Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways

doi: 10.12659/MSM.913384

Figure Lengend Snippet: Expression of death receptor-associated apoptotic markers in the cobalt chloride (CoCl 2 )-treated human uterosacral ligament fibroblasts (hUSLFs). ( A ) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Fas, decoy receptor 2 (DcR2), cellular FLICE inhibitory protein (c-FLIP), death receptor 5 (DR5), and cleaved caspase-8 protein levels by Western blot analysis in hUSLFs treated with the chemical inducer of hypoxia, cobalt chloride (CoCl 2 ), at 48 h. ( B ) Quantitative analysis of Fas, TRAIL, DcR2, c-FLIP, and DR5 expression standardized to β-actin. ( C ) Caspase-8 activity measured in hUSLFs treated with CoCl 2 for 48 h. ( D ) After 48 h, the effects of CoCl 2 on the levels of lactate dehydrogenase (LDH) in hUSLFs were measured. ( E ) Results of the MTT assay for cell viability following treatment with CoCl 2 for 48 h with or without the presence of 50 μM of the caspase-8 inhibitor, Z-IETD-FMK. * P <0.05, ** P <0.01. Con – control. C-cas-8 – cleaved caspase-8.

Article Snippet: After pretreated with and without 50 μM of the caspase-8 inhibitor, Z-IETD-FMK (MedChem Express, Monmouth Junction, NJ, USA), and treatment with and without 100 μM of the caspase-9 inhibitor, Z-LEHD-FMK (KeyGen Biotech Co. Ltd., Nanjing, China) for 2 h, cells were treated with CoCl 2 48 h later.

Techniques: Expressing, Western Blot, Activity Assay, MTT Assay, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways

doi: 10.12659/MSM.913384

Figure Lengend Snippet: Expression of mitochondrial-associated apoptotic markers in the cobalt chloride (CoCl 2 )-treated human uterosacral ligament fibroblasts (hUSLFs). ( A ) Bcl-2, Bax, cytochrome C, Bcl-2 interacting protein 3 (BNIP3), cleaved caspase-3, and cleaved caspase-9 protein levels were analyzed by Western blot in human uterosacral ligament fibroblasts (hUSLFs) that were treated with the chemical inducer of hypoxia, cobalt chloride (CoCl 2 ) at 48 h. ( B ) Quantitative analysis of Bcl-2/Bax, cytochrome C, BNIP3, cleaved caspase-3, and cleaved caspase-9 protein expression standardized to β-actin. ( C ) Caspase-3, and caspase-9 activity measured in hUSLFs that were treated with CoCl 2 for 48 h. ( D ) Results of the MTT assay for cell viability following treatment with CoCl 2 for 48 h, which were also exposed to 100 μM of the caspase-9 inhibitor, Z-LEHD-FMK, with or without the presence of 50 μM of the caspase-8 inhibitor, Z-IETD-FMK. ( E ) Photomicrograph of JC-1 fluorescence mitochondrial imaging used to study the change in mitochondrial membrane potential in the different groups. ( F ) The relative mitochondrial membrane potential level was evaluated by comparing the red fluorescence cells to green fluorescence cells in the groups. * P <0.05, ** P <0.01. Scale bar=100 μm. Con – control; C-cas-3 – cleaved caspase-3; C-cas-9 – cleaved caspase-9; Cyto c – cytochrome C.

Article Snippet: After pretreated with and without 50 μM of the caspase-8 inhibitor, Z-IETD-FMK (MedChem Express, Monmouth Junction, NJ, USA), and treatment with and without 100 μM of the caspase-9 inhibitor, Z-LEHD-FMK (KeyGen Biotech Co. Ltd., Nanjing, China) for 2 h, cells were treated with CoCl 2 48 h later.

Techniques: Expressing, Western Blot, Activity Assay, MTT Assay, Fluorescence, Imaging, Membrane, Control

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a , Phylogenetic analysis and chromosomal locus of human and mouse inflammatory caspases. b , c , CASP4 ( b ) and CASP5 ( c ) RNA expression summary in human tissues. From Human Protein Atlas; y-axis, normalized TPM (transcripts per million). d , qRT-PCR of indicated inflammatory caspase and inflammasome genes in human colonic intestinal epithelial cells (IEC) from biopsy samples (LC: left colon, RC: right colon), colonic organoids, and monocyte derived macrophages (MDM). e , Immunoblots of CASP5 in MDM, colonic or small intestinal organoid whole cell extracts (WCE) with catalytic domain-targeting antibody. f , Schematic of CASP5a-Myc-BioID2 construct and confocal Z-projection of a representative human colonic organoid. g , Top 30 proteins identified in CASP5a-Myc-BioID2 colonic organoids but not Myc-BioID2 controls (ranked by combined peptide-spectrum match (PSM) from untreated and S . Typhimurium-infected 2D differentiated colonic organoids). Dishevelled (DVL) proteins in bold. h , Enrichment analysis of top 60 proteins identified in CASP5a-Myc-BioID2 colonic organoids but not Myc-BioID2 controls (ranked by combined PSM). i , Post-immunoprecipitation (IP) with endogenous CASP5 using anti-CASP5 antibody or isotype control. j , Biotinylated DVL1–3 post-pull-down with NeutrAvidin beads upon Myc-BioID2 or catalytically inactive CASP5a (C315A)-Myc-BioID2 expression. k , l , Post-IP with ectopically expressed FLAG-tagged CASP5a, b and c ( k ), or FLAG-tagged DVL2 ( l ) using anti-FLAG antibody or isotype control. m , Immunoblots on anti-HA IPs of HA-tagged CASP5a (C315A) with co-IP FLAG-tagged DVL2 and domain-deleted forms of both proteins expressed in HEK293T cells. Results represent ≥3 independent experiments (colonic organoids from either of 2 donors/experiment) ( e,i-m ); colonic organoid results from donor 1483 ( e,f,i,j ) and donors 1483/1494 ( d ) are shown (donor details and colonic segment in Methods). n=11 for CASP5, n=13 for CASP4/1, n=12 for the rest (LC/RC in d ); n=3 (organoids/MDM in d ). One-way ANOVA with Tukey’s post-hoc test ( d ); Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: RNA Expression, Quantitative RT-PCR, Derivative Assay, Western Blot, Construct, Infection, Immunoprecipitation, Control, Expressing, Co-Immunoprecipitation Assay

