Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: A The expression levels of Pcna, Casp3, Tnf , and Tgfb1 in cholangiocytes. Female C57BL/6 mice were fed DDC diet for 4 weeks with vehicle or 1,25(OH) 2 D 3 (5 µg/kg, 2x week) treatment and primary cholangiocytes were isolated ( n = 4/group). B Relative mRNA levels of Vdr . Female C57BL/6 mice were treated with 1,25(OH) 2 D 3 (5 µg/kg, 2x week) for 4 weeks, and primary cholangiocytes, HSCs, liver macrophages, and LSECs were isolated ( n = 3/group). C Hepatic mRNA levels of Txnip . Female C57BL/6 mice were treated with 1,25(OH) 2 D 3 (5 µg/kg, 2x week) for 4 weeks ( n = 3/group). D Relative mRNA levels of Txnip . Female C57BL/6 mice were treated with 1,25(OH) 2 D 3 (5 µg/kg, 2x week) for 4 weeks, and primary hepatocytes, cholangiocytes, HSCs, liver macrophages, and LSECs were isolated ( n = 3/group). E qPCR analysis. BMOL cells were pre-incubated with control or Vdr siRNA for 24 h and treated with 1,25(OH) 2 D 3 (10 µM) for 24 h ( n = 4 biological replicates). F Luciferase assay. BMOL cells were pre-incubated with control or Vdr siRNA for 24 h and treated with 1,25(OH) 2 D 3 (10 µM) for 6 h ( n = 4 biological replicates). Values represent means ± SDs. n.s., not significant. Significance determined by one-way ANOVA followed by Tukey’s multiple comparisons test ( A , E , F ) and unpaired two-tailed t test ( B , C , D ). Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Expressing, Isolation, Incubation, Control, Luciferase, Two Tailed Test

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: Female Txnip fl/fl and Txnip ΔSox9 mice were fed DDC diet for 4 weeks. A Representative H&E staining ( n = 4/group). B IHC staining for pan-CK. Right: Numbers of pan-CK + cells ( n = 4/group). C Relative hepatic mRNA levels of Krt19 , Krt7 , and Sox9 ( Txnip fl/fl , n = 4; Txnip ΔSox9 , n = 3). D IF staining for F4/80 and Sirius-red staining. Right: The F4/80 + cell numbers and Sirius red-positive areas ( n = 4/group). Magnification: 200X. E , F qPCR analysis of genes involved in inflammation ( E ) and fibrosis ( F ) ( Txnip fl/fl , n = 4; Txnip ΔSox9 , n = 3). Values represent means ± SDs. n.s., not significant. Significance determined by unpaired two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Staining, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: Principal component analysis ( A ) and volcano map ( B ) for control and Txnip -overexpressing (OE) BMOL cells ( n = 3/group). C Top 20 enriched gene ontologies from DAVID analysis. D Heatmap of representative differentially expressed genes in control and Txnip OE BMOL cells. E Flow cytometric analysis of shRNA control and shRNA Txnip BMOL cells ( n = 3 biological replicates). F Effects of 1,25(OH) 2 D 3 on cell proliferation and apoptosis in shRNA control and shRNA Txnip BMOL cells, as assessed by Western blot analysis ( n = 3 biological replicates). G IF staining of pan-CK/BrdU and pan-CK/caspase-3 in liver tissues. Txnip fl/fl and Txnip ΔSox9 mice were DDC-fed for 4 weeks. Right: Percentages of pan-CK + BrdU + and pan-CK + caspase-3 + cells ( n = 4/group). Original magnifications: 400X and 800X. ( H ) qPCR analysis of cholangiocytes isolated from DDC-fed Txnip fl/fl and Txnip ΔSox9 mice ( n = 4/group). Values represent means ± SDs. n.s., not significant. Significance determined by unpaired two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Control, shRNA, Western Blot, Staining, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: A Representative images of IF staining for F4/80/pan-CK and α-SMA/pan-CK in DDC-fed mice. F4/80 + liver macrophages and α-SMA + HSCs were observed near pan-CK + cells (arrows) near portal regions (1) and in parenchymal regions (2). Original magnifications: 400X and 800X. B qPCR analysis. Primary KCs isolated from C57BL/6 mice were co-cultured with shRNA control or shRNA Txnip BMOL cells for 24 h ( n = 4 biological replicates). C mRNA levels of cytokines in shRNA control and shRNA Txnip BMOL cells ( n = 3 biological replicates). D Relative mRNA expression levels of pro-inflammatory genes in cholangiocytes isolated from DDC-fed Txnip fl/fl and Txnip ΔSox9 mice ( n = 4/group). E Levels of TNF-α in culture media of shRNA control or shRNA Txnip BMOL cells treated with or without TNF-α neutralizing antibody (5 µg/ml) for 24 h ( n = 4 biological replicates). F Expression of pro-inflammatory genes in KCs isolated from C57BL/6 mice and co-cultured with shRNA control or shRNA Txnip BMOL cells plus TNF-α neutralizing antibody ( n = 4 biological replicates). G Relative mRNA expression levels of fibrosis-related genes. HSCs isolated from C57BL/6 mice were co-cultured with shRNA control or shRNA Txnip BMOL cells ( n = 3 biological replicates). H Relative mRNA expression levels of Tgfb1 in shRNA control and shRNA Txnip BMOL cells ( n = 3 biological replicates). I qPCR analysis of fibrosis-related genes in cholangiocytes from DDC-fed Txnip fl/fl and Txnip ΔSox9 mice ( n = 4/group). J Levels of TGF-β in culture media from shRNA control or shRNA Txnip BMOL cells treated with or without TGF-β neutralizing antibody (2 µg/ml) for 24 hours ( n = 3 biological replicates). ( K ) Expression of fibrosis-related genes in primary HSCs co-cultured with shRNA control or shRNA Txnip BMOL cells with or without TGF-β neutralizing antibody ( n = 3 biological replicates). Values represent means ± SDs. n.s., not significant. Significance determined by unpaired two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Staining, Isolation, Cell Culture, shRNA, Control, Expressing, Two Tailed Test

