recombinant human bmp 4  (R&D Systems)

Journal: eBioMedicine

Article Title: USP25 regulates atherosclerosis by restricting RIPK1-mediated inflammatory responses

doi: 10.1016/j.ebiom.2026.106213

Figure Lengend Snippet: USP25 deficiency exacerbates atherosclerosis in HFD-fed ApoE −/− mice. (A) Representative Oil Red O staining of aortas from HFD-fed Usp25 +/+ ApoE −/− and Usp25 −/− ApoE −/− mice. Scale bar: 5 mm. (B) Data show the percentage of plaque area/total vessel area. AA, aortic arch; TA, thoracic aorta. ∗∗∗P < 0.001, unpaired 2-tailed Student's t test (n = 6). (C) Representative H&E staining of aortic root sections. Scale bar: 200 μm. (D) Quantification of lesion area (left) and percentages of necrotic core (right). ∗∗P < 0.01, unpaired 2-tailed Student's t test (n = 10). (E) Representative Oil Red O (top), anti-F4/80 immunofluorescence (middle), and anti-α-SMA immunofluorescence (bottom) staining of aortic root sections. Scale bar: 100 μm. (F) Percentages of Oil Red O (top), F4/80 + (middle), and α-SMA + (bottom) area in aortic root sections. ∗∗P < 0.01 , ∗∗∗P < 0.001, unpaired 2-tailed Student's t test (n = 5). (G) Protein levels of IL-6 and TNF-α in atherosclerotic lesions were analysed using ELISA. ∗∗P < 0.01 , ∗∗∗P < 0.001, unpaired 2-tailed Student's t test (n = 6). (H) Transcriptional levels of indicated genes in aortas were determined by qRT-PCR. Target gene expression was normalized to the level of Actb mRNA. ∗P < 0.05, ∗∗P < 0.01, unpaired 2-tailed Student's t test (n = 5).

Article Snippet: TNF-α (Cat#: 10291-TA) was purchased from R&D Systems (Minnesota, USA).

Techniques: Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Targeted Gene Expression

Journal: eBioMedicine

Article Title: USP25 regulates atherosclerosis by restricting RIPK1-mediated inflammatory responses

doi: 10.1016/j.ebiom.2026.106213

Figure Lengend Snippet: Deficiency of USP25 in hematopoietic cells exacerbates atherosclerosis in HFD-fed ApoE −/− mice. (A) Representative Oil Red O staining of aortas from Usp25 +/+ → ApoE −/− and Usp25 −/− → ApoE −/− mice fed a HFD for 16 weeks. Scale bar: 5 mm. (B) Data show the percentage of plaque area/total vessel area. ∗∗∗P < 0.001, unpaired two-tailed Student's t test (n = 6). (C) H&E staining of aortic root sections. Scale bar: 200 μm. (D) Quantitative analysis of lesion area (left) and percentages of necrotic cores (right). ∗∗ P < 0.01, unpaired two-tailed Student's t test (n = 10). (E) Representative Oil Red O (top), anti-F4/80 immunofluorescence (middle), and anti-α-SMA immunofluorescence (bottom) staining of aortic root sections. Scale bar: 100 μm. (F) Percentages of Oil Red O (top), F4/80 + (middle), and α-SMA + (bottom) area in aortic root sections. ∗∗P < 0.01, unpaired two-tailed Student's t test (n = 5). (G) Protein levels of IL-6 and TNF-α in atherosclerotic lesions were analysed using ELISA. ∗∗P < 0.01 , ∗∗∗P < 0.001, unpaired 2-tailed Student's t test (n = 6). (H) Aortas were isolated from Usp25 +/+ → ApoE −/− and Usp25 −/− → ApoE −/− mice after 16 weeks of HFD feeding. Transcriptional levels of indicated genes in aortas were determined by qRT-PCR. ∗∗P < 0.01, ∗∗∗P < 0.001, unpaired two-tailed Student's t test (n = 6).

Article Snippet: TNF-α (Cat#: 10291-TA) was purchased from R&D Systems (Minnesota, USA).