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a–c , TOPflash reporter activity, untreated (Ctrl) or stimulated with Wnt3a conditioned media (Wnt3a CM, 30% unless specified) ( a ), qRT-PCR of AXIN2 and TCF7 ( b ), and representative confocal micrographs ±Wnt3a CM (2 hr, white arrows, nuclear β-catenin) ( c ) in CASP5a, CASP5b, or CASP5c-expressing HEK293T cells. d , TOPflash reporter activity in CASP5c, CASP5c/a, or CASP5c/b-expressing HEK293T cells. e , Post-IP with FLAG-tagged DVL2 using anti-FLAG antibody. f , g , TOPflash activity in human colonic ( f ) and small intestinal ( g ) organoids with CASP5a, b or c-expression. h , Immunoblots of β-catenin and β-tubulin in cytosolic fractions from colonic organoids ectopically expressing CASP5c vs. Ctrl. i , j , qRT-PCR of indicated genes in CASP5a, b or c-expressing colonic organoids in differentiation media. k , l , Relative ATP levels ( k ) and representative confocal micrographs ( l ) of colonic organoids in differentiation media (5% Wnt3a CM). m , Phase-contrast micrographs and ATP measurements of Ctrl or CASP5c-expressing colonic organoids in differentiation media (Wnt-free) for 2 weeks. n , o , Immunoblots ( n ) and qRT-PCR ( o ) for CASP5 and CASP4 in colonic/small intestinal organoids in differentiation media without (Ctrl) or with 50% Wnt3a CM (Wnt3a). p – r , qRT-PCR of indicated genes in colonic organoids in expansion media, unirradiated or at 24/48 hr post-irradiation (6 Gy). Results represent ≥3 independent experiments ( a – r ); colonic organoid results from donor 1483 ( f , h , i , j , n left, o left), and 1494 ( k , l , m left, p – r ), and from both donors ( m right); small intestinal from donor HT-291 ( g , o right) and both donors ( n middle/right); a , b , d , f , g , h–j , p – r , n=3, c , n=322,452,325,334,470 (left to right), k , m , n=6, l , n=24,26,29,66 (diameter), n=44,39,45,55 (Ki-67), n=16,18,17,54 (EdU), n=50,37,47,61 (CK20), o , n=3 (left), n=5 (right). One-way ANOVA with Tukey’s post-hoc test ( a – d , g – l , q , r ); Two-way ANOVA with Tukey’s post-hoc test ( f , p ); Two-tailed unpaired t-test ( m , o ); Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Irradiation, Two Tailed Test

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a , Immunoblots of AXIN1 IP from control (Ctrl) untreated or Wnt3a CM treated (2 hr) HEK293T cells without (Ctrl) or with CASP5a, CASP5b, CASP5c, or CASP5c (C173A) expression. b , c , Immunoblots on AXIN1 IP from CASP5c-expressing or control HEK293T ( b ), and β-catenin IP from CASP5c and/or HA-tagged ubiquitin (HA-UB)-expressing or Ctrl HEK293T cells ( c ). HEK293T treated with 10 μM MG132 ( b , c ) and/or Wnt3a CM (2 hr). d , CASP5 activity from HEK293T cell culture medium expressing indicated CASP5 constructs, with or without GSDMD. e, Immunoblot of CASP5 in NeutrAvidin pull-down fractions from HEK293T cells expressing indicated CASP5 proteins treated with biotinylated (b)VAD-fmk. f , g , TOPflash activity in HEK293T cells expressing CASP5c, CASP5-ΔCARD, or CASP5c (C173A). h , Immunoblot of β-catenin on cytosolic fractions from HEK293T cells untreated or Wnt3a CM-treated (2 hr). i , Confocal micrographs of HEK293T cells transfected with CASP5c or CASP5c (C173A). White arrows, nuclear β-catenin. j , qRT-PCR of AXIN2 and TCF7 in HEK293T cells expressing indicated CASP5 proteins. k , Schematics of full-length and truncated APC. l , Immunoblot of APC from HEK293T cells expressing truncated APC or mutants with/without co-expression of CASP5c or CASP5c (C173A), treated 18 hr with indicated caspase inhibitors. m , Activity of recombinant CASP5c at increasing doses (left). Immunoblots for in vitro CASP5c cleavage of APC, enriched from transfected HEK293T cells (right). n , Immunoblots on AXIN1 IP from CRISPR-generated APC-KO HEK293T cells transfected with indicated plasmids, Wnt3a CM-treated (2 hr). o , Immunoblots on AXIN1 IP from WT or APC-KO HEK293T cells without (Ctrl) or with ectopic expression of APC-FL, APC-1–556, or APC-557–2843. Results represent ≥3 independent experiments; f , g , h , j , l , m , n=3, d , n=7,8,8,8,3,6,6,6,6,6,3 (left to right), i , n=205,551,818. One-way ANOVA with Tukey’s post-hoc test ( d , f–j , l , m ); Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Western Blot, Control, Expressing, Ubiquitin Proteomics, Activity Assay, Cell Culture, Construct, Transfection, Quantitative RT-PCR, Recombinant, In Vitro, CRISPR, Generated

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a , b , t-distributed stochastic neighbor embedding (t-SNE) plot ( a ) and single-cell 2D CASP5 , CASP4 and LGR5 expression maps ( b ) of IEC from healthy adult colonic tissues. c, Dot plots showing average expression and % expressed of LGR5, CASP4 and CASP5 in indicated colonic cell types. d , Distribution of CASP5 -expressing cells as % in colonic cell types. Color legend as in ( a ). a – d , Gene expression and cell type annotation from Smillie et al . 40 . e , Dot plots showing average expression and % expressed of LGR5, CASP4 and CASP5 in indicated cell types from healthy human small intestine (SI) or colon. Gene expression and cell type annotation from Burclaff et al . 41 . f , g , Representative BaseScope in situ hybridization images of total CASP5 transcripts ( f ) and isoforms co-detected with cell type/Wnt target genes ( g ) in colonic epithelium. Pseudocolored puncta for tissue-scale visualization; top insets show enlarged actual signals. f (right), h , Quantitative measurements of signal puncta positions and IHC-positive cell nuclei from 5–6 donors. Distances of signal puncta and nuclei of positive stained cells from crypt bottoms were normalized to crypt length (1 to several crypts/donor). i , Representative IHC images of OLFM4 (intestinal stem/progenitor cells), Ki-67 (TA cells), and CK20 (differentiated enterocytes) in colonic epithelium from 5–6 donors. j , Correlation between each CASP5 isoform and deconvoluted cell types from TCGA bulk RNA-sequencing of adjacent normal colonic tissues. k , CASP5 isoform expression from full-length single-cell RNA-sequencing data of adjacent normal colonic tissues. Each color indicates a different lineage based on pseudotime analysis. l , Proportion of CASP5 isoform-expressing cells by cell lineage. k , l , Data are from Li et al . 45 . f , n=559, h , n=161,157,167,705,590,182,409,395,399 (left to right). Box plots describe the median and interquartile range (IQR) of each gene, whiskers depict 10–90 percentile ( f , h ).