Journal: eLife

Article Title: UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling

doi: 10.7554/eLife.68348

Figure Lengend Snippet: ( A–L ) DU145 cells were transfected for 48 hr with the indicated siRNA (control, siCTRLpool or siCTRLsingle1; control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2). ( A,C,H ) Western blotting images of phospho-kinases spotted on the Proteome Profiler Human Phospho-Kinase Array in complete media (A) or under EGF stimulation (C, 50 ng/ml, 10 min) or serum-starved ( H ). Phospho-EGFR double spots are marked in red rectangles. ( B ) Immunoblot and quantification (n = 3 independent experiments) of p-EGFR (Y1068 or Y1086). p-EGFR levels were quantified by calculating the ratio between p-EGFR and EGFR, both normalized to loading control signal. Immunoblot of UBTD1 shows the level of siRNA depletion. ( D, E ) Immunoblot and quantification (n = 3 independent experiments) of pSTAT3, p-ERK, and p-AKT. p-STAT3, p-ERK, and p-AKT levels were quantified by calculating the ratio between phospho-protein and total-protein, both normalized to loading control signal. Immunoblot of UBTD1 shows the level of siRNA depletion. ( F ) Representative wide-field immunofluorescence images (left) and quantification (right) of pSTAT3 nuclear translocation corresponding to nuclei/cytoplasm mean intensity ratio of pSTAT3. ( G ) mRNA quantification of STAT3 target genes (bcl2, mmp2, mmp9 and hif2). ( I ) Quantification of EGFR mRNA level. ( J ) EGF secretion measured by ELISA. ( K ) mRNA quantification of EGFR ligands. ( L ) EGFR ligands secretion measured by ELISA. Scale bar = 10 µm. n ≥ 3 independent experiments; ns = non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ( F, I, J ) two-tailed t-test; ( B,C,E,G, K,L ) two-way ANOVA and Bonferroni’s multiple comparisons test; data are mean ± s.e.m. Figure 1—figure supplement 1—source data 1. Uncropped western blot for .

Article Snippet: Relative phosphorylation levels of 43 kinases and two related proteins were assessed using the Proteome Profiler Human Phospho-Kinase Array Kit (R and D Systems), according to the manufacturer’s instructions.

Techniques: Transfection, Control, Western Blot, Immunofluorescence, Translocation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Multi-omics analysis reveals potential regulatory roles of SPI1 in EHT (A) Heatmap of 575 expressed TFs during the differentiation of Pluripotent Stem Cells (PSC) into Hematopoietic Progenitor Cells (HPC). Four expression clusters (TC1-TC4) are identified and are denoted by color and number. (B) Line graph of variable accessible chromatin elements during PSC to HPC differentiation, again with four identified clusters (C1-C4). (C) Enriched TF motifs in peaks of clusters C1-C4. (D) Heatmap showing variations in TF activity among the top 50 active transcription factors during differentiation. (E) Pearson correlation analysis of TF activity and gene expression for five key TFs. (F) Visualization of ATAC-seq footprint for motifs of the five representative TFs across different developmental stages. See also Figure S2 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Biomarker Discovery, Expressing, Activity Assay, Gene Expression

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 at the in vitro -generated HECs stage severely impairs HPC generation (A) Schematic illustration of the in vitro differentiation approach. SPI1 siRNA or control siRNA were introduced on D4 of EHT, with evaluations from D5 to D8. (B) Flow cytometric sorting strategy for HECs treated with siRNA. (C) qPCR results showing SPI1 expression from D5 to D8, using two distinct siRNA sequences targeting SPI1 . (D) Western blot analysis of PU.1 expression in siCtrl and siSPI1_1/2 groups from D5 to D8, with GAPDH as the internal normalization control. (E) Representative flow cytometry density plots showing CD34 + CD43 + cells among total cells in siCtrl and siSPI1_1/2 groups, from D5 to D8. (F) Quantification of suspended CD34 + CD43 + HPCs from siCtrl and siSPI1_1/2 groups, from D5 to D8. (G) Microscopy images of cells on D8 for siCtrl and siSPI1 conditions, scale bar = 100 μm. (H) Representative flow cytometry density plots of CD34 + CD43 + HPCs in suspended cells for siCtrl and siSPI1_1/2 groups on D8. Data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S4 , Table S1 and Table S2 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, In Vitro, Generated, Control, Expressing, Western Blot, Flow Cytometry, Microscopy, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 during EHT does not alter HPC proliferation or apoptosis but affects differentiation potential (A) Flow cytometry sorting strategy for D8 suspended CD34 + CD43 + HPCs (FAM +/− ). (B and C) Representative flow cytometry density plots (B) and frequency plot (C) for cell cycle, G0/G1 (Edu − Hoechst low ), S (Edu + Hoechst low/high ) and G2/M (Edu − Hoechst high ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. (D and E) Representative flow cytometry density plots (D) and frequency plot (E) of early apoptosis (Annexin V + DAPI − ) and late apoptosis (Annexin V + DAPI + ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. Significance determined by two-tailed unpaired Student’s t-test. (F) Colony Forming Unit (CFU) assay of FAM − CD34 + CD43 + HPCs generated on D8 under siCtrl and siSPI1_1/2 conditions. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-way ANOVA. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figure S5 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, Flow Cytometry, Two Tailed Test, Colony-forming Unit Assay, Generated

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Transcriptional regulatory network suggests LYL1 and KLF1 as potential downstream targets of SPI1 (A) Heatmap displaying reordered normalized trans -regulation score (TRS) at the HPC stage. Identified modules from nonnegative matrix factorization (NMF) are represented in different colors. (B) Boxplots showing gene expression levels in different modules from A, comparing Endothelial Progenitor Cells (EPC) and HPC. p values, two-tailed paired Student’s t test. (C) All SPI1 Target Genes (TGs) are ordered descendingly according to their TRS, with a focus on the top 10 transcription factors (TFs) within these TGs. (D) Violin plots showing expression levels of the top 10 TFs from SPI1 TGs in both in vivo AGM hematopoiesis (left) and the in vitro system (right). (E) qPCR results showing the expression of the top 10 transcription factors from SPI1 target genes in D8 CD34 + CD43 + HPCs, excluding non-expressed genes SNAI3 and RUNX3 , in siCtrl and siSPI1_1/2 groups. Data are from n = 3 independent experiments, with significance assessed using two-tailed unpaired Student’s t test. (F) Enrichment analysis of Gene Ontology (GO) terms and KEGG signaling pathways of SPI1 ’s TGs that have p -value <0.001. Data are presented as means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance.

