gamma ( RORC ) Human Tagged ORF Clone, transcript variant 2, NM_001001523 , encoding the RORγt isoform) was obtained from OriGene (Cat. No. RC224338).

Techniques: Biomarker Discovery, Selection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Validation of the diagnostic performance of the three-gene signature: ( A ) Receiver operating characteristic curves for the three biomarkers in the training cohort GSE71220 . The area under the curve values are 0.777 for RORC , 0.771 for CLEC4D , and 0.752 for TCF7 . RORC is shown as a green stepped line, CLEC4D in red, and TCF7 in blue. ( B ) Receiver operating characteristic curves in the internal validation cohort GSE42057 . The area under the curve values are 0.662 for RORC , 0.644 for CLEC4D , and 0.680 for TCF7 . Color coding follows the same scheme as in panel A. ( C ) Nomogram for predicting COPD probability based on the expression levels of the three-gene signature. Each gene contributes a score corresponding to its expression value; the total score is summed to estimate the predicted probability. As an example, a randomly selected patient with a total score of 156 corresponds to a predicted probability of 0.983. ( D ) Decision curve analysis evaluating the clinical utility of the three-gene signature. The curves represent the net benefit of clinical decisions based on RORC , TCF7 , and CLEC4D , and the combined nomogram across a range of threshold probabilities.

Article Snippet: The human RORC ORF clone (ROR gamma ( RORC ) Human Tagged ORF Clone, transcript variant 2, NM_001001523 , encoding the RORγt isoform) was obtained from OriGene (Cat. No. RC224338).

Techniques: Biomarker Discovery, Diagnostic Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: External validation of the three-gene signature across independent cohorts: ( A ) Validation in the peripheral blood cohort GSE56766 . The violin plots show the expression levels of the three biomarkers in patients with COPD and healthy controls. CLEC4D was significantly upregulated, while RORC and TCF7 did not show statistically significant differences. The receiver operating characteristic curves for each biomarker are shown to the right, with the area under the curve values ranging from 0.602 to 0.654. RORC is depicted in blue, CLEC4D in orange, and TCF7 in green. ( B ) Validation in the peripheral blood cohort GSE306950 . The violin plots show that RORC and TCF7 were significantly downregulated, whereas CLEC4D was significantly upregulated in the COPD samples compared to controls. The corresponding ROC curves yielded area under the curve values of 0.976 for RORC , 0.776 for CLEC4D , and 0.776 for TCF7 . Color coding follows the same scheme as in panel ( A ). ( C ) Validation in the lung tissue cohort GSE106986 . The violin plots show that both RORC and TCF7 were significantly downregulated in COPD lung tissue, while CLEC4D did not reach statistical significance. The receiver operating characteristic analysis revealed area under the curve values of 0.886 for RORC , 0.571 for CLEC4D , and 0.814 for TCF7 . Color coding is consistent with previous panels. Significance: * p < 0.05, *** p < 0.001.

Article Snippet: The human RORC ORF clone (ROR gamma ( RORC ) Human Tagged ORF Clone, transcript variant 2, NM_001001523 , encoding the RORγt isoform) was obtained from OriGene (Cat. No. RC224338).

Techniques: Biomarker Discovery, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Gene set enrichment analysis and regulatory network construction: ( A ) Gene set enrichment analysis of the three biomarkers. The GSEA enrichment plots display the top five significantly enriched pathways for RORC , CLEC4D , and TCF7 . Positive enrichment for RORC in neuroactive ligand–receptor interaction (NES = 3.05) and negative enrichment in spliceosome and ubiquitin−mediated proteolysis pathways (NES < −2.7) are shown. For CLEC4D , positive enrichment in spliceosome, ubiquitin−mediated proteolysis, and proteasome pathways, and negative enrichment in neuroactive ligand–receptor interaction and olfactory transduction are displayed. For TCF7 , positive enrichment in the ribosome and primary immunodeficiency pathways (NES = 2.14) and negative enrichment in the complement and coagulation cascades are presented. ( B ) Transcription factor−miRNA−mRNA regulatory network for TCF7 and RORC . The network was constructed using the GSE24709 dataset and visualized with Cytoscape. The nodes represent transcription factors (triangles), miRNAs (diamonds), and target genes (circles). IRF4 , FOXM1 , and EGR1 are shown as transcription factors connecting TCF7 and RORC . The network is presented in a blue color theme. ( C ) Gene–gene interaction network for TCF7 , CLEC4D , and RORC . Interactions were retrieved from GeneMANIA, integrating evidence from multiple public databases and published studies. Edge colors represent seven interaction types: physical interactions (77.64%), co−expression (8.01%), predicted interactions (5.37%), co−localization (3.63%), genetic interactions (2.87%), pathway (1.88%), and shared protein domains (0.60%). ( D ) Transcription factor−miRNA−mRNA regulatory network highlighting miRNA connections. hsa−miR−485−5p is indicated as a key miRNA connecting TCF7 and RORC . The network follows the same blue color theme as panel ( B ).

Article Snippet: The human RORC ORF clone (ROR gamma ( RORC ) Human Tagged ORF Clone, transcript variant 2, NM_001001523 , encoding the RORγt isoform) was obtained from OriGene (Cat. No. RC224338).

Techniques: Ubiquitin Proteomics, Olfactory, Transduction, Coagulation, Construct, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Immune landscape and biomarker associations: ( A ) Heatmap of ssGSEA-based quantification for 28 immune cell types in patients with COPD versus healthy controls. Red indicates higher relative abundance, and blue indicates lower abundance. ( B ) Correlation heatmap of differentially abundant immune cell subpopulations. Spearman correlations are represented by color intensity (red: positive; blue: negative). ( C ) Heatmap of immune−related functional pathway enrichment scores in COPD versus controls. ( D – F ) Correlation heatmaps illustrating the associations between the three diagnostic biomarkers ( CLEC4D , RORC , and TCF7 ) and ( D ) immune cell subpopulations, ( E ) functional pathways, and ( F ) HLA gene expression levels. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical significance is denoted as follows: ****, p < 0.0001.

Article Snippet: The human RORC ORF clone (ROR gamma ( RORC ) Human Tagged ORF Clone, transcript variant 2, NM_001001523 , encoding the RORγt isoform) was obtained from OriGene (Cat. No. RC224338).