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Single Cell, Expressing, Gene Expression, In Situ Hybridization, Staining, RNA Sequencing

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a–c , CASP5 isoform correlation with ( a ) genes in full-length single-cell sequencing of normal colonic tissues (positive correlation, Venn diagram), ( b ), transcription factor enrichment analysis of genes, ( c ) Waterfall plots of genes ordered by Pearson’s correlation coefficient. Wnt-associated genes from HALLMARK in black/red (p<0.05). d , qRT-PCR of CASP5 isoforms and LGR5, MKI67 , ALPI over time in colonic organoids switched from expansion to differentiation media (Wnt3a withdrawal; d0.5-d3). e , Immunoblots of colonic organoids switched to differentiation media (Wnt3a withdrawal; left), or BMP differentiation media (Wnt3a withdrawal, –Noggin+BMP2/4; right). f,i , Immunoblot of ubiquitin (Ub) and indicated proteins in anti-β-catenin IP from d1 vs. d3 organoids treated with 10 μM MG132 (2 hr). g , Caspase activity from WT or CASP5c-expressing organoids differentiated for 1 or 3 days. h , TOPflash activity in CASP5a/b and CASP5a/b/c siRNA colonic organoids. j , Immunoblots of FLAG-DVL2 and CASP5 in anti-FLAG IP from d1 vs. d3 differentiated FLAG-DVL2-expressing organoids. k , Schematic of CASP5 isoform roles in Wnt regulation in the intestinal epithelium. l , Representative confocal micrographs of CASP5a/b or CASP5a/b/c siRNA colonic organoids in differentiation media ±5% Wnt3a CM. m , qRT-PCR of indicated genes in CASP5a/b or CASP5a/b/c siRNA colonic organoids in differentiation media. n,o , qRT-PCR of indicated genes in IEC from healthy donors vs. Crohn’s disease (CD) and ulcerative colitis (UC) patients. Results represent ≥3 independent experiments (colonic organoids from 2 donors/experiment) ( d – j , l – m ); colonic organoid results from donor 1483 ( d – j ) and 1494 ( l , m ); d , h , i , j , m , n=3, g , n=9 (Ctrl), n=6 (CASP5c), l , n= n=94,49,62 (Diameter), n=53,12,24 (EdU), n=41,32,38 (Ki-67), n=6,6,6 (ATP), n , n=42,45,45,42,45,44,42,43,43 (left to right), o , n=24,39,39 ( AXIN2 ), n=23,20,42 ( OLFM4 ), n=35,21,42 ( ZNF277 ). One-way ANOVA with Tukey’s post-hoc test ( l , m , o ); Two-way ANOVA with Tukey’s post-hoc test ( h , n ); Two-tailed unpaired t-test ( g , i , j ); Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Single Cell, Sequencing, Quantitative RT-PCR, Western Blot, Ubiquitin Proteomics, Activity Assay, Expressing, Two Tailed Test

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: The National Library Basic Local Alignment Search Tool protein blast tool was used to conduct pairwise alignment with dots for identities between human CASP4 (UniProt P469662), human CASP5 (UniProt P51878 ), and mouse CASP11 (UniProt P70343 ). a , Human CASP4 and CASP5 amino acid sequences highlighting the CARD domain and LPS binding domains as indicated, as well as percent identity (74%) between CASP4 and CASP5 using CASP4 as the query sequence and CASP5 as the subject sequence. b , Mouse CASP11 protein sequence. c , Percent identity (60%) between human CASP4 and mouse CASP11 using CASP4 as the query sequence and CASP11 as the subject sequence; And percent identity (54%) between human CASP5 and mouse CASP11 using CASP5 as the query sequence and CASP11 as the subject sequence. d , Percent identity (55.84%) of human CASP4 and CASP5 CARD domain amino acid sequences using the CASP4 CARD as the query sequence and CASP5 CARD as the subject sequence. e , Percent identity (40%) of human CASP4 59 amino acid LPS binding region (shown in bold in a ) as the query sequence and the CASP5 N-terminal 59 amino acid as the subject sequence.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Sequencing, Binding Assay

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a, b , Phase-contrast ( a top left) and two-photon ( a remainder) micrographs of three-dimensional (3D) cultured, and phase-contrast ( b top) and confocal ( b bottom) micrographs of two-dimensional (2D) cultured human colonic organoids used for assessing IEC-specific inflammasome activation upon bacterial infection. Organoids were grown in differentiation media and stained with cell fate markers (MUC2–Goblet cells; CHGA–Enteroendocrine cells; Ki-67–Proliferating cells (indicating stem cell niche)). c , 2D cultured colonic organoids grown in differentiation media were stimulated with different Gram-negative bacteria for 16 hr, and biochemical hallmarks of non-canonical inflammasome activation (CASP4/5 sensing cytosolic LPS) were assessed: IL-18 and GSDMD cleavage, which could only be observed in WT S. Typhimurium and S. flexneri ΔOspC3 stimulated cells given their ability to enter the IEC cytosol, while WT S. flexneri virulence factor OspC3 inhibited caspase-4 activity 51 . d , CASP4 and CASP5 shRNA knockdown validation and subsequent stimulation with S. typhimurium and S. flexneri ΔOspC3 (MOI 1) induced GSDMD (N-terminal), IL-18 cleavage in colonic organoids. AIEC 2A (MOI 10) was included as a negative control. e , Colony-forming unit (CFU) of intrahcellular bacteria in 2D cultured colonic organoids with CASP4 or CASP5 siRNA knockdown 16 hr after S. typhimurium or S. flexneri ΔOspC3 (MOI 1) stimulation. Quantification of outcome on intracellular bacterial burdens: CFU increase with CASP4 knockdown (impaired clearance) but not CASP5 knockdown. The N-terminal fragment of GSDMD, generated upon cleavage, directly lyses intracellular bacteria by forming pores in their membranes 52 . f , MDM were stimulated with different Gram-negative bacteria at indicated MOI for 16 hr, and CASP4, CASP1, IL-1β, IL-18, and GSDMD (N-terminal) cleavage were selected as markers of inflammasome activation and analyzed by immunoblotting. g , CFU of intracellular bacteria in MDM with CASP1, CASP4, or CASP5 (isoforms a, b, and c) siRNA knockdown 16 hr after F. novicida (MOI 2.5) stimulation. h , CASP4 and CASP5 isoforms a, b, and c (si CASP5 a/b/c) or isoforms a and b (si CASP5 a/b) were knocked down by siRNA in MDM. Cells were then stimulated with E. coli DH5α (MOI 20) for 16 hr. i , j , CASP4 ( i ) and CASP5 ( j ) RNA expression summary in different cell types. Y-axis represents normalized TPM (transcripts per million). Figures adopted from the Human Protein Atlas database ( https://www.proteinatlas.org ). Immunoblots in c,d,f,h show levels of pro-CASP1, pro-CASP4, pro-IL-1β, pro-IL-18, GSDMD, and cleaved GSDMD (N-terminal) in WCE, and the cleavage of CASP4, CASP1, IL-1β and IL-18 in concentrated supernatants. Cell death evaluated by LDH release, and IL-1β, IL-18 and IL-6 secretion measured by ELISA. AIEC 2A, an intestinal adherent invasive E. coli strain enriched in patients with Crohn’s Disease-associated spondyloarthritis. E. coli DH5α, virulence factor mutant S. typhimurium ΔSpi1/2, and S. flexneri BS103 are avirulent Gram-negative bacteria of counterpart virulent E. coli 11775, S. typhimurium , and S. flexneri . S. flexneri ΔOspC3 is a mutant that retains the ability to invade IEC but lacks OspC3 which inhibits caspase-4 activity 51 . Colonic organoids were differentiated in differentiation media for 3 days. WCE, whole cell extract. Sup., supernatant. *, indicates the position of cleaved proteins. Results represent at least 3 independent experiments (MDM from different donors, colonic organoids from either of the two donors in each experiment); colonic organoid results from donor 1483 ( a–e ) are shown (donor details and intestinal segment in methods); n=3 ( e , g , h ). One-way ANOVA with Tukey’s post-hoc test ( e , g , h ); Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Activation Assay, Bacteria, Cell Culture, Infection, Staining, Activity Assay, shRNA, Knockdown, Biomarker Discovery, Negative Control, Generated, Western Blot, RNA Expression, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a , Amino acid sequences of CASP5 isoforms a, b, and c, showing the regions of different domains as indicated. b , Domain structures and sequence differences of CASP5 isoforms a, b, and c. Open blue and green triangles are a schematic representation of the location of the CASP5 amino acids shown. c , Schematic of CASP5 domain structure from InterPro. Vertical arrows indicate si CASP5a/b and si CASP5a/b/c target sites. d , Immunoblots of CASP5 in MDM or colonic organoid WCE on day 3 post-CASP5 siRNA knockdown. e , Immunoblots of indicated proteins in human MDM or colonic organoid WCE 16 hr after treatment with 20 ng/mL IFN-β or IFN-γ, 100 ng/mL LPS, or Salmonella Typhimurium (MOI=1). f , qRT-PCR of indicated genes in unstimulated or S . Typhimurium-stimulated (MOI=1) colonic organoids and MDM. g , qRT-PCR of indicated inflammatory caspases in small intestinal organoids. h, Anti-Myc immunoblots for ectopically expressed Myc-BioID2 and CASP5a-Myc-BioID2 on human colonic organoid WCEs using anti-Myc antibody (bottom panel shows a longer exposure). i , CASP5 immunoblots for ectopically expressed CASP5 isoforms in HEK293T (transient transfection with 100 ng expression plasmid in one 24 well plate well) or HCT116 (stably transduced cell line) as indicated. j – l , TOPflash luciferase reporter activity in HCT116 ( j ) or HEK293T ectopically expressing indicated caspases ( k , l ). m , CASP5 immunoblots for ectopically co-expressed CASP5 isoforms as indicated in HEK293T (transient transfection with 100 ng expression plasmid in one 24 well plate well). d , e , f , Colonic organoids differentiated for 3 days. Results represent at least 3 independent experiments ( d,e,h-m ); colonic organoid results from donor 1483 ( d , e,h ) and donors 1483/1494 ( f ) are shown (donor details and colonic segment in Methods). f,g,j,k,l , n=3. One-way ANOVA with Tukey’s post-hoc test ( f , g,j ); Two-way ANOVA with Tukey’s post-hoc test ( k , l ); Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Expressing, Sequencing, Western Blot, Knockdown, Quantitative RT-PCR, Transfection, Plasmid Preparation, Stable Transfection, Luciferase, Activity Assay