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Gene Expression, Two Tailed Test, Expressing, In Vivo, In Vitro, Protein-Protein interactions

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Overexpression of LYL1 and KLF1 can rescue SPI1 knockdown phenotypes (A) Co-transfection strategy of SPI1 siRNA with KLF1 / LYL1 overexpression during EHT to assess D8 HPC differentiation potential. (B) Western blot analysis of KLF1/LYL1 from D6 differentiated cells in the siCtrl+OE-Ctrl, siSPI1, and siSPI1+OE-KLF1/LYL1 groups, normalized to GAPDH. (C and D) Flow cytometry analysis of suspended CD34 + CD43 + HPCs on D8: (C) Representative density plot and (D) quantification in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. n = 3 independent experiments. (E) Colony-forming units (CFU) assay on D8 suspended CD34 + CD43 + HPCs across siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups, with significance assessed using two-way ANOVA. (F) Schematic description of lymphoid differentiation. (G) Representative flow cytometry density plots of the distribution of CD45 -/mid/+ CD5 + CD7 + cells under the normal conditions at D29. (H–I) Analysis of CD45 + CD5 + CD7 + cells: (H) Representative flow cytometry density plots (H) and frequency plot (I) of CD45 + CD5 + CD7 + cells in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figures S6 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Over Expression, Knockdown, Cotransfection, Western Blot, Flow Cytometry, Colony-forming Unit Assay, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet:

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Recombinant, Transfection, Plasmid Preparation, Reverse Transcription, In Vivo, In Vitro, Biomarker Discovery, Software

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 2. TGFβ induces TGFβ2 expression in GBM and non-GBM cell lines. A, qRT-PCR of TGFB 1 , TGFB 2 , and TGFB 3 in LN229 cells treated with TGFβ1 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, qRT-PCR of TGFB 2 in LN229 cells treated with TGFβ1, TGFβ2, and TGFβ3 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in culture supernatant from LN229 cells treated with TGFβ for 72 hours. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGF b 2 in LN229 cells treated with TGFβ1 and/or the TβRI inhibitor (TβRI inh.) LY-2109761 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. E, qRT-PCR of TGFB 2 in GBM and non-GBM cell lines treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 3. CREB1 regulates the autocrine induction of TGFβ2 by TGFβ. A, nucleotide sequence of the proximal region of the TGFB 2 promoter. The SBEs and CREB1 site (CRE) are indicated relative to the transcription start site. ClustalW sequence alignment for 3 animal species [ Homo sapiens ( H.s .), Pan troglodytes ( P.t. ), and Mus musculus ( M.m .)] shows the conservation of the binding sites. B, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an shRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an siRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGFβ2 in LN229 cells expressing ICER treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. The molecular weights are shown.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, shRNA, Control, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 5. PI3K and RSK regulate the TGFβ- mediated induction of TGFβ2 through CREB1. A, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with TGFβ for 3 hours and the PI3K inhibitor (inh) LY-294002 for 24 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with increasing amounts of the RSK inhibitor BI-D1870 for 24 hours and TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with TGFβ for 48 hours and the PI3K inhibitor for 72 hours. *, P < 0.05, using the Student t test; data, mean ± SD. D, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with the RSK inhibitor BI-D1870 for 72 hours and TGFβ for 48 hours. *, P < 0.05, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Western Blot, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 6. TGFβ2 correlates with CREB1 expression in GBM patient tumors. A and B, graphs showing the correlation between CREB1 and TGFB 1 (B) or TGFB 2 (A) mRNA levels in patient GBM tumor samples. Data obtained from the REMBRANDT database. A Spearman test was used, and the correlation coeffi cient (ρ) and the two-tailed P value are shown. C, graph showing the correlation between p-CREB1 and TGFβ2 protein levels in tissue microarrays (TMA) from patient GBM samples. Not all spots were evaluable in all stainings. A Spearman test was used, and the correlation coeffi cient (ρ) and the two- tailed signifi cance are shown. Representative images from the TMAs are shown; scale bar, 50 μm. D, Kaplan–Meier curves showing the OS of patients with TGFB2 mRNA levels upregulated ≥3-fold and CREB1 mRNA levels upregulated ≥2-fold. Statistical signifi cance was assessed by the log-rank test. Data obtained from the REMBRANDT database.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Two Tailed Test

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 7. CREB1 regulates TGFβ2 expression in PDX models. A, scheme showing the experimental procedure. B, IHC of p-CREB1 and TGFβ2 from mouse tumors 60 days after inoculation with neurospheres expressing shRNAs targeting CREB1 and control shRNAs; scale bar, 50 μm (C). Kaplan–Meier survival curves from mice in B. D, the TGFβ2 malignant autocrine loop. In GBM, TGFβ collaborates with the PI3K and RSK pathways through a CREB1– SMAD3 transcriptional complex to induce TGFβ2 expression. This leads to the generation of an autocrine loop and accumulation of TGFβ2 in the tumor, hyperactivation of TGFβ, and tumor progression.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Paraffin-embedded Immunohistochemistry, Control

Journal: Frontiers in Immunology

Article Title: The C-Terminal Effector Domain of Non-Structural Protein 1 of Influenza A Virus Blocks IFN-β Production by Targeting TNF Receptor-Associated Factor 3

doi: 10.3389/fimmu.2017.00779

Figure Lengend Snippet: NS1/126-225 inhibits the activation of IRF3. (A) 293T cells were transfected with an IRF3 reporter plasmid and pRL-TK in the presence or absence of RIG-I(N). Cells were cotransfected with plasmids expressing NS1 or NS1/126-225, or an empty vector as a control. Luciferase activity within lysates was determined 24 h posttransfection. (B,C) 293T cells were transfected with an empty vector, NS1, or NS1/126-225 along with or without RIG-I(N) for 24 h. Lysates were then analyzed for levels of IRF3 dimers by western blotting using non-reduced SDS-PAGE (B) , and levels of p-IRF3(S396) and total IRF3 in the cytoplasmic and nuclear lysates (C) . GAPDH and Lamin A/C were used as cytoplasmic and nuclear fraction markers, respectively. (D) A549 cells were transfected with empty vector, NS1, or NS1/126-225 for 24 h. IFN-β production in response to RIG-I(N) was measured by ELISA. (E) Representative fluorescence micrographs of VSV-GFP in 293T cells transfected with an empty vector, NS1, or NS1/126-225 along with RIG-I(N) for 24 h followed by infection with VSV-GFP for 12 h. All experiments were performed at least three times with similar results. ** P < 0.01 by Student’s t -test.

Article Snippet: Then 100 μl of the cleared medium or the IFN-β standard was used in duplicate for detection of IFN-β using a human IFN-β ELISA Kit (Elabscience, E-EL-H0085c), according to the manufacturer’s instructions.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Control, Luciferase, Activity Assay, Western Blot, SDS Page, Enzyme-linked Immunosorbent Assay, Fluorescence, Infection

Journal: Frontiers in Immunology

Article Title: The C-Terminal Effector Domain of Non-Structural Protein 1 of Influenza A Virus Blocks IFN-β Production by Targeting TNF Receptor-Associated Factor 3

doi: 10.3389/fimmu.2017.00779

Figure Lengend Snippet: NS1/126-225 inhibits the IFN-β signaling pathway between the level of MAVS and TBK1. (A) 293T cells plated in 24-well plates were transfected with IFN-β-Luc, pRL-TK and empty vector, RIG-I(N), MAVS, TBK1, IKKε, or IRF3(5D) expression plasmids along with a control vector, NS1, or NS1/126-225. Luciferase activity and the transcription of IFN-β were measured at 24 h after transfection. (B) 293T cells plated in 12-well plates were transfected with an empty vector or a vector expressing NS1 or NS1/126-225 along with TBK1 or IRF3(5D), then IFN-β, OASL, PKR, and Mx1 mRNA levels were determined by real-time PCR. The transcript level of each gene was normalized to the expression of β-actin. (C) Production of IFN-β by TBK1 in the presence of NS1/126-225. A549 cells plated in 12-well plates were transfected with an empty vector, NS1, or NS1/126-225 along with TBK1. After 24 h, the amount of IFN-β in the supernatants was measured by ELISA. Western blot analysis of lysates was performed. The data are presented as mean ± SD derived from three repeat experiments. * P < 0.05 or ** P < 0.01, *** P < 0.001 (Student’s t -test).