Techniques: Staining, Two Tailed Test, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR

Journal: eBioMedicine

Article Title: USP25 regulates atherosclerosis by restricting RIPK1-mediated inflammatory responses

doi: 10.1016/j.ebiom.2026.106213

Figure Lengend Snippet: USP25 deletion enhances TNF-α-induced inflammatory responses in macrophages. (A) Plot of the TNF signalling network among 12 cell populations in human atherosclerotic lesions based on GSE159677 . The right panel shows the intensity of intercellular signal communication. (B) BMDMs isolated from Usp25 +/+ and Usp25 −/− mice were treated with or without TNF-α (50 ng/mL) for 30 min. Cell lysates were analysed by Western blot analysis with indicated antibodies. (C–D) Representative immunofluorescence staining (C) and quantitation (D) of p65 nuclear translocation in BMDMs treated with or without TNF-α (50 μg/mL). Scale bar: 50 μm ∗∗∗P < 0.001, two-way ANOVA with Bonferroni post hoc test (n = 3). (E) BMDMs were stimulated with or without TNF-α (50 ng/mL) for 6 h. The mRNA levels of the indicated genes were detected by qRT-PCR. ∗P < 0.05, two-way ANOVA with Bonferroni post hoc test (n = 3).

Article Snippet: TNF-α (Cat#: 10291-TA) was purchased from R&D Systems (Minnesota, USA).

Techniques: Isolation, Western Blot, Immunofluorescence, Staining, Quantitation Assay, Translocation Assay, Quantitative RT-PCR

Journal: eBioMedicine

Article Title: USP25 regulates atherosclerosis by restricting RIPK1-mediated inflammatory responses

doi: 10.1016/j.ebiom.2026.106213

Figure Lengend Snippet: USP25 affects atherosclerosis by regulating RIPK1. (A) Experimental flowchart for the establishment of atherosclerosis in AAV-infected bone marrow chimeric mice. (B) Representative Oil Red O staining of aortas from indicated mice fed a HFD for 16 weeks. Scale bar: 5 mm. (C) Data show the percentage of plaque area/total vessel area. ∗∗∗P < 0.001, unpaired two-tailed Student's t test (n = 6). (D) H&E staining of aortic root sections. Scale bar: 200 μm. (E) Quantification of lesion area (left) and percentages of necrotic core (right). ∗∗∗P < 0.001, unpaired 2-tailed Student's t test (n = 10). (F) Representative Oil Red O staining of aortic root sections. Scale bar: 100 μm. (G) Percentages of Oil Red O area in aortic root sections. ∗∗P < 0.01, unpaired two-tailed Student's t test (n = 5). (H–I) Representative immunofluorescence staining (H) and quantification (I) of F4/80 in aortic root sections. Scale bar: 100 μm ∗∗∗P < 0.001, unpaired two-tailed Student's t test (n = 5). (J) Protein levels of TNF-α and IL-6 in atherosclerotic lesions were analysed using ELISA. ∗∗P < 0.01, unpaired two-tailed Student's t test (n = 6). (K) Transcriptional levels of indicated genes in aortas were determined by qRT-PCR. ∗P < 0.05, ∗∗P < 0.01, unpaired two-tailed Student's t test (n = 5).

Article Snippet: TNF-α (Cat#: 10291-TA) was purchased from R&D Systems (Minnesota, USA).

Techniques: Infection, Staining, Two Tailed Test, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: eBioMedicine