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a , Phase-contrast micrographs of three-dimensional (3D)-cultured human small intestinal organoids from ileum (left) and duodenum (right). b , Representative confocal micrographs of human ileum organoids grown in expansion or differentiation media as indicated and labeled for Phalloidin (cytoskeleton, yellow), Ki-67 (TA cells, green), MUC2 (Goblet cells, blue), or Nucspot (nuclei, red). c , d , CASP5 immunoblots for ectopically expressed CASP5 isoforms in primary human colonic and small intestinal organoid cells (Tet-On stable colonic organoid lines induced with 1 ng/mL doxycycline). Comparatively lower endogenous CASP5 in control (Ctrl) not visible at exposure shown. e , TOPflash luciferase reporter activity at 16 hr post-stimulation with Wnt3a CM or no stimulation, as indicated, of Ctrl or CASP5a, b or c expressing Tet-On inducible human colonic organoids. f , TOPflash luciferase reporter activity at 16 hr post-stimulation with Wnt3a CM or no stimulation, as indicated, of Ctrl or CASP5a, b or c expressing human small intestinal organoids. g , h , Ctrl or CASP5a, b, or c expressing 2D-cultured colonic organoids grown in differentiation media (No Wnt) and Ctrl organoids stimulated with Wnt3a CM for 2 hr (positive control). Representative confocal micrographs of organoids labeled for β-catenin (red) and Nuclei (DAPI, blue), white arrows indicate representative nuclear β-catenin signals, bar scales=10 μm ( g ), and quantification of nuclear β-catenin signal granularity analyzed using CellProfiler software ( h ). i , RT-qPCR of Wnt-responsive AXIN2 , TCF7 , LGR5 , OLFM4 , and CASP5c transcripts in Ctrl or CASP5c expressing small intestinal organoids grown in differentiation media for 1 day. CASP5c significantly upregulated AXIN2 : 3.1-fold; TCF7 : 3.0-fold; LGR5 : 7.1-fold; OLFM4 : 73.0-fold in small intestinal organoids. j , Representative confocal micrographs of small intestinal organoids labeled for Phalloidin (cytoskeleton), Ki-67 (TA cells), Villin (differentiated IEC), DAPI (nuclei), and EdU incorporation (S-phase cells). Bar scales=50 μm. Bar graphs show quantification, organoid size and labeling measurements using ImageJ software (Fiji); 30% larger size, % Ki-67 + 5.3-fold higher and % EdU + 3.4-fold higher in CASP5c vs control small intestinal organoids. k , IncuCyte live cell imaging derived representative phase-contrast images of Ctrl or CASP5c expressing human small intestinal organoids. l , Phase-contrast micrographs and ATP levels of Ctrl or CASP5c expressing small intestinal organoids grown in differentiation media for 3 days; ATP levels elevated 1.2-fold in CASP5c vs control organoids. m , Phase contrast micrographs of colonic and small intestinal organoids grown in Wnt-free or Wnt-expansion media for 14 days and labeled with Sytox Green. n , Confocal micrographs of colonic organoids in Wnt-free or Wnt-expansion media for 14 days. o , Phase contrast and confocal micrographs of colonic organoids switched to Wnt-expansion medium for 48 hr or 7 days after 14-day culture in Wnt-free media. Labeling for Phalloidin (cytoskeleton), Ki-67 (TA cells) and DAPI (nuclei) ( n , o ). Bar graphs, quantification of Ki-67 + cells and ATP levels from cultures in n , o . Results represent ≥3 independent experiments; colonic organoid results from donor 1483 ( d , e,m,n,o ) and 1494 ( g , h ); small intestinal organoid results from ileum (HT-375, a , c ) or duodenum (HT-291, a , b , d , f , i , j , k – m ). Donor details and intestinal segments in methods; e , f , i , l n=3; h , n=322,452,325,334,470 (left to right); j , n=16,26,17,26 (Diameter), n=16,26,17,26 (Ki-67), n=22,26,25,37 (Edu), n=16,16,18,30 (Villin); n , n=34,27,16,41 (Ki-67), n=3 (ATP); o , n=16,23 (Ki-67), n=3 (ATP). One-way ANOVA with Tukey’s post-hoc test ( e , f , h , j,n ), Two-tailed unpaired t-test ( i , l, and o ); Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Cell Culture, Labeling, Western Blot, Control, Luciferase, Activity Assay, Expressing, Positive Control, Software, Quantitative RT-PCR, Live Cell Imaging, Derivative Assay, Two Tailed Test