Article Snippet: Then 100 μl of the cleared medium or the IFN-β standard was used in duplicate for detection of IFN-β using a human IFN-β ELISA Kit (Elabscience, E-EL-H0085c), according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay

Journal: Frontiers in Immunology

Article Title: The C-Terminal Effector Domain of Non-Structural Protein 1 of Influenza A Virus Blocks IFN-β Production by Targeting TNF Receptor-Associated Factor 3

doi: 10.3389/fimmu.2017.00779

Figure Lengend Snippet: NS1/126-225 disrupts the formation of MAVS–TRAF3 complex. (A) Schematic diagram of the TRAF3 protein and the following deletion mutants: Flag-TRAF3-ΔTD (AA1 to 346) and Flag-TRAF3-TD (AA347 to 568). (B) Co-IP and western blotting of 293T cells transfected with HA-tagged NS1/126-225 along with a vector expressing the indicated Flag-tagged TRAF3 truncations or full-length TRAF3. An empty vector was used as a negative control. (C) 293T cells were transfected with Flag-TRAF3 and HA-MAVS together with either HA-NS1/126-225 or HA-NS1 and Myc-RIG-I(N) for 36 h. (D) 293T cells were transfected with Flag-TRAF3 and HA-MAVS together with or without Myc-RIG-I(N) for 24 h, then infected with 0.1 MOI HM virus for 12 h. Cell lysates were harvested, then subjected to immunoprecipitation using an anti-Flag antibody, and analyzed by western blotting. (E) 293T cells were transfected with HA-NS1/126-225 as well as Flag-IKKε and HA-MAVS for 24 h. Cell lysates were collected 12 h post-infection with Sev. Co-IP was performed with anti-Flag, and the precipitates were probed with anti-Flag and anti-HA antibodies. (F) A549 cells in 12-well plates were transfected with NS1/126-225, NS1 or an empty vector along with MAVS and TRAF3, together with or without RIG-I(N) for 36 h; NS1/126-225 or NS1 cotransfected with MAVS and IKKε for 24 h, then cells were infected with Sev or mock infected for 12 h. IFN-β concentrations in the supernatants were measured by ELISA. All experiments were performed at least three times. Statistical significance was analyzed with a two-tailed student’s t -test (* P < 0.05 or ** P < 0.01).

Article Snippet: Then 100 μl of the cleared medium or the IFN-β standard was used in duplicate for detection of IFN-β using a human IFN-β ELISA Kit (Elabscience, E-EL-H0085c), according to the manufacturer’s instructions.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Negative Control, Infection, Virus, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature communications

Article Title: A mouse model for a partially inactive obesity-associated human MC3R variant.

doi: 10.1038/ncomms10522

Figure Lengend Snippet: Figure 8 | Maintained adipose tissue function in MC3RhDM/hDM mice. Gonadal fat was isolated from fasted female mice fed a high-fat diet for (a) quantitative real-time PCR (MC3RhWT/hWT n ¼ 5; MC3RhDM/hDM n ¼ 8/group), (b–d) western blotting analysis (MC3RhWT/hWT n ¼ 5; MC3RhDM/hDM n ¼ 4), and (e–l) FACS analysis (MC3RhWT/hWT n ¼ 4; MC3RhDM/hDM n ¼ 5) in MC3RhWT/hWT (open bars) and MC3RhDM/hDM (closed bars) mice. (a) The expression levels of genes related to macrophage infiltration, inflammation and adipose tissue metabolism were normalized for b-actin expression. Western protein expression results for (b) peroxisome proliferator-activated receptor gamma (PPARg), (c) adiponectin and (d) phosphospecific 50-adenosine monophosphate- activated protein kinase (p-AMPK), divided by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and normalized relative to average for MC3RhWT/hWT. The stromal vascular fraction was isolated from gonadal fat (B2 g) from MC3RhWT/hWTand MC3RhDM/hDMmice. For detecting macrophages by flow cytometry, a gating strategy was used to enrich samples from MC3RhWT/hWTand MC3RhDM/hDMmice for adipose tissue macrophages by selecting cells in the gate 1 area (e,g). The gate 1 area was stained with a F4/80 antibody (f and h) and the dot plots depict forward scatter (FSC) and side scatter (SSC) (left). For detecting neutrophils, whole cells (non-gated) were stained with CD11b and Gr-1 (i,j). Bar graphs show the average values±s.e.m. for percentage of cells in the stromal vascular fraction that were (k) macrophages and (l) neutrophils. *Po0.05 MC3RhDM/hDMversus MC3RhWT/hWT. (m,n) Haematoxylin and eosin stained sections showed no apparent differences in adipocyte morphology between groups. Groups were compared by Student’s t-tests (two-tailed) (a–d and k–i). Scale bar, 100 mm. Data are represented as mean±s.e.m. for a-d, k, and l. FACS, fluorescence-activated cell sorting

Article Snippet: Other metabolites or hormones were measured using the indicated kits; Insulin (SRI-13K, Linco Research, St Charles, MO), corticosterone (ADI-900-097; Enzo Life science, Farmingdale, NY), IGF-1 (22-IGF-R21, ALPCO, Salem, NH), Adiponectin (DRP 300 for human plasma samples obtained by venipuncture and MRP300 for mouse serum, R&D SYSTEMS, Minneapolis, MN), Leptin (MOB00, R&D SYSTEMS), triglycerides (337-B; Sigma), and non-esterified fatty acids (13831175; Roche Molecular Biochemicals, Indianapolis, IN).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Cytometry, Staining, Two Tailed Test, FACS