Techniques: Biomarker Discovery, Functional Assay, Diagnostic Assay, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Single-cell transcriptomic landscape and intercellular communication analysis: ( A ) UMAP visualization of the GSE249584 single-cell RNA-seq dataset. The cells were clustered into eight distinct immune cell types based on marker gene expression. ( B ) UMAP plots showing the expression distribution of CLEC4D , RORC , and TCF7 in the control and COPD samples. CLEC4D was predominantly expressed in myeloid populations, whereas RORC and TCF7 were largely restricted to lymphoid lineages. ( C ) Violin plots comparing the expression levels of the three biomarkers between control and COPD groups across different cell types. CLEC4D showed upregulation in monocytes, macrophages, and dendritic cells in the COPD samples. RORC expression was downregulated in T cells and natural killer cells, whereas TCF7 exhibited elevated expression in natural killer and B cell clusters in COPD compared to controls. * p < 0.05, ** p < 0.01. ( D ) CellChat network analysis showing the overall communication strength and weight among immune cell populations in control and COPD conditions. Edge thickness represents the probability of intercellular communication. ( E ) Bubble plot displaying significant ligand–receptor pairs between cell populations in control and COPD groups. Circle size indicates communication probability, and color intensity represents relative expression levels.

Article Snippet: The human RORC ORF clone (ROR gamma ( RORC ) Human Tagged ORF Clone, transcript variant 2, NM_001001523 , encoding the RORγt isoform) was obtained from OriGene (Cat. No. RC224338).

Techniques: Single Cell, RNA Sequencing, Marker, Gene Expression, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Molecular docking analysis of RORC with candidate drugs: ( A ) Three−dimensional structure of RORC (green) in complex with Lovastatin (blue). The protein is depicted as a green ribbon diagram, with the ligand shown in blue. The residues involved in ligand binding are highlighted in pink, and hydrogen bonds are represented by red dashed lines. Interacting residues include His479 (hydrogen bond distance 2.2 Å), His323 (3.3 Å), and Gln286 (2.1 Å). The binding affinity is −9.0 kcal/mol. ( B ) Three−dimensional structure of RORC (green) in complex with Celecoxib (blue). The protein is shown in green, the ligand in blue, binding residues in pink, and hydrogen bonds as red dashed lines. Interacting residues include Glu359 (2.8 Å), Gly470 (2.4 Å), Leu472 (2.8 Å), and Arg473 (1.9 Å). The second conformation of Celecoxib is shown. The binding affinity for this conformation is −8.8 kcal/mol.

Article Snippet: The human RORC ORF clone (ROR gamma ( RORC ) Human Tagged ORF Clone, transcript variant 2, NM_001001523 , encoding the RORγt isoform) was obtained from OriGene (Cat. No. RC224338).

Techniques: Ligand Binding Assay, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: qRT−PCR validation of the three−gene signature in CSE−stimulated (COPD−like) cellular models. Expression levels of CLEC4D , RORC , and TCF7 were measured by quantitative real−time PCR in THP−1−derived macrophages (for CLEC4D ) and Jurkat T cells (for RORC and TCF7 ) following 24 h of stimulation with cigarette smoke extract: ( A ) CLEC4D expression was significantly upregulated in CSE−stimulated macrophages compared to control ( p < 0.001). In contrast, ( B , C ) RORC and TCF7 expression were significantly downregulated in CSE−stimulated Jurkat T cells compared to control ( p < 0.001 for both). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using a two−tailed Student’s t -test. Significance: *** p < 0.001.

Article Snippet: The human RORC ORF clone (ROR gamma ( RORC ) Human Tagged ORF Clone, transcript variant 2, NM_001001523 , encoding the RORγt isoform) was obtained from OriGene (Cat. No. RC224338).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Pharmacological intervention restores RORC protein expression under inflammatory stress in macrophages: ( A ) Representative Western blot images of RORC and internal control β−tubulin in the THP−1−derived macrophages across five experimental conditions: Control, Model (LPS + CSE challenge), Model + Lovastatin, Model + Lovastatin + RORC overexpression (Model + Drug + OE), and OE alone. ( B ) Quantitative densitometric analysis of the RORC protein levels normalized to β−tubulin. The inflammatory model significantly suppressed RORC expression compared to the Control. Pharmacological treatment (Model + Lovastatin) successfully and significantly restored RORC protein levels. Data are presented as mean ± SD ( n = 3 per group). Statistical significance was assessed using Student’s t −test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The human RORC ORF clone (ROR gamma ( RORC ) Human Tagged ORF Clone, transcript variant 2, NM_001001523 , encoding the RORγt isoform) was obtained from OriGene (Cat. No. RC224338).

Techniques: Expressing, Western Blot, Control, Derivative Assay, Over Expression

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A Expression of RORC (RORγ) in normal human kidneys (from the Kidney Interactive Transcriptomics database). B RT-PCR ( n = 6 biological replicates) was performed to detect the expression of RORγ in selected mouse renal cells, including mouse podocytes (MPC), glomerular endothelial cells (GEC), TECs, and fibroblasts (MF). C The mRNA level ( n = 5 biological replicates) and protein levels of RORγ in the hTECs (human TECs) and mTECs (mouse TECs) were incubated with HGPA (final concentration 30 mmol/L glucose and 100 μM PA) for 18 h or 36 h. D RORγ transcript levels in kidney tubules from healthy living donors (HLD) and patients with diabetic kidney disease (DKD) (Nephroseq database; healthy living donors [HLD], n = 31; DKD, n = 17). The gene expression of RORγ in tublnt from HLD and patients with DKD. Spearman correlation analysis (two-sided) of the transcription levels of RORγ in tublnt and eGFR in HLD and patients with DKD. E Reanalysis of the data obtained from the GEO database ( GSE30122 ; HLD, n = 12; DKD, n = 10). The gene expression of RORγ in tublnt. Spearman correlation analysis (two-sided) of the transcription levels of RORγ in tublnt and eGFR in HLD and patients with DKD. F : Representative Masson’s trichrome and RORγ immunohistochemical staining of kidney sections from a minimal change disease control and DKD patients. Scale bar, 100 µm. G : Quantification of collagen deposition and RORγ-positive area ( n = 8 DKD patients; 3 fields/patient). H Representative images of RORγ immunohistochemical staining in the kidney sections from STZ-induced diabetic mice are shown (scale bar = 100 μm). I The mRNA levels of Rorγ, TNF-α , and KIM-1 in the kidney of STZ-induced diabetic mice ( n = 6 mice). J Western blot analysis of the expression of RORγ, KIM-1, TNF-α, and tubulin. Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by two-tailed Student’s unpaired t-test ( D , E , and G ); by one-way ANOVA with Bonferroni’s correction ( B , C ); Spearman’s correlation analysis ( D and E ).