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a–d, Colonic organoids grown in expansion media and either not irradiated (No irrad.) or at 24 and 48 hr post-irradiation (6 Gy). a – c , qRT-PCR of GADD45A , CDK1A (DNA damage response markers) ( a ), CASP5c in colonic organoids grown in No Wnt3a CM, 5% Wnt3a CM or 50% Wnt3a CM (expansion media) as indicated ( b ), or CASP5c in Ctrl or CASP5c colonic organoids ( c ). d , Representative confocal micrographs of Ctrl or CASP5c colonic organoids grown in expansion media labeled for Phalloidin, Ki-67 and DAPI. Bar scales=50 μm. Bar graph quantification of Ki-67 labeling using ImageJ software (Fiji). e , Amino acid sequences and DEP domain alignment of mouse DVL1 and human DVL2 proteins. Positively charged aspartic acids in the DEP domain of mouse DVL1 and corresponding residues in human DVL2 are highlighted. f , Tertiary structures of mouse DVL1 protein DEP domain showing positions of D449 and D452 within the DEP electric dipole (boxes). g , Immunoblots for FLAG or HA tagged proteins in anti-FLAG IPs (IP FLAG) or WCE pre-IP (Input). FLAG-tagged DVL2 or mutant DVL2 (D457A, D460A) and/or HA-tagged CASP5a proteins ectopically expressed in HEK293T cells prior to anti-FLAG IP and immunoblotting. h , Quantification of Fig. 3a AXIN IP indicated immunoblot relative band intensities from control (Ctrl) untreated or Wnt3a CM treated (2 hr) HEK293T cells without (Ctrl) or with ectopic expression of CASP5a, CASP5b, CASP5c, or CASP5c (C173A). Band intensities were calculated with ImageJ. i , j , Quantification of indicated immunoblot band intensities in Fig. 3b ( i ) and 3c ( j ). 3 independent experiments were quantified, each symbol is one experiment ( h – j ). Results represent at least 3 independent experiments; colonic organoid results from donor 1483 ( a – d ) are shown. One-way ANOVA with Tukey’s post-hoc test ( a , h – j ); Two-way ANOVA with Tukey’s post-hoc test ( b , d ); Student’s t-test ( c) ; Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Expressing, Irradiation, Quantitative RT-PCR, Labeling, Software, Western Blot, Mutagenesis, Control

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a , CASP5 and FLAG immunoblot of HEK293T expressing various CASP5 constructs as indicated with or without GSDMD at 24 hr post transfection. b , CASP5 activity in HEK293T cell culture medium expressing indicated CASP5 isoforms or 2-fold (1/2) or 4-fold (1/4) less CASP5c and co-expressing GSDMD. c , TOPflash luciferase reporter activity (left panel) and corresponding immunoblots (right panels) in HEK293T cells expressing indicated CASP5 isoforms or 2-fold (1/2) or 4-fold (1/4) less CASP5c. d , CASP5 activity in culture medium of HEK293T cells treated with or without 20 μM WEHD, LEVD or Q-VD-OPh inhibitor for 18 hr. e , f , h , j , Immunoblots for CASP5 in WCE from CASP5a, CASP5b, CASP5c, or CASP5c (C173A) expressing HEK293T cells ( e ), or CASP5 isoform expressing HEK293T cells without (Ctrl) or treated with 20 μM WEHD, LEVD,VX765 or Q-VD-OPh inhibitor for 18 hr ( f , h , j ). Vector indicates an empty vector without the insertion of the CASP5 gene ( f , h , j ). Autocleavage products manifested in the appearance of N-terminal fragments CASP5a-N (38 kDa), CASP5b-N (31 kDa), and a smaller CASP5c-N (22 kDa), depended on catalytic activity, and were absent upon transfection of mutant CASP5c (C173A). The sizes of the autocleavage products were concordant with predicted proteolytic cleavage sites 4 . g , i , k , Schematics of predicted CASP5a, CASP5b and CASP5c auto-cleavage steps and cleavage fragments. CASP5a, b, and c share a common domain structure composed of caspase protease domain which is composed of large (p20) and small (p10) subunits separated by an interdomain linker (IDL). The N-terminal CARD (blue) in CASP5a and CASP5b is connected to the protease domain via a CARD domain linker (CDL). l , Immunoblots for CASP5 in WCE from CASP5c or CASP5-ΔCARD expressing HEK293T cells. m , Amino acid sequences of CASP5a and CASP5c highlighting the location of the C residue that were mutated to A in this study: C315A in CASP5a and the equivalent C173A in CASP5c. n , CASP5 substrate prediction among Wnt signaling pathway proteins and regulators using the PeptideCutter software ( https://web.expasy.org/peptide_cutter/ ), caspase-1 was employed as the probe in the prediction. o , Immunoblotting analysis of truncated APC cleavage by CASP5c in HEK293T cells. p , Schematics showing the truncated APC relative to full length APC and predicted fragments post cleavage at D566. q , Quantification of Fig. 3n AXIN IP indicated immunoblot relative band intensities from Wnt3a CM treated (2 hr) APC-KO HEK293T cells with ectopic expression of APC-FL or APC-D556A and CASP5c as indicated, Band intensities were calculated with ImageJ. r, TOPflash luciferase reporter activity from APC-KO HEK293T cells with ectopic expression as indicated, harvested 48 hr post-transfection after overnight Wnt3a CM treatment. s , Quantification of Fig. 3o AXIN IP indicated immunoblot relative band intensities from APC-KO HEK293T cells without (Ctrl) or with ectopic expression of APC-FL, APC-1–556, or APC-557–2843. Band intensities were calculated with ImageJ. t , TOPflash luciferase reporter activity from WT or APC-KO HEK293T cells expressing indicated plasmids for 48 hr. a , c , e , f , h , j , l , o , ACTB is β-actin loading control. All CASP5 encoding plasmids transfected at 100 ng/24 well plate and WCE or supernatants prepared at 24 hr post-transfection. b , n=6, c , d , r – t , n=3, q ,n=2–3. One-way ANOVA with Tukey’s post-hoc test ( b – d , q – t ); Two-tailed unpaired t-test ( q ); Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Activity Assay, Western Blot, Expressing, Construct, Transfection, Cell Culture, Luciferase, Plasmid Preparation, Mutagenesis, Residue, Software, Control, Two Tailed Test