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Schematic of the urea cycle. b , Related metabolites in BMDMs treated with VSV for 6 h or not (NT), assayed by LC–MS/MS. log 2 fold changes are relative to NT. Citrulline, argininosuccinate, fumarate, aspartic acid, putrescine and glutamine increased significantly ( P < 0.05, two-tailed Student’s t -test) ( n = 3). c , Heatmap of gene expression in BMDMs treated with VSV for 6 h or not (NT), assessed by qPCR. log 2 fold changes are relative to NT. SMS , SAT1 , SMOX , ASS1 , IDH3b and CS increased significantly ( P < 0.05, two-tailed unpaired Student’s t -test) ( n = 3). d , IFNβ expression in BMDMs pretreated with indicated metabolites overnight and infected with VSV for 6 h or not (NT), assessed by qPCR ( n = 3). e , IFNβ expression in BMDMs pretreated with DMF (5 µM), MMF (20 µM), FHINI (20 µM) or DMSO (Ctrl) overnight, followed by VSV infection for 6 h or not (NT), determined by qPCR ( n = 3). f , Ki67 + BMDM percentage (left) and survival rate (right) after culture in fumarate (2 mM), DMF (5 µM) or MMF (20 µM) overnight, measured by FACS ( n = 3). g , IFNβ levels in BMDM supernatants pretreated overnight with DMF (5 µM) or DMSO followed by VSV infection for 6 h or not (NT), determined by ELISA ( n = 3). h , VSV transcript in BMDMs pretreated overnight with DMF (5 µM) or DMSO (−) followed by VSV (+) infection for 6 h, analysed by qPCR ( n = 3). i – n , Mice treated with DMF or DMSO for 7 days before the VSV challenge. Serum fumarate levels ( i ; n = 3) and IFNβ mRNA ( j ), serum IFNβ protein ( k ), VSV mRNA ( l ), lung histology ( m ), and ASS1 and IFNβ expression in peritoneal macrophages ( n ) were assessed ( j – n ; n = 5). The scale bar (200 µm) applies to all images in m . Statistical analysis: two-tailed Student’s t -test ( b , c , f , h and i ) or two-way ANOVA ( d , e , g , j , k , l and n ). Data are the mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. CPS1, carbamoyl phosphate synthetase 1; OTC, ornithine transcarbamylase; ASL, argininosuccinate lyase; ARG1, arginase 1; ODC, ornithine decarboxylase; ASS1, argininosuccinate synthase.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Gene Expression, Expressing, Infection, Enzyme-linked Immunosorbent Assay

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Heatmap of gene expression in VSV-infected BMDMs. Log2 fold changes are relative to uninfected BMDMs (0 h) (n = 3). b - c , IFN-β transcripts in iBMDMs pretreated with metabolites, and then infected with VSV for 6 h or not (NT) (n = 3). d , VSV plaque morphology in Vero cells after DMF (20 μM) or DMSO treatment for 6 h (n = 4). e - g , iBMDMs pretreated with DMF (5 µM) or DMSO, followed by VSV infection for 6 h or not (NT). IFN-β and ASS1 transcripts ( e ), VSV expression ( f ), and IFN-β protein ( g ) (n = 3). h , ASS1 enzymatic activity in BMDMs infected with VSV for 6 h or not (−) (n = 3). i , Enzymatic activity of ASS1, purified from HEK293 cells infected with VSV or not, was analyzed (n = 3). j , Expression of OTC, ASS1, ASL and ARG1 in BMDMs treated with VSV or not (−), measured by Western blot. k , NO levels in ASS1 +/+ and ASS1 −/− BMDMs infected with VSV or not (−) (n = 3). l-n , ASS1 transcripts in BMDMs treated with IFN-β (10 pg/ml) for different time points ( l ), treated with increasing concentrations of IFN-β for 6 h ( m ), and infected with VSV or not in the presence of IFN-β ( n ) (n = 3). o , IFN-β protein levels in supernatants of BMDMs infected with VSV or not (−), determined by ELISA (n = 3). p , ASS1 expression in BMDMs infected with VSV or not (−) in the presence of IFN-β (100 pg/ml) or not (−) for 6 h, measured by qPCR (n = 3). Statistical analysis: two-tailed Student’s t -test ( a , d , f , h , i, l , m , o and p ) or two-way ANOVA ( b , c, e, g and k ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Gene Expression, Infection, Expressing, Activity Assay, Purification, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Fumarate, malate and aspartate levels in BMDMs derived from macrophage-specific ASS1 knockout ( ASS1 −/− ) mice and corresponding wild-type ( ASS1 +/+ ) mice pretreated with AOAA (5 mM) or not (−), followed by VSV infection for 6 h or not (−), measured by LC–MS/MS/MS ( n = 3). b , Atom-transition map showing that the isotope carbon-13 ( 13 C) transfers from [U- 13 C 6 ]glutamine through the AAS shunt. The open circles represent carbon-12 ( 12 C); the green circles indicate 13 C from [U- 13 C 6 ]glutamine. c – e , Incorporation of 13 C atoms into fumarate ( c ), aspartate ( d ) and malate ( e ) in BMDMs from ASS1 +/+ and ASS1 −/− mice cultured with [U- 13 C 6 ]glutamine and treated with VSV or not (−) for 6 h, determined by LC–MS/MS ( n = 3). f , g , Expression of ASS1, IFNβ and VSV ( f ) in BMDMs pretreated overnight with DMF (5 µM) or DMSO (−) followed by VSV infection for 6 h or not (−), determined by qPCR and IFNβ protein levels by ELISA ( g ) ( n = 3). h , OCR analysis of BMDMs from ASS1 +/+ and ASS1 −/− mice infected with VSV for 6 h or not (−). Basal respiration, maximal respiration, proton leak and reserve respiratory capacity were analysed ( n = 4). i , Schematic illustration of the respiratory chain. j , Expression of respiratory chain genes in BMDMs infected with VSV for 6 h or not (NT), analysed by qPCR. log 2 fold changes are relative to BMDMs not infected with VSV (NT). Significant decreases in NDUFA1 , NDUFA3 , NDUFA4 and others ( n = 3). k , OCR analysis of ASS1 +/+ ( n = 4) and ASS1 −/− ( n = 3) BMDMs treated with VSV infection for 6 h or not (−) in the presence or absence of 1 mM succinate. Statistical analysis: two-tailed Student’s t -test ( a and j ) or two-way ANOVA ( c – g ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. CoQ, coenzyme Q; Cyt c, cytochrome complex; Suc, Succinate. Panel i created with BioRender.com .