Article Snippet: Rorγ flox/flox (C57BL/6 JCya- Rorc em1flox /Cya; Strain ID: CKOCMP-19885-Rorc-B6J-VA), Cab39 flox/flox (C57BL/6JCya- Cab39 em1flox /Cya; Strain ID: CKOCMP-12283-Cab39-B6J-VA), Sting1 -/- (C57BL/6NCya -Sting1 em1 / Cya; Strain ID: KOCMP-72512-Sting1-B6N-VA), C57BL/6J mice, and Cdh16-Cre mice (Cat#: C001452) were obtained from Cyagen Biosciences (Guangzhou) Inc (Guangzhou, Guangdong, China).

Techniques: Expressing, Transcriptomics, Reverse Transcription Polymerase Chain Reaction, Incubation, Concentration Assay, Gene Expression, Immunohistochemical staining, Staining, Control, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A Rorγ mRNA levels in multiple tissues of WT and RTKO mice ( n = 6 mice). B Schematic of kidney sampling in diabetic kidney disease (DKD) model mice. C Kidney weight/body weight ratio, urinary albumin-to-creatinine ratio (UACR), blood urea nitrogen (BUN), and kidney KIM-1 mRNA in DKD model mice ( n = 6 mice). D Representative H&E staining (left) and quantification of tubular injury (right) in DKD model kidneys. E Kidney cholesterol content and mRNA levels of cholesterol synthesis genes in DKD model mice ( n = 6 mice). F Left: Renal inflammatory gene ( Cx3cl1, Mcp1, Cxcl10, IL-1β, Ccr 2) mRNA in DKD model mice ( n = 6 mice). Right: Representative F4/80 immunofluorescence staining. G Left: Kidney fibrotic gene ( Tgfβ1, Col1a1, Col3a1, Fn1, α-SMA ) mRNA in DKD model mice ( n = 6 mice). Right: Representative Sirius red staining. ( H ) Schematic of kidney sampling in aging model mice. I Representative H&E staining (left) and quantification of tubular injury (right) in aging model kidneys ( n = 6 mice). J UACR and renal *KIM-1* mRNA in aging model mice ( n = 6 mice). K Kidney cholesterol content and synthesis gene mRNA in aging model mice ( n = 6 mice). L Left: Representative Sirius red staining in aging model kidneys. Right: Renal fibrotic gene ( Tgfβ1, Col1a1, Col3a1 ) mRNA ( n = 6 mice). M Left: Representative F4/80 immunofluorescence staining in aging model kidneys. Right: Renal inflammatory gene ( Cx3cl1, Mcp1, IL-1β ) mRNA ( n = 6 mice). N Kidney senescence marker (p21, p16) mRNA in aging model mice ( n = 6 mice). Source data are provided as a Source Data file. Data are mean ± SD. P values calculated by two-tailed unpaired t-test (A, I , J , K , L , M , N ) or one-way ANOVA with Bonferroni correction ( C , D , E , F , G). Panels B and H were created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: Rorγ flox/flox (C57BL/6 JCya- Rorc em1flox /Cya; Strain ID: CKOCMP-19885-Rorc-B6J-VA), Cab39 flox/flox (C57BL/6JCya- Cab39 em1flox /Cya; Strain ID: CKOCMP-12283-Cab39-B6J-VA), Sting1 -/- (C57BL/6NCya -Sting1 em1 / Cya; Strain ID: KOCMP-72512-Sting1-B6N-VA), C57BL/6J mice, and Cdh16-Cre mice (Cat#: C001452) were obtained from Cyagen Biosciences (Guangzhou) Inc (Guangzhou, Guangdong, China).

Techniques: Sampling, Staining, Immunofluorescence, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A GSEA showing the enrichment of Cytosolic DNA-sensing pathway. B WT or Rorγ KO TECs were incubated with or without HGPA (30 mmol/L glucose and 200 μM PA) for 24 h. Left panel: immunoblot of whole-cell lysates with indicated antibodies. Right panel: ISGs were determined by qPCR ( n = 5 biological replicates). C Left panel: Rorγ KO TECs were transfected with Ad-null, Ad- Rorγ , or Ad- Rorγ E502Q (E502Q) for 12 h, replaced with fresh medium culture for 48 h, and then cultured with HGPA for an additional 24 h. Middle panel: immunoblot analysis. Right panel: ISGs were determined by qPCR ( n = 5 biological replicates). D , E TECs were isolated from WT, Rorγ KO , Rorγ KO Sting1 KO , or Rorγ KO cGas KO mice. Left panel: Immunoblot analysis. Right panel: ISGs were determined by qPCR ( n = 5 biological replicates). F , G F: Rorγ KO Sting KO TECs were transfected with Ad-null, Ad- Sting WT ( Sting WT ), Ad- Sting Y239S (Y239S), or Ad- Sting S365A (S365A) for 12 h, replaced with fresh medium culture for 48 h (WT or Rorγ KO TECs were transfected with Ad-null as a control), and then incubated with HGPA for 24 h. G Left panel: Immunoblot analysis. Right panel: heat map showing the expression of ISGs. H Immunoblot analysis of kidney lysates. I Representative Images of H&E and Sirius red staining in kidney sections from mice are shown. J KW/BW, UACR, BUN, KIM-1 mRNA levels, and tubular injury score of mice ( n = 6 mice). K Kidney cholesterol content (left panel) and mRNA levels of CSGs and ISGs (right panel) of kidneys from mice ( n = 6 mice). Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by one-way ANOVA with Bonferroni’s correction. Panels C and F were created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: Rorγ flox/flox (C57BL/6 JCya- Rorc em1flox /Cya; Strain ID: CKOCMP-19885-Rorc-B6J-VA), Cab39 flox/flox (C57BL/6JCya- Cab39 em1flox /Cya; Strain ID: CKOCMP-12283-Cab39-B6J-VA), Sting1 -/- (C57BL/6NCya -Sting1 em1 / Cya; Strain ID: KOCMP-72512-Sting1-B6N-VA), C57BL/6J mice, and Cdh16-Cre mice (Cat#: C001452) were obtained from Cyagen Biosciences (Guangzhou) Inc (Guangzhou, Guangdong, China).