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a, Oncoplot of seven mutations from TCGA COAD and READ 576 samples. Data were collected and visualized from cBioportal ( https://www.cbioportal.org ). The upper plot indicates samples from COAD (blue) or READ (red). The type of genetic alteration in each tumor sample is represented by a different color in the central plot. The 3% CASP5 mutation rate may be an underestimate because detecting small in/del mutations particularly in long mononucleotide repeats, such as A10 in genes including CASP5 , is challenging because of sequence read alignment artifacts, reducing mutation calling accuracy. b , Mutual exclusivity of WNT-associated mutations ( APC , AXIN1 , AXIN2 , ZNRF3 , RNF43 , and CTNNB1 ) and CASP5 in TCGA COAD and READ 576 samples. Only significant pairs (p-value < 0.05) are shown. c , Oncoplot of CASP5 (A) 10 and APC (A) 7 mutation from 53 MSI-H patients. Samples having APC and CASP5 sequencing information were used. The upper plot represents number of mutations per each sample, and right bar plot indicates percentage of mutated samples. Mutation status is classified by color (black, mutant; gray, wildtype). d , CASP5 methylation probe annotation of Infinium Human Methylation 450K BeadChip from UCSC database (upper) and boxplot of b-value for CASP5 methylation probes from TCGA COAD and READ samples (lower). Each dot from box plot indicates b-value from each sample. 396 samples were classified by MSI-H status (MSI-H, MSI-L, and MSS) or normal status as performed previously 54 . Samples without MSI-H information were excluded. P-values were calculated by two-sided Student’s t test.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Methylation, Mutagenesis, Sequencing

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a , Dot plots showing cell type annotation based on the expression of the genes listed on top. Gene expression and cell type annotation data mined from Smillie et al . 40 . b , Feature plots showing the distribution of CASP5 and CASP4 as indicated on tSNE plots of IEC from healthy adult colonic tissues shown in Fig. 4a . c , Proportion of IEC in healthy adult, pediatric or fetal subjects expressing indicated genes. d , Proportion of CASP5 , CASP4 , and LGR5 transcript expressing cells in each cell type in healthy adult and fetal intestinal epithelium. e , Proportion of IEC in healthy adult, pediatric or fetal subjects expressing indicated genes. c – e , Datasets mined from Elmentaite et al . 42 . f , Proportion of CASP4 + , CASP5 + or LGR5 + epithelial cells in healthy adult subjects or in non-inflamed or inflamed intestinal epithelium from ulcerative colitis adult subjects expressing indicated genes. g , Proportion of CASP5 , CASP4 , and LGR5 transcript expressing cells in each epithelial cell type in indicated healthy or ulcerative colitis disease status. f , g , Datasets mined in healthy and ulcerative colitis adult subjects were from Smillie et al . 40 . h , Percentage of overlaps of the CASP5 isoform positions to the indicated cell type marker gene and Wnt target gene positions. 10–90 percentile were used to calculate the overlaps. i, Heatmap of qRT-PCR-based gene expression levels of Lgr5 , Ki-67 , Fgfpb1 , Vil1 , and caspase-11 isoforms 1/4 and 2 in FACS sort-purified EpCAM + GFP + or GFP – crypt, EpCAM – CD45 + hematopoietic, or EpCAM + GFP – cell fractions from the duodenum, jejunum, ileum and colon epithelium of LGR5-GFP reporter mice. Data from 3 experiments as indicated. j , Schematic illustration of Wnt and BMP gradients along the colonic crypt-lumen axis (left) and depiction of major cell types in the colonic epithelium (right). c , e , f , Box plots describe the median and interquartile range (IQR) of each gene. Whiskers depict the 1.5× IQR. P values in c , f , from a Wilcoxon rank-sum test and e , Kruskal-Wallis test. f , Healthy n=24, UC non-inflamed n=21, and UC inflamed, n=16. Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Expressing, Gene Expression, Marker, Quantitative RT-PCR, Purification

Journal: Nature

Article Title: Caspase-5c amplifies Wnt via APC cleavage to promote intestinal homeostasis

doi: 10.1038/s41586-026-10343-8

Figure Lengend Snippet: a , b , qRT-PCR of CASP5 isoforms after siRNA knockdown in colonic ( a ) and small intestinal ( b ) organoids. c , TOPflash luciferase reporter activity in human small intestinal organoids 3 days post CASP5 knockdown by si CASP5a/b and si CASP5a/b/c siRNAs. d , Representative confocal micrographs of CASP5a/b or CASP5a/b/c siRNA knockdown small intestinal organoids grown in differentiation media supplemented with 5% Wnt3a CM (low Wnt) labeled for EdU incorporation (S-phase TA cells), Villin (enterocytes), Ki-67 (proliferative TA cells), Phalloidin (cytoskeleton), and DAPI (nuclei). Bar scales=50 μm. Bar graphs show quantifications, organoid size and labeling measurements using ImageJ software (Fiji). CASP5a/b knockdown increased organoid size (80%), Ki-67 (2.9-fold), and EdU incorporation (4.1-fold) compared to control siRNA organoids, whereas CASP5a/b/c knockdown restored basal levels. e , ATP levels measured for same organoids used for imaging show 3.7-fold increase in CASP5a/b knockdown vs control siRNA organoids and 3.5-fold increase vs CASP5a/b/c knockdown organoids. f , qRT-PCR of AXIN2 , LGR5 , MKI67, and ZNF277 in small intestinal organoids with CASP5a/b or CASP5a/b/c siRNA knockdown grown in differentiation media. Fold increases in CASP5a/b siRNA vs control siRNA organoids: AXIN2 : 5.5-fold; LGR5 : 1.5-fold; MKI67 : 1.6-fold; ZNF277 : 1.8-fold. Results represent at least 3 independent experiments (colonic and small intestinal organoids from either of the two donors in each experiment); colonic organoid results from both donor 1483 and 1494 ( a ) are shown; small intestinal organoid results from duodenum (HT-291) ( b – f ) are shown; Donor details and intestinal segment in methods. a , n=4, b , c , n=3, d , n=18,14,15 (Diameter), n=12,8,8 (EdU), n=45,15,23 (Ki-67), n=13,12,15 (Villin), e , n=6, f , n=2 ( AXIN2 ), n=3 (rest). One-way ANOVA with Tukey’s post-hoc test ( d – f ), Two-way ANOVA with Tukey’s post-hoc test ( a – c ); Error bars, mean ± s.e.m.

Article Snippet: To validate assay specificity, we treated HEK293T cells with 20 μM CASP5 inhibitors WEHD (MCE, HY-P0111), LEVD (MCE, HY-128707) and pan-caspase inhibitor Q-VD-OPh (AdooQ BioScience, A14915) for 18 hr.

Techniques: Quantitative RT-PCR, Knockdown, Luciferase, Activity Assay, Labeling, Software, Control, Imaging

Journal: Cells

Article Title: High Throughput Screening Targeting the Dengue NS3-NS5 Interface Identifies Antivirals against Dengue, Zika and West Nile Viruses

doi: 10.3390/cells11040730

Figure Lengend Snippet: Summary of IC 50 data from AlphaScreen analysis.