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Knock-Out, Infection, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a - e , The incorporation of 13 C atoms into α-ketoglutarate ( a ), succinate ( b ), isocitrate ( c ), aconitate ( d ) and citrate ( e ) in BMDMs from ASS1 +/+ and ASS1 −/− mice cultured in medium containing 2 mM [U- 13 C 6 ]-glutamine and treated with VSV or not (−) for 6 h was determined by LC-MS/MS. The isotopic labeling of each metabolite is denoted as m + n, where n is the number of 13 C atoms (n = 3). f , Luciferase assay analysis of IFN-β promoter activity in HEK293 cells transfected with control siRNA (siCtrl) or ASS1 siRNA (siASS1) and then treated with VSV for a further 6 h (n = 3). Protein expression was analyzed by Western blot. g , HEK293 cells transfected with an increasing amount of Flag-tagged ASS1 plasmids for 24 h. The expression of IFN-β (left) and ASS1 (right) was determined by qPCR (n = 3). h , IFN-β transcript in BMDMs pretreated with DMF (5 µM) overnight, then followed by VSV infection for 6 h in the presence or absence of AOAA (5 mM) was determined by qPCR (n = 3). i , ASS1 +/+ and ASS1 −/− BMDMs treated with VSV for 6 h or not (−) were used for OCR analysis (n = 4). Relative to Fig. . j , IFN-β expression in BMDMs treated with DECA (10 µM) or not (PBS) followed by VSV infection for 6 h was analyzed by qPCR (n = 3). k , ASS1 +/+ (n = 4) and ASS1 −/− (n = 3) BMDMs treated with VSV or not (−) for 6 h in the presence or absence of 1 mM succinate were used for OCR analysis. Relative to Fig. . Statistical analysis: two-tailed Student’s t -test ( f - h ) or two-way ANOVA ( a-e and i-k ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Cell Culture, Liquid Chromatography with Mass Spectroscopy, Isotopic Labeling, Luciferase, Activity Assay, Transfection, Control, Expressing, Western Blot, Infection, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Luciferase reporter assays for IFNβ promoter activity in HEK293 cells transfected with indicated vectors, treated with DMF (10 µM) or DMSO for 4 h, followed by measurement of IFNβ promoter activity and western blot analysis ( n = 3). b , ASS1 +/+ and ASS1 −/− BMDMs pretreated with DMF (5 µM) or DMSO (−) overnight, followed by VSV infection for 6 h or not (−), analysed by Western blot. c , HEK293 cells transfected with GST-MAVS plasmids were treated with VSV for 6 h or not (−), and MAVS succination was assessed by immunoblotting. d , e , Immunoprecipitated Flag-MAVS from HEK293 cells treated with DMF (10 μM) for 6 h was subjected to mass spectrometry for MAVS succination. Cysteine residues at positions 46 ( d ) and 283 ( e ) in MAVS are susceptible to succination by fumarate. f , HEK293 cells transfected with Flag-MAVS or mutants, treated with 10 μM DMF for 6 h and analysed for MAVS succination by immunoblotting. g , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) were treated with VSV for 6 h or not (−) and analysed for MAVS succination. h , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) treated VSV for 6 h and IFNβ and VSV expression analysed by qPCR (left) ( n = 3). Protein expression measured by western blot (right). i , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) treated with DMF (5 μM) or not for 6 h and analysed for MAVS succination by immunoblotting. j – m , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) were pretreated with DMF (5 μM) or not (−), followed by VSV infection for 6 h. IFNβ, MAVS and VSV transcripts were determined by qPCR ( j ) ( n = 3). Cell lysates were analysed by SDS–PAGE ( k and m ), and MAVS aggregation was examined by SDD–AGE ( l ). Statistical analysis: two-tailed Student’s t -test ( a and h , right) or two-way ANOVA ( h , left, and j ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. Luc, luciferase; WB, western blot.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Luciferase, Activity Assay, Transfection, Western Blot, Infection, Immunoprecipitation, Mass Spectrometry, Transduction, Mutagenesis, Expressing, SDS Page, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Protein expression of BMDMs pretreated with DMF (5 μM) overnight or not (−), followed by VSV infection, was determined by Western blot. b , Protein expression in BMDMs pretreated overnight with the indicated metabolites followed by VSV infection or not (−) was determined by Western blot. c , HEK293 cells transfected with GST-MAVS plasmids were treated with MMF (20 μM), DMF (10 μM or 20 μM), fumarate (200 μM) respectively or not (−) for 6 h. MAVS succination was analyzed by immunoblotting (left), and densitometry quantified the succinated-to-total MAVS ratio (right). d , Schematic of RIG-I-like Receptor (RLR) signaling. e , MAVS-deficient iBMDMs transduced with human MAVS were transfected with siRNAs targeting RIG-I and MDA5 or vector control (siCtrl), followed by VSV infection and DMF (5 μM) treatment for 6 h. Whole cell lysates and anti-MAVS immunoprecipitants were analyzed by immunoblotting. f , MAVS-deficient iBMDMs transduced with human MAVS were transfected with both siRNAs targeting RIG-I and MDA5 or vector control (siCtrl), followed by VSV infection or not (−) for 6 h. Whole cell lysates were analyzed by immunoblotting. g , MAVS-deficient iBMDMs transduced with human MAVS or mutant MAVS (2CS) were transfected with siRNAs targeting RIG-I and MDA5, or vector control (siCtrl, -), followed by VSV infection for 6 h. IFN-β expression(left) and protein levels were measured (n = 3). h , Purified Flag-MAVS protein from HEK293 cells was used in in vitro assays to assess succination and aggregation of MAVS in the presence or absence of fumarate (1 mM) for 6 h. i , Purified Flag-MAVS and MAVS (2CS) proteins from HEK293 cells were used in in vitro assays to assess succination and aggregation of MAVS. Statistical analysis: two-tailed Student’s t -test ( c right) or two-way ANOVA ( g ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Western Blot, Transfection, Transduction, Plasmid Preparation, Control, Mutagenesis, Purification, In Vitro, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a - b , ASS1 +/+ and ASS1 −/− BMDMs were infected with SeV or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β and SeV in were analyzed by qPCR, and fumarate levels were analysed by LC-MS/MS ( a ) (n = 3). Whole cell lysates and anti-MAVS immunoprecipitants were analyzed for MAVS succination by immunoblotting ( b ). c - d , ASS1 +/+ and ASS1 −/− BMDMs were infected with IAV or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β and IAV in were analyzed by qPCR, and fumarate levels were analyzed by LC-MS/MS ( c ) (n = 3). Whole cell lysates and anti-MAVS immunoprecipitants were analyzed for MAVS succination by immunoblotting ( d ). e - f , ASS1 +/+ and ASS1 −/− BMDMs were infected with HSV-1 or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β and HSV-1 were analyzed by qPCR, and fumarate levels were analyzed by LC-MS/MS ( e ) (n = 3). Whole cell lysates and anti-MAVS immunoprecipitants were analyzed for MAVS succination by immunoblotting ( f ). g , Lysates from ASS1 +/+ and ASS1 −/− BMDMs infected with SeV (left), IAV (middle), or HSV-1 (right) or left uninfected (−) for 6 h was analyzed by western blot. h , IFN-β expression in MAVS-deficient iBMDMs transduced with human MAVS or mutant MAVS(2CS) infected for 6 h with SeV (left), IAV (middle), or HSV-1 (right) or left uninfected (−) was measured by qPCR (n = 3). i , MAVS-deficient iBMDMs transduced with human MAVS or mutant MAVS(2CS) were infected with SeV, IAV, or HSV-1 or left uninfected (−) for 6 h. Whole cell lysates and anti-MAVS immunoprecipitants were analyzed by immunoblotting. Statistical analysis: two-tailed Student’s t -test (the fourth section of a , c and e ) or two-way ANOVA (the first three part of a , c and e , and h ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Infection, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Transduction, Mutagenesis, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Expression of IFNβ in hMDMs expressing sgRNA-Ctrl ( ASS1 +/+ hMDMs) and hMDMs expressing sgRNA-ASS1 ( ASS1 −/− hMDMs) not infected or infected with VSV (left) or SeV in the presence or absence of DMF (10 µM) for 6 h was determined by qPCR analysis ( n = 3). b , Expression of VSV in ASS1 +/+ and ASS1 −/− hMDMs infected with VSV or SeV in the presence or absence of 10 µM DMF for 6 h was measured by qPCR analysis ( n = 3). c , Fumarate levels in ASS1 +/+ and ASS1 −/− hMDMs infected for 6 h with VSV or SeV, or uninfected (NT), were measured by LC–MS analysis ( n = 3). d , ASS1 +/+ and ASS1 −/− hMDMs in the presence or absence of 10 µM DMF were infected with VSV or left uninfected (−) for 6 h. Whole-cell lysates and anti-MAVS immunoprecipitants were analysed by immunoblotting. e , Whole lysates and immunoprecipitants with anti-MAVS from ASS1 +/+ and ASS1 −/− hMDMs in the presence or absence of DMF (10 µM) infected with SeV or not (−) were analysed by western blot. Statistical analysis: two-way ANOVA ( a – c ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Lysates from ASS1 +/+ and ASS1 −/− hMDMs infected with VSV or SeV or not (−) for 6 h in the presence or absence of DMF (10 µM) were analyzed by Western blot. b , ASS1 enzymatic activity in hMDMs treated with MDLA (10 mM) or not (−) for 6 h (left). Protein expression was also determined by immunoblotting (right) (n = 3). c , Fumarate levels in hMDMs treated with or without MDLA (10 mM) for 6 h was measured by LC-MS/MS (n = 3). d-f , hMDMs treated with MDLA (10 mM) or not (−) were infected with VSV ( d ), SeV ( e ), IAV ( f ), or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β, VSV, SeV and IAV were measured by qPCR respectively (n = 3). g , Lysates from hMDMs treated with MDLA (10 mM, +) or not (−) were infected with VSV, SeV, IAV, or left uninfected (−) were analyzed by Western blot. Statistical analysis: two-tailed Student’s t -test ( b-c ) or two-way ANOVA ( d-f ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Infection, Western Blot, Activity Assay, Expressing, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , b , In silico bioinformatic analysis of the expression of ASS1 using public databases. Expression of ASS1 was analysed between healthy people (Healthy) and patients infected with EVD using public databases ( GSE83565 ) ( a ). Box plots showing ASS1 expression in primary hMDMs treated with EBOV or MOCK, using data from the GSE84188 database ( b ). The box plot shows the minimum, maximum, median and interquartile range (IQR; 25th to 75th percentiles). Whiskers extend to 1.5 × IQR, and outliers are shown as individual points. c – g , ASS1 +/+ ( n = 5) and ASS1 −/− ( n = 5) mice were treated daily for seven consecutive days with 50 mg kg −1 DMF (dissolved in 10% DMSO) or 10% DMSO (−) by oral gavage, followed by intraperitoneal injection with VSV (2 × 10 7 PFU per mouse, +) or not (−). Mice were killed 6 h post-infection. c , d , mRNA levels of IFNβ ( c ) and VSV ( d ) in the lung and spleen were determined by qPCR analysis. e , Serum IFNβ protein levels were determined by ELISA analysis. f , IFNβ expression in splenic macrophage cells was analysed by qPCR (left, n = 3), and endogenous MAVS succination in splenic macrophage cells was measured by western blot (right). g , H&E staining of lung sections (scale bar, 100 μm). The scale bar applies to all images in g . h , Survival of ASS1 +/+ and ASS1 −/− mice intraperitoneally injected with VSV in the absence (VSV- ASS1 +/+ , n = 9; VSV -ASS1 −/− , n = 9) or presence of DMF dissolved in 10% DMSO (50 mg kg −1 , by oral gavage for seven consecutive days) (VSV, DMF- ASS1 +/+ , n = 10; VSV, DMF- ASS1 −/− , n = 12). i , Schematic illustrating the metabolic remodelling and upregulation of ASS1 to form the AAS cycle induced by viral infection to produce fumarate in the cytosol, promoting the antiviral response. Statistical analysis: two-tailed Student’s t -test ( a and b ), two-way ANOVA ( c – f ) or Kaplan–Meier survival analysis ( h ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. Arg, arginine; Orn, ornithine; Cit, citrulline; Asp, aspartate; Mal, malate; Suc, succinate. Panel i created with BioRender.com .