Techniques: Incubation, Western Blot, Transfection, Cell Culture, Isolation, Control, Expressing, Staining

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A WT or Rorγ KO TECs were subjected to an Opti-Prep gradient ultracentrifugation. Ten fractions were collected, diluted, and subjected to immunoprecipitation using an anti-STING. ERGIC-53, ERGIC marker; CALR, ER marker; GM130, Golgi marker. This experiment in this figure was repeated independently 3 times with similar results. B WT or Rorγ KO TECs were subjected to homogenization and cell fractionation using gradient centrifugation. Immunoblotting analyses were performed with the indicated antibodies. Calnexin, ER marker; Golgin97, Golgi marker. This experiment in this figure was repeated independently 3 times with similar results. C , D WT or Rorγ KO TECs were treated with HGPA as indicated in the figure. At different time points, the cells were subjected to immunofluorescence staining for STING1 and ERGIC-53, followed by colocalization analysis (for each group, three biological replicates were set up, and four random fields of view were selected for statistical analysis). E – H qPCR analysis of the expression of cholesterol-synthesis genes and ISGs in WT, Rorγ KO , Rorγ KO Srebf2 KD and Rorγ KO Srebf2 KD TECs reconstituted with shRNA-resistant SREBP2 wild type (FL) or shRNA-resistant transcription-inactive mutants (L511A/S512A, ΔbHLH) ( n = 5). ( I, J ) WT or Rorγ KO TECs were incubated with Trip (14 μM) or vehicle, or transfected with Ad-null or Ad Insig1 as indicated. The total cell lysates and nuclear fractions were prepared and subjected to western blotting using the indicated antibodies (left). (Right) qPCR analysis of the expression of CSGs and ISGs in cells ( n = 5). K HK2 cells were transfected with INSIG1 shRNA (sh INSIG1 ), Ad- RORγ , Ad- INSIG1 WT (shRNA-resistant), or Ad- INSIG1 D205A (shRNA-resistant) as indicated (upper panel). The total cell lysates and nuclear fractions were prepared and subjected to western blotting using the indicated antibodies (bottom panel). This experiment was repeated independently 3 times with similar results. Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by one-way ANOVA with Bonferroni’s correction. G and K were created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: Rorγ flox/flox (C57BL/6 JCya- Rorc em1flox /Cya; Strain ID: CKOCMP-19885-Rorc-B6J-VA), Cab39 flox/flox (C57BL/6JCya- Cab39 em1flox /Cya; Strain ID: CKOCMP-12283-Cab39-B6J-VA), Sting1 -/- (C57BL/6NCya -Sting1 em1 / Cya; Strain ID: KOCMP-72512-Sting1-B6N-VA), C57BL/6J mice, and Cdh16-Cre mice (Cat#: C001452) were obtained from Cyagen Biosciences (Guangzhou) Inc (Guangzhou, Guangdong, China).

Techniques: Immunoprecipitation, Marker, Homogenization, Cell Fractionation, Gradient Centrifugation, Western Blot, Immunofluorescence, Staining, Expressing, shRNA, Incubation, Transfection

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A , B WT or Rorγ KO TECs were treated with CHX (100 ng/ml) or MG132 (10 µM) as indicated, followed by immunoblot analysis. C Co-IP of INSIG1 was performed from lysates of WT or Rorγ KO TECs with immunoblotting. D WT TECs were transfected with the indicated siRNAs and analyzed by immunoblotting. E Co-IP of INSIG1 was performed from lysates of WT or Yod1 KO TECs with immunoblotting. F HK2 cells were transfected with Ad-Flag-YOD1 WT or C160S mutant. Right: Co-IP of INSIG1 was performed. G Schematic of potential RORγ post-translational modification sites from database. H Left: Diagram of INSIG1 cytosolic lysines. Right: HK2 cells were transfected with shYOD1 and Ad-Flag-INSIG1 WT or K → R mutants (K33R, K156R, K158R, K273R) for immunoblot analysis. I Left: Conservation of INSIG1 K156/K158. Right: HK2 cells were transfected with shYOD1 and Ad-Flag-INSIG1 WT or DKR mutant (K156R/K158R) for Co-IP with anti-Flag. J Left: Co-IP of INSIG1 from mouse kidney lysates. Middle/Right: WT TECs were co-transfected with Ad-HA-Yod1 and/or Ad-Flag-Insig1 WT for Co-IP with anti-Flag. K Left: Predicted transmembrane topology of INSIG1. Right: IP of INSIG1 truncation mutants expressed in HK2 cells was performed. L Bottom: Immunoblot analysis of WT or Rorγ KO TECs. Top: Yod1 mRNA was analyzed by qPCR ( n = 5 biological replicates). M YOD1 promoter sequence with predicted RORγ binding sites (RORE1, RORE2) and mutations (RORE1m, RORE2m). N Luciferase activity of truncated YOD1 promoter fragments was measured in HEK293T cells ( n = 3 biological replicates). O , P HK2 cells were transfected with Ad-RORγ. Left: ChIP was performed using anti-RORγ antibody at the YOD1 promoter. Right: qPCR quantification ( n = 5 biological replicates). Q Luciferase assay of YOD1 promoter with mutated RORE sites was performed in HEK293T cells co-transfected with Ad-Rorγ ( n = 3 biological replicates). R Co-IP of INSIG1 from mouse kidney lysates was performed. Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by two-tailed Student’s unpaired t -test ( M ). P values calculated by one-way ANOVA with Bonferroni’s correction ( N , P , and Q ). Panels F , H , and K were created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: Rorγ flox/flox (C57BL/6 JCya- Rorc em1flox /Cya; Strain ID: CKOCMP-19885-Rorc-B6J-VA), Cab39 flox/flox (C57BL/6JCya- Cab39 em1flox /Cya; Strain ID: CKOCMP-12283-Cab39-B6J-VA), Sting1 -/- (C57BL/6NCya -Sting1 em1 / Cya; Strain ID: KOCMP-72512-Sting1-B6N-VA), C57BL/6J mice, and Cdh16-Cre mice (Cat#: C001452) were obtained from Cyagen Biosciences (Guangzhou) Inc (Guangzhou, Guangdong, China).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Modification, Sequencing, Binding Assay, Luciferase, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A GSEA showing enrichment of the AMPK signaling pathway. B Co-IP of LKB1 from WT or Rorγ KO TECs transfected with control (siCtrl) or Cab39 siRNA, followed by immunoblotting. C LKB1 kinase activity (commercial assay) and RORγ protein levels in WT or Rorγ KO TECs ( n = 6 biological replicates). D Immunoblot analysis in WT or Rorγ KO TECs transfected as indicated in figure. CAB39 protein and mRNA levels ( n = 5 biological replicates) in ( E ) WT vs. Rorγ KO TECs and F WT TECs transfected with Ad-null or Ad-RORγ. G Luciferase activity of truncated CAB39 promoter fragments in HEK293T cells ( n = 3 biological replicates). H ChIP-qPCR analysis of RORγ binding to the CAB39 promoter in HK2 cells transfected with Ad-RORγ ( n = 5 biological replicates). I CAB39 promoter sequence (left) and transcriptional activity of the WT or mutant versions in HEK293T cells (right) ( n = 5 biological replicates). J Immunoblot analysis of TECs from WT, Cab39 TKO , or Rorγ TKO Cab39 TKO (RCKO) mice. K Immunoblot analysis of WT or Rorγ KO TECs co-transfected with Ad-INSIG1 and Ad-Flag-GP78. L Co-IP of Flag-GP78 in WT or Rorγ KO TECs co-transfected with Ad-Flag-GP78 and Ad-INSIG1. M Co-IP of Flag-INSIG1 in WT or Rorγ KO TECs transfected with Ad-Flag-INSIG1. N Co-IP of Flag-INSIG1 in HK2 cells transfected as indicated in the figure. O , P Co-IP of INSIG1 from kidney lysates of Yod1 KO mice treated with AAV-RORγ and/or the AMPK agonist CDC (10 mg/kg). Q Immunoblot analysis of mouse kidney lysates. R Representative H&E and Sirius red staining of mouse kidney sections. S KW/BW, UACR, BUN, renal cholesterol, KIM-1 mRNA, and tubular injury score in mice ( n = 6 mice). T Kidney mRNA levels of CSGs and ISGs in mice ( n = 6 mice). These experiments were repeated independently 3 times with similar results ( B , D , J , Q ). Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by two-tailed Student’s unpaired t -test ( C , E , and F ). P values calculated by one-way ANOVA with Bonferroni’s correction ( G , H , I , S , and T ). Panel D was created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: Rorγ flox/flox (C57BL/6 JCya- Rorc em1flox /Cya; Strain ID: CKOCMP-19885-Rorc-B6J-VA), Cab39 flox/flox (C57BL/6JCya- Cab39 em1flox /Cya; Strain ID: CKOCMP-12283-Cab39-B6J-VA), Sting1 -/- (C57BL/6NCya -Sting1 em1 / Cya; Strain ID: KOCMP-72512-Sting1-B6N-VA), C57BL/6J mice, and Cdh16-Cre mice (Cat#: C001452) were obtained from Cyagen Biosciences (Guangzhou) Inc (Guangzhou, Guangdong, China).