Article Snippet: I-OMe-AG538 (α-Cyano-(3-methoxy-4-hydroxy-5-iodocinnamoyl)-(3′,4′-dihydroxyphenyl)ketone; sc-300821) and SHS (8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino(4-methyl-3,1-phenylene) carbonylimino]]bis-1,3,5-naphthalenetrisulfonic acid, hexasodium salt–sc-200833) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Amplified Luminescent Proximity Homogenous Assay, Binding Assay

Journal: Cells

Article Title: High Throughput Screening Targeting the Dengue NS3-NS5 Interface Identifies Antivirals against Dengue, Zika and West Nile Viruses

doi: 10.3390/cells11040730

Figure Lengend Snippet: Summary of EC 50 data for from plaque assay for I-OMe-AG538 and SHS.

Article Snippet: I-OMe-AG538 (α-Cyano-(3-methoxy-4-hydroxy-5-iodocinnamoyl)-(3′,4′-dihydroxyphenyl)ketone; sc-300821) and SHS (8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino(4-methyl-3,1-phenylene) carbonylimino]]bis-1,3,5-naphthalenetrisulfonic acid, hexasodium salt–sc-200833) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Plaque Assay, Virus

Journal: Scientific Reports

Article Title: Dysregulation of adipocytokines via the hsa-miR-548ay-3p/PPARγ signaling pathway leads to cardiac fibrosis

doi: 10.1038/s41598-025-11324-z

Figure Lengend Snippet: EAT CM from patients with AF-induced activation of CFs. (A) the representative images (100× magnification) of immunofluorescence staining PPARγ in EAT from patients with SR or AF, and the quantitative analysis of mean fluorescence intensity (SR, n=15; AF, n=15). (B) and (C) the concentration of omentin-1 and TGF-β1 in EAT CM of patients with SR or AF determined by ELISA (SR, n=10; AF, n=10). (D) the representative images (50× magnification) of scratch assay of CFs treated with different EAT CM, and the analysis of wound closure rate. (E) the results of CCK-8 of CFs treated with different EAT CM. (F), (G), and (H) the representative images (100× magnification) of immunofluorescence staining α-SMA, COL1, and COL3 in CFs treated with different EAT CM, and the quantitative analysis of mean fluorescence intensity. (I) the mRNA expression levels of α-SMA, COL1, and COL3 in CFs treated with different EAT CM detected by RT-qPCR. ###, p < 0.001 vs. SR group; **, p < 0.01 vs. SR CM group; ***, p < 0.001 vs. SR CM group

Article Snippet: The concentration of omentin-1, TGF-β1, and adiponectin in the CM or mouse plasma was measured using ELISA Kit (Omentin ELISA Kit, EIA-OME-1, Ray-Biotech; TGF-β1 ELISA Kit, EK981, Multi sciences Biotech; adiponectin ELISA Kit, EK295, Multi sciences Biotech) according to the manufacturer’s recommended procedures.

Techniques: Activation Assay, Immunofluorescence, Staining, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, CCK-8 Assay, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Dysregulation of adipocytokines via the hsa-miR-548ay-3p/PPARγ signaling pathway leads to cardiac fibrosis

doi: 10.1038/s41598-025-11324-z

Figure Lengend Snippet: Inhibition of PPARγ led to adipocytokine dysregulation, which activated CFs. (A) the protein expression levels of PPARγ, omentin-1, and TGF-β1 in adipocytes treated without or with T0070907 detected using western blotting and quantitative analysis. (B) and (C) the concentration of omentin-1 and TGF-β1 in CM of adipocyte treated without or with T0070907 determined using ELISA (CON CM, n=5; T007 CM, n=5). (D) the representative images (50× magnification) of scratch assay of CFs treated with different adipocyte CM, and the analysis of wound closure rate. (E) the results of CCK-8 of CFs treated with different adipocyte CM. (F), (G), and (H) the representative images (100× magnification) of immunofluorescence staining α-SMA, COL1, and COL3 in CFs treated with different adipocyte CM, and the quantitative analysis of mean fluorescence intensity. (I) the mRNA expression levels of α-SMA, COL1, and COL3 in CFs treated with different adipocyte CM detected by RT-qPCR. (J) the intersection of miRNAs targeting PPARγ predicted by TargetScan and differentially expressed miRNAs. (K) and (L) the miRNA expression levels of hsa-miR-548ay-3p and hsa-miR-627-3p in EAT from patients with SR or AF detected by RT-qPCR (SR, n=25; AF, n=25). ##, p < 0.01 vs. CON group; ###, p < 0.001 vs. CON group; **, p < 0.01 vs. CON CM group; ***, p < 0.001 vs. CON CM group; ***, p < 0.001; ns, no significance

Article Snippet: The concentration of omentin-1, TGF-β1, and adiponectin in the CM or mouse plasma was measured using ELISA Kit (Omentin ELISA Kit, EIA-OME-1, Ray-Biotech; TGF-β1 ELISA Kit, EK981, Multi sciences Biotech; adiponectin ELISA Kit, EK295, Multi sciences Biotech) according to the manufacturer’s recommended procedures.

Techniques: Inhibition, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, CCK-8 Assay, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Dysregulation of adipocytokines via the hsa-miR-548ay-3p/PPARγ signaling pathway leads to cardiac fibrosis

doi: 10.1038/s41598-025-11324-z

Figure Lengend Snippet: Overexpression of hsa-miR-548ay-3p inhibited adipocyte differentiation, altered adipocytokine expression, and activated CFs. (A) the miRNA expression levels of hsa-miR-548ay-3p in adipocytes infected with different lentivirus detected using RT-qPCR. (B) the protein expression level of PPARγ in preadipocytes infected with different lentivirus detected by western blotting and the quantitative analysis. (C) the protein expression level of ACC, FAS, PLIN-1, PPARγ, omentin-1, TGF-β1, and FABP4 in adipocytes infected with different lentivirus and treated without or with rosiglitazone detected by western blotting and the quantitative analysis. (D) and (E) the concentration of omentin-1 and TGF-β1 in CM of adipocyte infected with different lentivirus and treated without or with rosiglitazone determined by ELISA (CON CM, n=5; Vector CM, n=5; miR-548 OE CM, n=5; miR-548 OE+RO, n=5). (F) the mRNA expression levels of FAS, PLIN-1, PPARγ, omentin-1, TGF-β1, and FABP4 in adipocytes infected with different lentivirus and treated without or with rosiglitazone detected by RT-qPCR. (G) the representative images (50× magnification) of scratch assay of CFs treated with different adipocyte CM, and the analysis of wound closure rate. (H) the results of CCK-8 of CFs treated with different adipocyte CM. (I), (J), and (K) the representative images (100× magnification) of immunofluorescence staining α-SMA, COL1, and COL3 in CFs treated with different adipocyte CM, and the quantitative analysis of mean fluorescence intensity. (L) the mRNA expression levels of α-SMA, COL1, and COL3 in CFs treated with different adipocyte CM detected by RT-qPCR. *, p < 0.05 vs. Vector group; **, p < 0.01 vs. Vector group; ***, p < 0.001 vs. Vector group; #, p < 0.05 vs. miR-548 OE group; ##, p < 0.01 vs. miR-548 OE group; ###, p < 0.001 vs. miR-548 OE group; +, p < 0.05 vs. Vector CM group; ++, p < 0.01 vs. Vector CM group; +++, p < 0.001 vs. Vector CM group; $, p < 0.05 vs. miR-548 OE CM group; $$, p < 0.01 vs. miR-548 OE CM group; $$$, p < 0.001 vs. miR-548 OE CM group