Article Title: USP25 regulates atherosclerosis by restricting RIPK1-mediated inflammatory responses

doi: 10.1016/j.ebiom.2026.106213

Figure Lengend Snippet: USP25 overexpression alleviates atherosclerosis in HFD-fed ApoE −/− mice. (A) Representative Oil Red O staining of aortas from Usp25 +/+ → ApoE −/− and Usp25 Tg → ApoE −/− mice fed a HFD for 16 weeks. Scale bar: 5 mm. (B) Data show the percentage of plaque area/total vessel area. ∗∗∗P < 0.001, unpaired two-tailed Student's t test (n = 6). (C) Aortas from HFD-fed Usp25 +/+ → ApoE −/− and Usp25 Tg → ApoE −/− mice were lysed for protein isolation. The lysates were immunoprecipitated with anti-RIPK1 antibody, followed by Western blot analysis with indicated antibodies. (D) H&E staining of aortic root sections. Scale bar: 200 μm. (E) Quantification of lesion area (left) and percentages of necrotic core (right). ∗∗P < 0.01, unpaired 2-tailed Student's t test (n = 10). (F) Representative Oil Red O staining of aortic root sections. Scale bar: 100 μm. (G) Percentages of Oil Red O area in aortic root sections. ∗∗P < 0.01, unpaired two-tailed Student's t test (n = 5). (H–I) Representative immunofluorescence staining (H) and quantification (I) of F4/80 in aortic root sections. Scale bar: 100 μm ∗∗P < 0.01, unpaired two-tailed Student's t test (n = 5). (J) Protein levels of TNF-α and IL-6 in atherosclerotic lesions were analysed using ELISA, ∗∗P < 0.01, unpaired two-tailed Student's t test (n = 6). (K) Transcriptional levels of indicated genes in aortas were determined by qRT-PCR. ∗P < 0.05, ∗∗P < 0.01, unpaired two-tailed Student's t test (n = 5).

Article Snippet: TNF-α (Cat#: 10291-TA) was purchased from R&D Systems (Minnesota, USA).

Techniques: Over Expression, Staining, Two Tailed Test, Isolation, Immunoprecipitation, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: TNFSF13 insufficiency disrupts human colonic epithelial cell growth and associated B cell dynamics

doi: 10.1172/JCI186032

Figure Lengend Snippet: ( A ) ELISA for secreted TNFSF13 in colonoid conditioned media ( n = 3 patient lines for control and VEO-IBD; n = 3 passages for variant). ( B ) Representative TNFSF13 RNAscope in colonoids from control and variant participants (scale bar: 50 μm; n = 3 patient lines for control; n = 3 passages for variant). ( C ) Costaining of TNFSF13 and FAS RNAscope probes with Ki67 antibody in colon biopsies. Arrows indicate cells accumulated outside epithelial crypts ( n = 3 patients for control; n = 3 tissue blocks for variant). ( D ) Representative images of colonoid formation assays at day 6 after seeding (scale bar: 300 μm). ( E ) Quantification of newly formed colonoids by size at day 6. Each passage included 2 technical replicates ( n = 4 patient lines for control and VEO-IBD; n = 4 passages for variant). ( F ) TNFSF13 and FAS RNAscope with E-cadherin immunostaining in WT and variant iPSC-derived colon organoids at day 7 (scale bar: 50 μm; n = 3 passages). ( G ) Representative colonoid formation in WT and variant iPSC-derived organoids at day 9 (scale bar: 400 μm; n = 3 passages, each with ≥ 2 technical replicates). ( H ) Quantification of colonoid formation rate and area at day 9. Colonoid size calculated by maximum vertical projection area. ( I and J ) Percentage of EdU + cells following IgG or TNFSF13 neutralizing antibody (nTNFSF13) treatment in ( I ) control tissue–derived colonoids ( n = 3 patient lines) or ( J ) WT iPSC-organoids at day 7 ( n = 3 passages). 2-way ANOVA with multiple comparisons was used for ( A and E ); 2-tailed Student’s t test for ( H – J ). P values shown on graphs unless P > 0.05.