Techniques: Co-Immunoprecipitation Assay, Transfection, Control, Western Blot, Activity Assay, Luciferase, ChIP-qPCR, Binding Assay, Sequencing, Mutagenesis, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A Top: Immunoblot analysis of kidney lysates from DKD and control (Ctrl) mice. Bottom: Kidney Ctcf mRNA levels ( n = 5 mice). B CTCF expression in kidney tubules from healthy living donors (HLD, n = 31) and DKD patients ( n = 17) in the Nephroseq and GEO GSE30122 (HLD n = 12, DKD n = 10) databases. C Left: Immunoblot analysis of WT TECs transfected with Ctcf siRNA (si Ctcf ) and treated with HGPA or 5-aza (10 µM, 3 days). Right: Rorγ mRNA levels (qPCR, n = 5 biological replicates). D ChIP-qPCR of CTCF binding to the RORγ promoter in HK2 cells treated with HGPA or 5-aza ( n = 5 biological replicates). E Methylation levels of the RORγ promoter region ( n = 5 biological replicates). F Rorγ mRNA levels in WT TECs treated with HGPA over time (qPCR, n = 5 biological replicates). G Immunoblot analysis of total and nuclear fractions from TECs treated with HGPA for 16 h. H ChIP-qPCR of RORγ binding to the YOD1 promoter in TECs treated with HGPA ± Act-D (5 µg/mL). I Immunofluorescence of RORγ in TECs treated with HGPA or Act-D. Scale bar, 50 µm. J Co-IP of Flag-RORγ co-transfected with indicated kinases (IKKα, JNK2, etc.) in HK2 cells, followed by immunoblotting. K Co-IP of Flag-RORγ WT or S510A in HK2 cells transfected with AMPKα1 siRNA, followed by immunoblotting. L Top: Co-IP of RORγ from mouse kidney lysates. Bottom: Co-IP of Flag-RORγ co-transfected with HA-AMPKα1 in HK2 cells. M Left: Schematic of HK2/shAMPKα1 cell transfection with siRNA-resistant AMPKα1 constructs. Right: Co-IP of Flag-RORγ. N Left: Schematic of RORγ deletion mutants. Right: Co-IP of Flag-RORγ deletion mutants co-expressed with HA-AMPKα1 in HEK293T cells. Data are representative of three biological replicates with similar results ( G , I , J – N ). Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by two-tailed Student’s unpaired t -test ( A , B , D , E , and H ). P values calculated by one-way ANOVA with Bonferroni’s correction ( C and F ). Panel M was created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: Rorγ flox/flox (C57BL/6 JCya- Rorc em1flox /Cya; Strain ID: CKOCMP-19885-Rorc-B6J-VA), Cab39 flox/flox (C57BL/6JCya- Cab39 em1flox /Cya; Strain ID: CKOCMP-12283-Cab39-B6J-VA), Sting1 -/- (C57BL/6NCya -Sting1 em1 / Cya; Strain ID: KOCMP-72512-Sting1-B6N-VA), C57BL/6J mice, and Cdh16-Cre mice (Cat#: C001452) were obtained from Cyagen Biosciences (Guangzhou) Inc (Guangzhou, Guangdong, China).

Techniques: Western Blot, Control, Expressing, Transfection, ChIP-qPCR, Binding Assay, Methylation, Immunofluorescence, Co-Immunoprecipitation Assay, Construct, Two Tailed Test