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: In Silico, Expressing, Infection, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , ASS1 +/+ and ASS1 −/− mice were intraperitoneally injected with VSV (2×10 7 pfu per mouse) or not (−). Mice were sacrificed 6 h post-infection. Endogenous MAVS succination was analyzed in the lungs of mice by Western blot. b , ASS1 +/+ and ASS1 −/− mice were treated daily for seven consecutive days with 50 mg/kg DMF (dissolved in 10% DMSO) or 10% DMSO (−) by oral gavage, followed by intraperitoneal injection with VSV (2 × 10 7 pfu per mouse) or not (−). Mice were sacrificed 6 h post-infection. Endogenous MAVS succination was analyzed in the lungs of mice by Western blot. c-j , ASS1 +/+ and ASS1 −/− BMDMs were infected with VSV for different time points as indicated. ASS1 transcripts ( c ), IFN-β protein levels ( d ), VSV ( e ) and IFN-β ( f ) transcripts, fumarate levels ( g ), phosphorylation of TBK1 and IRF3 ( h ), ASS1 activity ( i ), FH transcripts ( j ) were determined respectively (n = 3). k - l , Aspartate and citrulline levels in BMDM infected with VSV at different time points were measured by LC-MS/MS (n = 3). Statistical analysis: two-tailed Student’s t -test ( i, k and l ) or two-way ANOVA ( c-g and j ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Injection, Infection, Western Blot, Phospho-proteomics, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: Establishment of an EMT imaging system. (A) Schematic overview of EMTimage, expressing RFP under the control of the E‐cadherin promoter. Differential interference contrast (DIC)/RFP fluorescence overlay images of living E‐cadherin ‐RFP/Py2T cells during EMT/MET. EMT was induced by 5 ng·mL −1 TGFβ1 stimulation for 7 days and MET was induced by withdrawal of TGFβ1 for 7 days. Scale bar, 50 μm. (B) Relative expression of epithelial and mesenchymal genes normalized to Gapdh in E‐cadherin ‐RFP/Py2T cells responding to TGFβ for the indicated time periods. Average values and SD from n = 3 biological replicates, each with technical triplicates, and P ‐values (** P < 0.01, *** P < 0.001) after two‐tailed paired Student’s t ‐test. (C) Immunoblot of parental Py2T and E‐cadherin ‐RFP/Py2T cells for epithelial and mesenchymal proteins, and β‐actin as a loading control, along with molecular size markers (representative of n = 3 independent experiments). For original images, see Fig. . (D) Representative imaging of DIC, RFP, immunofluorescence staining (green) of epithelial and mesenchymal proteins and nuclear DAPI (blue) during EMT (TGFβ) and MET (TGFβ withdrawal) in E‐cadherin ‐RFP/Py2T cells ( n = 3 independent experiments). Scale bars, 50 μm.