Article Snippet: To evaluate the function of TNFSF13 in B cells, recombinant Human TNFSF13 (HEK293-expressed) protein (Cat #5860-AP-010, R&D Systems, Minnesota, USA) at a varying concentration gradient (0, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 1.5, 2 μg/mL) was added at 4h post plating.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Variant Assay, RNAscope, Immunostaining, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: TNFSF13 insufficiency disrupts human colonic epithelial cell growth and associated B cell dynamics

doi: 10.1172/JCI186032

Figure Lengend Snippet: Percentage of FAS + and HVEM + cells by flow cytometry at day 7 after seeding in ( A ) colonoids ( n = 3 patient lines for control and VEO-IBD; n = 3 passages for variant) or ( B ) iPSC-organoids ( n = 3 passages per line). ( C ) Representative Western blot for FAS from coimmunoprecipitation supernatant in control and VEO-IBD colonoids at day 7. TNFSF13 antibody used for capture. Protein ladder lane (M) not shown in overlay ( n ≥ 4 patient lines for control and VEO-IBD). ( D ) SPR binding analyses of FASL-FAS (upper) and TNFSF13-FAS (lower) interactions at various concentrations ( n = 3 independent experiments). ( E ) Costaining of TNFSF13 and FAS RNAscope probes in control colonoids at day 7. Arrows indicate coexpression (scale bar: 100 μm; n = 3 experiments). ( F ) Co staining of TNFSF13 and FAS RNAscope probes with Ki67 antibody in control colonoids at day 7. Arrows indicate triple-positive cells (scale bar: 100 μm; n = 3 experiments). ( G ) Costaining of TNFSF13 and FAS RNAscope probes with FABP2 antibody in control colonoids at day 7. Arrows indicate triple-positive cells (scale bar: 100 μm; n = 3 experiments). ( H and I ) Percentage of EdU+ cells in control colonoids (left) or WT iPSC-organoids (right) at day 7 following treatment with ( H ) IgG or FAS neutralizing antibody (nFAS) or ( I ) IgG or recombinant human FAS ligand (rFASL). Two-way ANOVA with multiple comparisons for ( A and B ); 2-tailed Student’s t test for ( H and I ). P values shown unless P > 0.05.

Article Snippet: To evaluate the function of TNFSF13 in B cells, recombinant Human TNFSF13 (HEK293-expressed) protein (Cat #5860-AP-010, R&D Systems, Minnesota, USA) at a varying concentration gradient (0, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 1.5, 2 μg/mL) was added at 4h post plating.

Techniques: Flow Cytometry, Control, Variant Assay, Western Blot, Binding Assay, RNAscope, Staining, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: TNFSF13 insufficiency disrupts human colonic epithelial cell growth and associated B cell dynamics

doi: 10.1172/JCI186032

Figure Lengend Snippet: ( A ) UMAP showing the expression pattern of TNFSF13 and FAS in scRNA-seq data from human tissue–derived colonoids. ( n = 2 lines of colonoids from 2 different patients for control and VEO-IBD, n = 2 independent passages of colonoids for variant.) Dot plot indicating the relative expression pattern of selected genes of TNFSF13 family and related receptors and enterocyte markers among annotated clusters for human colonoids scRNA-seq data. ( B ) Dot plot with relative expression of top 5 changed genes for each annotated cluster for scRNA-seq datasets in human colonoids. ( C ) Color scale indicates group with higher percentage of cells within a given cluster in each comparison. The color indicates the condition with higher percentage of a cluster in each pairwise comparison. ( D ) Dot plot with relative expression of selected genes of TNFSF13 family and related receptors and enterocyte markers among control, VEO-IBD and variants in human colonoids. ( E ) qPCR for ALDOB in colonoids on d7 after seeding. n = 3 lines of colonoids from 3 different patients for control and VEO-IBD, n = 3 passages of colonoids for variant. One-way ANOVA (with multiple comparisons) was used for statistical analysis. ( F ) Representative IF images for FABP2 and E-cadherin in human colonoids. White arrows denote FABP2 + cells. Scale bar: 50 μm. P values are shown on bar graphs unless P > 0.05.

Article Snippet: To evaluate the function of TNFSF13 in B cells, recombinant Human TNFSF13 (HEK293-expressed) protein (Cat #5860-AP-010, R&D Systems, Minnesota, USA) at a varying concentration gradient (0, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 1.5, 2 μg/mL) was added at 4h post plating.

Techniques: Expressing, Derivative Assay, Control, Variant Assay, Comparison