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A ChIP-qPCR of RORγ binding to the YOD1 promoter in HK2 cells transfected with RORγ-S510D and treated with HGPA ± Act-D for 16 h ( n = 5 biological replicates). B Left: Database schematic of RORγ post-translational modification sites. Right: Cross-species conservation of RORγ K37. C Co-IP of Flag-RORγ from HK2 cells treated with HGPA, followed by immunoblotting. D Co-IP of Flag-RORγ WT, K37R, or K37Q from HK2 cells treated with HGPA, followed by immunoblotting. E Left: Co-IP of Flag-RORγ WT or K37R from HK2 cells treated with or without HGPA. Right: ChIP-qPCR of Flag-RORγ (S510D or S510D + K37R) binding to the YOD1 promoter ( n = 5 biological replicates). F Co-IP of Flag-RORγ co-transfected with different SIRTs in HK2 cells treated with HGPA, followed by immunoblotting. G Co-IP of endogenous RORγ from WT TECs transfected with SIRT1 or siSIRT1 and treated with HGPA, followed by immunoblotting. H Co-IP of RORγ from mouse kidney lysates, followed by immunoblotting. I Co-IP of Flag-RORγ co-transfected with HA-SIRT1 in HK2 cells, followed by immunoblotting. J Cross-species conservation of the RORγ LEDLL motif. K HK2 cells co-transfected with HA-SIRT1 and Flag-RORγ WT or LEDAA mutant, then treated with HGPA. Left: Co-IP of Flag-RORγ. Right: ChIP-qPCR of RORγ binding to the YOD1 promoter ( n = 5 biological replicates). L Co-IP of Flag-RORγ from WT TECs transfected with siSIRT1 and treated with HGPA, followed by immunoblotting. M Co-IP of Flag-RORγ co-transfected with HA-SIRT1 WT or catalytically dead H363Y mutant in HK2 cells, followed by immunoblotting. N Co-IP of RORγ from WT or Rorγ KO TECs treated with the AMPK agonist CDC, followed by immunoblotting. O SIRT1 mRNA levels and enzymatic activity in WT or Rorγ KO TECs treated with CDC ( n = 5 biological replicates). These experiments were repeated independently 3 times with similar results ( C , D , F – I , L – M ). Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by two-tailed Student’s unpaired t -test ( A ). P values calculated by one-way ANOVA with Bonferroni’s correction ( E , K , and O ). Panel M was created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: Rorγ flox/flox (C57BL/6 JCya- Rorc em1flox /Cya; Strain ID: CKOCMP-19885-Rorc-B6J-VA), Cab39 flox/flox (C57BL/6JCya- Cab39 em1flox /Cya; Strain ID: CKOCMP-12283-Cab39-B6J-VA), Sting1 -/- (C57BL/6NCya -Sting1 em1 / Cya; Strain ID: KOCMP-72512-Sting1-B6N-VA), C57BL/6J mice, and Cdh16-Cre mice (Cat#: C001452) were obtained from Cyagen Biosciences (Guangzhou) Inc (Guangzhou, Guangdong, China).

Techniques: ChIP-qPCR, Binding Assay, Transfection, Modification, Co-Immunoprecipitation Assay, Western Blot, Mutagenesis, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A Exosome construction schematic diagram. B Left panel: Representative transmission electron microscope (TEM) image of exosomes is shown. Right panel: Nanoparticle Tracking Analysis of exosomes is shown. This experiment was repeated independently 3 times with similar results. C Human or mouse TECs were treated with exoRORγ as indicated. The protein levels of RORγ determined by Western blot. D , E This experiment was repeated independently 3 times with similar results. HK2 cells incubated with exoRORγ as indicated. D : Left panel: the mRNA levels of CAB39 and YOD1 were detected by qPCR ( n = 5 biological replicates). Right panel: luciferase reporter assays showing the activity of YOD1 promoter fragments in HEK293T cells ( n = 5 biological replicates). E : total cell lysates were prepared and subjected to western blotting using the indicated antibodies. F A schematic representation of the experimental design for kidney sampling in male WT DKD mice treated with exoRORγ or not (exoRORγ was administered via renal in situ injection). G Left panel: the total cell lysates from the kidneys of mice were prepared, and western blotting was performed using the indicated antibodies. Right panel: the mRNA levels of Cab39 and Yod1 in the kidneys of mice were detected by qPCR ( n = 5). H Representative images of H&E and Sirius red staining in kidney sections from mice are shown. I The KW/BW, UACR, BUN, renal cholesterol, KIM-1 mRNA levels, and tubular injury score of mice were measured ( n = 6). J The expression of ISGs and cholesterol synthesis genes in the kidneys from mice were detected by qPCR ( n = 6). Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by two-tailed Student’s unpaired t -test ( D ). P values calculated by one-way ANOVA with Bonferroni’s correction ( G , I , and J ). Panels A and F were created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: Rorγ flox/flox (C57BL/6 JCya- Rorc em1flox /Cya; Strain ID: CKOCMP-19885-Rorc-B6J-VA), Cab39 flox/flox (C57BL/6JCya- Cab39 em1flox /Cya; Strain ID: CKOCMP-12283-Cab39-B6J-VA), Sting1 -/- (C57BL/6NCya -Sting1 em1 / Cya; Strain ID: KOCMP-72512-Sting1-B6N-VA), C57BL/6J mice, and Cdh16-Cre mice (Cat#: C001452) were obtained from Cyagen Biosciences (Guangzhou) Inc (Guangzhou, Guangdong, China).

Techniques: Transmission Assay, Microscopy, Western Blot, Incubation, Luciferase, Activity Assay, Sampling, In Situ, Injection, Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A Predicted structural model of GMNT (Ganodermanontriol) (red dashed box) bound to the LBD and zf-C4 domain (black dashed box) of RORγ. B Molecular docking between the GMNT and RORγ. Green represents hydrogen bonding, pink represents PI-PI stacking, and green amino acids at the periphery for van der Waals forces. C RORγ mRNA (qPCR) and protein (immunoblot) levels in human or mouse TECs treated with GMNT for 24 h ( n = 5 biological replicates). D Luciferase activity of the YOD1 promoter in HEK293T cells ( n = 5 biological replicates). E , F HK2 cells treated with GMNT (10 or 20 µM) for 24 h. E: CAB39 and YOD1 mRNA levels (qPCR, n = 5 biological replicates). F ChIP-qPCR of RORγ binding to the CAB39 or YOD1 promoter ( n = 3 biological replicates). G Immunoblot analysis of WT or Rorγ KO TECs treated with HGPA and/or GMNT (10 µM) for 24 h. This experiment was repeated independently 3 times with similar results. H Left: Schematic of RORγ deletion mutants. Right: Pull-down of Flag-RORγ deletion mutants with GMNT-Biotin in 293 T cells. This experiment was repeated independently 3 times with similar results. I Schematic of kidney sampling in WT DKD mice treated with or without GMNT. J Left: the total cell lysates from kidney of mice were prepared and subjected to western blotting using the indicated antibodies. Right: the mRNA levels of Cab39 and Yod1 in the kidneys of mice were detected by qPCR ( n = 5). K Representative images of H&E and Sirius red staining in kidney sections from mice are shown. L The KW/BW, UACR, BUN, renal cholesterol, KIM-1 mRNA levels, and tubular injury score of mice were measured ( n = 6 mice). M The expression of ISGs and cholesterol synthesis genes in the kidneys from mice ( n = 6 mice). Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by two-tailed Student’s unpaired t -test (F). P values calculated by one-way ANOVA with Bonferroni’s correction ( C , D , E , J , L , and M ). Panel I was created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: Rorγ flox/flox (C57BL/6 JCya- Rorc em1flox /Cya; Strain ID: CKOCMP-19885-Rorc-B6J-VA), Cab39 flox/flox (C57BL/6JCya- Cab39 em1flox /Cya; Strain ID: CKOCMP-12283-Cab39-B6J-VA), Sting1 -/- (C57BL/6NCya -Sting1 em1 / Cya; Strain ID: KOCMP-72512-Sting1-B6N-VA), C57BL/6J mice, and Cdh16-Cre mice (Cat#: C001452) were obtained from Cyagen Biosciences (Guangzhou) Inc (Guangzhou, Guangdong, China).