Article Snippet: The concentration of omentin-1, TGF-β1, and adiponectin in the CM or mouse plasma was measured using ELISA Kit (Omentin ELISA Kit, EIA-OME-1, Ray-Biotech; TGF-β1 ELISA Kit, EK981, Multi sciences Biotech; adiponectin ELISA Kit, EK295, Multi sciences Biotech) according to the manufacturer’s recommended procedures.

Techniques: Over Expression, Expressing, Infection, Quantitative RT-PCR, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Wound Healing Assay, CCK-8 Assay, Immunofluorescence, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Dysregulation of adipocytokines via the hsa-miR-548ay-3p/PPARγ signaling pathway leads to cardiac fibrosis

doi: 10.1038/s41598-025-11324-z

Figure Lengend Snippet: Inhibition of hsa-miR-548ay-3p promoted adipocyte differentiation, altered adipocytokine expression, and inhibited TGF-β1-induced activation of CFs. (A) the miRNA expression levels of hsa-miR-548ay-3p in adipocytes infected with different lentivirus detected by RT-qPCR. (B) the protein expression level of PPARγ in preadipocytes infected with different lentivirus detected by western blotting and quantitative analysis. (C) the protein expression level of ACC, FAS, PLIN-1, PPARγ, omentin-1, TGF-β1, and FABP4 in adipocytes infected with different lentivirus and treated without or with T0070907 detected by western blotting and quantitative analysis. (D) and (E) the concentration of omentin-1 and TGF-β1 in CM of adipocyte infected with different lentivirus and treated without or with T0070907 determined by ELISA (CON CM, n=5; Vector CM, n=5; miR-548 inhibit CM, n=5; miR-548 inhibit+T007, n=5). (F) the mRNA expression levels of FAS, PLIN-1, PPARγ, omentin-1, TGF-β1, and FABP4 in adipocytes infected with different lentivirus and treated without or with T0070907 detected by RT-qPCR. (G) the results of dual luciferase reporter assay. (H) the representative images (50× magnification) of scratch assay of CFs treated with different adipocyte CM, and the analysis of wound closure rate. (I) the results of CCK-8 of CFs treated with different adipocyte CM. (J), (K), and (L) the representative images (100× magnification) of immunofluorescence staining α-SMA, COL1, and COL3 in CFs treated with different adipocyte CM, and the quantitative analysis of mean fluorescence intensity. (M) the mRNA expression levels of α-SMA, COL1, and COL3 in CFs treated with different adipocyte CM detected by RT-qPCR. *, p < 0.05 vs. Vector group; **, p < 0.01 vs. Vector group; ***, p < 0.001 vs. Vector group; #, p < 0.05 vs. miR-548 inhibit group; ##, p < 0.01 vs. miR-548 inhibit group; ###, p < 0.001 vs. miR-548 inhibit group; $, p < 0.05 vs. Blank group; $$, p < 0.01 vs. Blank group; $$$, p < 0.001 vs. Blank group; ++, p < 0.01 vs. TGF-β1+Vector CM group; +++, p < 0.001 vs. TGF-β1+ Vector CM group; &&&, p < 0.001 vs. TGF-β1+miR-548 inhibit CM group; ***, p < 0.001; ns, no significance

Article Snippet: The concentration of omentin-1, TGF-β1, and adiponectin in the CM or mouse plasma was measured using ELISA Kit (Omentin ELISA Kit, EIA-OME-1, Ray-Biotech; TGF-β1 ELISA Kit, EK981, Multi sciences Biotech; adiponectin ELISA Kit, EK295, Multi sciences Biotech) according to the manufacturer’s recommended procedures.

Techniques: Inhibition, Expressing, Activation Assay, Infection, Quantitative RT-PCR, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Luciferase, Reporter Assay, Wound Healing Assay, CCK-8 Assay, Immunofluorescence, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Dysregulation of adipocytokines via the hsa-miR-548ay-3p/PPARγ signaling pathway leads to cardiac fibrosis

doi: 10.1038/s41598-025-11324-z

Figure Lengend Snippet: Overexpression of hsa-miR-548ay-3p in mice adipose tissue induced adipose tissue remodeling, adipocytokine dysregulation, and cardiac fibrosis. (A) the miRNA expression levels of hsa-miR-548ay-3p in mice adipose tissue infected with different AAV-9 detected by RT-qPCR. (B), (C), and (D) the representative images (100× magnification) of immunofluorescence staining α-SMA, COL1, and COL3 in mice inguinal adipose tissue infected with different AAV-9 and treated with or without rosiglitazone, and the quantitative analysis of mean fluorescence intensity. (E) and (F) the concentration of omentin-1 and TGF-β1 in the plasma of mice infected with different AAV-9 and treated without or with rosiglitazone determined by ELISA. (G) the mRNA expression levels of FAS, PLIN-1, PPARγ, omentin-1, TGF-β1, and FABP4 in mice inguinal adipose tissue infected with different AAV-9 and treated without or with rosiglitazone detected by RT-qPCR. (H) the representative images of Masson staining of mice heart tissues (100× magnification) and analysis of interstitial fibrosis area. (I) the protein expression level of α-SMA in mice heart tissues detected by western blotting and quantitative analysis. (J), (K), (L), and (M) the representative images (100× magnification) of immunofluorescence staining COL1, COL3, adiponectin, and TGF-β1 in mouse heart tissues, and the quantitative analysis of mean fluorescence intensity. CON, n=15; Vector, n=15; miR-548 OE, n=15; miR-548 OE+RO, n=15. *, p < 0.05 vs. Vector group; ***, p < 0.001 vs. Vector group; #, p < 0.05 vs. miR-548 OE group; ###, p < 0.001 vs. miR-548 OE group

Article Snippet: The concentration of omentin-1, TGF-β1, and adiponectin in the CM or mouse plasma was measured using ELISA Kit (Omentin ELISA Kit, EIA-OME-1, Ray-Biotech; TGF-β1 ELISA Kit, EK981, Multi sciences Biotech; adiponectin ELISA Kit, EK295, Multi sciences Biotech) according to the manufacturer’s recommended procedures.

Techniques: Over Expression, Expressing, Infection, Quantitative RT-PCR, Immunofluorescence, Staining, Fluorescence, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Plasmid Preparation