Techniques: Western Blot, Luciferase, Activity Assay, ChIP-qPCR, Binding Assay, Sampling, Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A GSEA showing the enrichment of Cytosolic DNA-sensing pathway. B WT or Rorγ KO TECs were incubated with or without HGPA (30 mmol/L glucose and 200 μM PA) for 24 h. Left panel: immunoblot of whole-cell lysates with indicated antibodies. Right panel: ISGs were determined by qPCR ( n = 5 biological replicates). C Left panel: Rorγ KO TECs were transfected with Ad-null, Ad- Rorγ , or Ad- Rorγ E502Q (E502Q) for 12 h, replaced with fresh medium culture for 48 h, and then cultured with HGPA for an additional 24 h. Middle panel: immunoblot analysis. Right panel: ISGs were determined by qPCR ( n = 5 biological replicates). D , E TECs were isolated from WT, Rorγ KO , Rorγ KO Sting1 KO , or Rorγ KO cGas KO mice. Left panel: Immunoblot analysis. Right panel: ISGs were determined by qPCR ( n = 5 biological replicates). F , G F: Rorγ KO Sting KO TECs were transfected with Ad-null, Ad- Sting WT ( Sting WT ), Ad- Sting Y239S (Y239S), or Ad- Sting S365A (S365A) for 12 h, replaced with fresh medium culture for 48 h (WT or Rorγ KO TECs were transfected with Ad-null as a control), and then incubated with HGPA for 24 h. G Left panel: Immunoblot analysis. Right panel: heat map showing the expression of ISGs. H Immunoblot analysis of kidney lysates. I Representative Images of H&E and Sirius red staining in kidney sections from mice are shown. J KW/BW, UACR, BUN, KIM-1 mRNA levels, and tubular injury score of mice ( n = 6 mice). K Kidney cholesterol content (left panel) and mRNA levels of CSGs and ISGs (right panel) of kidneys from mice ( n = 6 mice). Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by one-way ANOVA with Bonferroni’s correction. Panels C and F were created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: In antibody reaction buffer (PBS plus 1 % BSA, 0.3 % Triton X-100 at pH 7.4), samples were stained with primary antibodies against RORγ (1:50, Merck, Cat. No. ZRB1398) antibody overnight at 4 °C.

Techniques: Incubation, Western Blot, Transfection, Cell Culture, Isolation, Control, Expressing, Staining

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A WT or Rorγ KO TECs were subjected to an Opti-Prep gradient ultracentrifugation. Ten fractions were collected, diluted, and subjected to immunoprecipitation using an anti-STING. ERGIC-53, ERGIC marker; CALR, ER marker; GM130, Golgi marker. This experiment in this figure was repeated independently 3 times with similar results. B WT or Rorγ KO TECs were subjected to homogenization and cell fractionation using gradient centrifugation. Immunoblotting analyses were performed with the indicated antibodies. Calnexin, ER marker; Golgin97, Golgi marker. This experiment in this figure was repeated independently 3 times with similar results. C , D WT or Rorγ KO TECs were treated with HGPA as indicated in the figure. At different time points, the cells were subjected to immunofluorescence staining for STING1 and ERGIC-53, followed by colocalization analysis (for each group, three biological replicates were set up, and four random fields of view were selected for statistical analysis). E – H qPCR analysis of the expression of cholesterol-synthesis genes and ISGs in WT, Rorγ KO , Rorγ KO Srebf2 KD and Rorγ KO Srebf2 KD TECs reconstituted with shRNA-resistant SREBP2 wild type (FL) or shRNA-resistant transcription-inactive mutants (L511A/S512A, ΔbHLH) ( n = 5). ( I, J ) WT or Rorγ KO TECs were incubated with Trip (14 μM) or vehicle, or transfected with Ad-null or Ad Insig1 as indicated. The total cell lysates and nuclear fractions were prepared and subjected to western blotting using the indicated antibodies (left). (Right) qPCR analysis of the expression of CSGs and ISGs in cells ( n = 5). K HK2 cells were transfected with INSIG1 shRNA (sh INSIG1 ), Ad- RORγ , Ad- INSIG1 WT (shRNA-resistant), or Ad- INSIG1 D205A (shRNA-resistant) as indicated (upper panel). The total cell lysates and nuclear fractions were prepared and subjected to western blotting using the indicated antibodies (bottom panel). This experiment was repeated independently 3 times with similar results. Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by one-way ANOVA with Bonferroni’s correction. G and K were created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: In antibody reaction buffer (PBS plus 1 % BSA, 0.3 % Triton X-100 at pH 7.4), samples were stained with primary antibodies against RORγ (1:50, Merck, Cat. No. ZRB1398) antibody overnight at 4 °C.

Techniques: Immunoprecipitation, Marker, Homogenization, Cell Fractionation, Gradient Centrifugation, Western Blot, Immunofluorescence, Staining, Expressing, shRNA, Incubation, Transfection

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A , B WT or Rorγ KO TECs were treated with CHX (100 ng/ml) or MG132 (10 µM) as indicated, followed by immunoblot analysis. C Co-IP of INSIG1 was performed from lysates of WT or Rorγ KO TECs with immunoblotting. D WT TECs were transfected with the indicated siRNAs and analyzed by immunoblotting. E Co-IP of INSIG1 was performed from lysates of WT or Yod1 KO TECs with immunoblotting. F HK2 cells were transfected with Ad-Flag-YOD1 WT or C160S mutant. Right: Co-IP of INSIG1 was performed. G Schematic of potential RORγ post-translational modification sites from database. H Left: Diagram of INSIG1 cytosolic lysines. Right: HK2 cells were transfected with shYOD1 and Ad-Flag-INSIG1 WT or K → R mutants (K33R, K156R, K158R, K273R) for immunoblot analysis. I Left: Conservation of INSIG1 K156/K158. Right: HK2 cells were transfected with shYOD1 and Ad-Flag-INSIG1 WT or DKR mutant (K156R/K158R) for Co-IP with anti-Flag. J Left: Co-IP of INSIG1 from mouse kidney lysates. Middle/Right: WT TECs were co-transfected with Ad-HA-Yod1 and/or Ad-Flag-Insig1 WT for Co-IP with anti-Flag. K Left: Predicted transmembrane topology of INSIG1. Right: IP of INSIG1 truncation mutants expressed in HK2 cells was performed. L Bottom: Immunoblot analysis of WT or Rorγ KO TECs. Top: Yod1 mRNA was analyzed by qPCR ( n = 5 biological replicates). M YOD1 promoter sequence with predicted RORγ binding sites (RORE1, RORE2) and mutations (RORE1m, RORE2m). N Luciferase activity of truncated YOD1 promoter fragments was measured in HEK293T cells ( n = 3 biological replicates). O , P HK2 cells were transfected with Ad-RORγ. Left: ChIP was performed using anti-RORγ antibody at the YOD1 promoter. Right: qPCR quantification ( n = 5 biological replicates). Q Luciferase assay of YOD1 promoter with mutated RORE sites was performed in HEK293T cells co-transfected with Ad-Rorγ ( n = 3 biological replicates). R Co-IP of INSIG1 from mouse kidney lysates was performed. Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by two-tailed Student’s unpaired t -test ( M ). P values calculated by one-way ANOVA with Bonferroni’s correction ( N , P , and Q ). Panels F , H , and K were created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: In antibody reaction buffer (PBS plus 1 % BSA, 0.3 % Triton X-100 at pH 7.4), samples were stained with primary antibodies against RORγ (1:50, Merck, Cat. No. ZRB1398) antibody overnight at 4 °C.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Modification, Sequencing, Binding Assay, Luciferase, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A Exosome construction schematic diagram. B Left panel: Representative transmission electron microscope (TEM) image of exosomes is shown. Right panel: Nanoparticle Tracking Analysis of exosomes is shown. This experiment was repeated independently 3 times with similar results. C Human or mouse TECs were treated with exoRORγ as indicated. The protein levels of RORγ determined by Western blot. D , E This experiment was repeated independently 3 times with similar results. HK2 cells incubated with exoRORγ as indicated. D : Left panel: the mRNA levels of CAB39 and YOD1 were detected by qPCR ( n = 5 biological replicates). Right panel: luciferase reporter assays showing the activity of YOD1 promoter fragments in HEK293T cells ( n = 5 biological replicates). E : total cell lysates were prepared and subjected to western blotting using the indicated antibodies. F A schematic representation of the experimental design for kidney sampling in male WT DKD mice treated with exoRORγ or not (exoRORγ was administered via renal in situ injection). G Left panel: the total cell lysates from the kidneys of mice were prepared, and western blotting was performed using the indicated antibodies. Right panel: the mRNA levels of Cab39 and Yod1 in the kidneys of mice were detected by qPCR ( n = 5). H Representative images of H&E and Sirius red staining in kidney sections from mice are shown. I The KW/BW, UACR, BUN, renal cholesterol, KIM-1 mRNA levels, and tubular injury score of mice were measured ( n = 6). J The expression of ISGs and cholesterol synthesis genes in the kidneys from mice were detected by qPCR ( n = 6). Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by two-tailed Student’s unpaired t -test ( D ). P values calculated by one-way ANOVA with Bonferroni’s correction ( G , I , and J ). Panels A and F were created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: In antibody reaction buffer (PBS plus 1 % BSA, 0.3 % Triton X-100 at pH 7.4), samples were stained with primary antibodies against RORγ (1:50, Merck, Cat. No. ZRB1398) antibody overnight at 4 °C.

Techniques: Transmission Assay, Microscopy, Western Blot, Incubation, Luciferase, Activity Assay, Sampling, In Situ, Injection, Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

doi: 10.1038/s41467-026-69724-2

Figure Lengend Snippet: A Predicted structural model of GMNT (Ganodermanontriol) (red dashed box) bound to the LBD and zf-C4 domain (black dashed box) of RORγ. B Molecular docking between the GMNT and RORγ. Green represents hydrogen bonding, pink represents PI-PI stacking, and green amino acids at the periphery for van der Waals forces. C RORγ mRNA (qPCR) and protein (immunoblot) levels in human or mouse TECs treated with GMNT for 24 h ( n = 5 biological replicates). D Luciferase activity of the YOD1 promoter in HEK293T cells ( n = 5 biological replicates). E , F HK2 cells treated with GMNT (10 or 20 µM) for 24 h. E: CAB39 and YOD1 mRNA levels (qPCR, n = 5 biological replicates). F ChIP-qPCR of RORγ binding to the CAB39 or YOD1 promoter ( n = 3 biological replicates). G Immunoblot analysis of WT or Rorγ KO TECs treated with HGPA and/or GMNT (10 µM) for 24 h. This experiment was repeated independently 3 times with similar results. H Left: Schematic of RORγ deletion mutants. Right: Pull-down of Flag-RORγ deletion mutants with GMNT-Biotin in 293 T cells. This experiment was repeated independently 3 times with similar results. I Schematic of kidney sampling in WT DKD mice treated with or without GMNT. J Left: the total cell lysates from kidney of mice were prepared and subjected to western blotting using the indicated antibodies. Right: the mRNA levels of Cab39 and Yod1 in the kidneys of mice were detected by qPCR ( n = 5). K Representative images of H&E and Sirius red staining in kidney sections from mice are shown. L The KW/BW, UACR, BUN, renal cholesterol, KIM-1 mRNA levels, and tubular injury score of mice were measured ( n = 6 mice). M The expression of ISGs and cholesterol synthesis genes in the kidneys from mice ( n = 6 mice). Source data are provided as a Source Data file. Data are presented as the mean ± SD. P values calculated by two-tailed Student’s unpaired t -test (F). P values calculated by one-way ANOVA with Bonferroni’s correction ( C , D , E , J , L , and M ). Panel I was created in BioRender. Shu Yang. (2025) https://biorender.com .

Article Snippet: In antibody reaction buffer (PBS plus 1 % BSA, 0.3 % Triton X-100 at pH 7.4), samples were stained with primary antibodies against RORγ (1:50, Merck, Cat. No. ZRB1398) antibody overnight at 4 °C.

Techniques: Western Blot, Luciferase, Activity Assay, ChIP-qPCR, Binding Assay, Sampling, Staining, Expressing, Two Tailed Test