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Imaging, Expressing, Control, Fluorescence, Two Tailed Test, Western Blot, Immunofluorescence, Staining

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: TGFβ promotes 3D mammosphere growth. (A) Mammosphere size after treatment or not of 4000 EMTimage cells with TGFβ1, BMP7, or BMP4 for 4 days in a 96‐well round bottom low‐attachment plate to obtain single spheres per well. Graph bars are colour‐coded (red, TGFβ1 (5 ng·mL −1 ); dark blue, BMP7 (100 ng·mL −1 ); light blue, BMP4 (100 ng·mL −1 )) and show average values and SD from n = 13 biological replicates, each with technical triplicates, and P ‐values (**** P < 0.0005) after two‐tailed paired Student’s t ‐test. (B) Representative DIC and RFP fluorescence overlay images of E‐cadherin ‐RFP/Py2T cells from passage 1, in three consecutive 3D cultures ( n = 4 independent experiments). Scale bar, 100 μm. (C) Numbers of mammospheres in the three consecutive passages of the mammosphere cells. The cells were treated under the same conditions as in panel A. Graph bars are colour‐coded (red or blue) and show average values and SD from n = 3 biological replicates, each with technical triplicates, and P ‐values (**** P < 0.0005) after two‐tailed paired Student’s t ‐test. (D) Immunoblot of E‐cadherin ‐RFP/Py2T cells cultured under 3D conditions and collected after stimulation with TGFβ1 (5 ng·mL −1 ) or BMP7 (100 ng·mL −1 ) for 3 and 5 days, followed by analyses for RFP, and epithelial, mesenchymal and stem cell proteins, as well as β‐actin as a loading control, along with molecular size markers (representative of n = 3 independent experiments). For original images, Fig. .

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Two Tailed Test, Fluorescence, Western Blot, Cell Culture, Control

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: TGFβ increases EMTimage stem cell frequency. (A) Schematic drawing of the experiment. Upon TGFβ‐induced EMT of E‐cadherin ‐RFP/Py2T cells (marked with a red circle) cultured under 2D conditions, RFP low mesenchymal cells (marked by light pink triangle) were cultured in 3D. In the absence of TGFβ in 3D, the mammosphere cells underwent partial MET and became enriched in RFP high cells (marked by red polka‐dotted hexagon). In the presence of TGFβ in 3D, the mammospheres remained in an RFP low state and grew larger (marked by light pink striped hexagon). Note that the same colour‐coded symbols are used in panels B‐E. (B‐E) 2D cells were pre‐treated with 5 ng·mL −1 TGFβ1 (red symbols) or 100 ng·mL −1 BMP7 (blue symbols) for 7 days and then analyzed (2D), or trypsinized and seeded in 3D cultures in the absence or presence of 5 ng·mL −1 TGFβ1 or 100 ng·mL −1 BMP7 for 5 days (3D). Representative DIC and RFP fluorescence overlay images of E‐cadherin ‐RFP/Py2T cells in 2D and 3D culture ( n = 3 biological replicates). Scale bar, 100 μm. (C) ELDA measuring sphere‐forming frequency upon TGFβ1 or BMP7 stimulation for 7 days. The number of wells devoid of spheres (fraction nonresponding) is plotted against the number of plated cells per well (from 200 to 1 cell). Average stem cell frequency values are fitted into straight lines. Steeper slopes indicate higher frequencies of sphere‐forming cells. A table indicates average stem cell frequency per condition and associated P ‐values ( n = 3 biological replicates, technical octaplicates; ** P < 0.01, *** P < 0.001, **** P < 10 ‐6 ) after χ 2 ‐test followed by P ‐value test performed to assess goodness of fit. (D) Immunoblot of E‐cadherin ‐RFP/Py2T cells cultured and stimulated as explained in panel B, as indicated, and analyzed for RFP, and epithelial, mesenchymal and stem cell proteins, as well as β‐actin as a loading control, along with molecular size markers (representative of n = 3 independent experiments). For original images, Fig. . (E) Relative expression of the indicated genes normalized to Gapdh in E‐cadherin ‐RFP/Py2T cells cultures as explained in panel B, shown as average values and SD from n = 3 biological replicates, each with technical triplicates, and P ‐values (* P < 0.05, ** P < 0.01, *** P < 0.001) after two‐tailed paired Student’s t ‐test. Graph bars are colour‐coded as explained in panels A and B.

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Cell Culture, Fluorescence, Western Blot, Control, Expressing, Two Tailed Test

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: Partial EMT, EpCAM low /CD51 + and EpCAM low /CD51 + /CD61 + cells, are preferentially induced by TGFβ under 3D culture conditions. (A) EMT‐score analysis by flow cytometry of cell surface proteins in the indicated two biological conditions and with detailed analysis in Fig. . EpCAM high or EpCAM low populations were gated. Three biological replicates were used for the analysis. (B) Pie charts illustrating the indicated colour‐coded cell populations as percentage of the total under the two biological conditions used. The cell type analysis was performed by flow cytometry of the indicated surface proteins in the EpCAM low population. Detailed analysis is shown in Fig. . (C) Impact of TGFβ on EMT‐scores under 3D culture conditions. The data are identical to those of panel B ( n = 3 biological replicates). Triple‐negative (TN) cells represent early EMT and correspond to EpCAM low /CD51 ‐ /CD61 ‐ /CD106 ‐ ; triple‐positive (TP) cells represent complete EMT and correspond to EpCAM low /CD51 + /CD61 + /CD106 + . (D) The EpCAM low /triple‐positive cell population generated after TGFβ stimulation under 3D conditions is graphed relative to the control (no TGFβ) condition, which is normalized to 1. The data source is identical to that in panels B and C ( n = 3 biological replicates). (E) Partial EMT‐score analysis by flow cytometry in the indicated two biological conditions. CD24 low cells were gated first, and CD44 and CD104 cell surface expression was analyzed using the indicated fluorescently‐conjugated antibodies. Three biological replicates were used for the analysis. (F) The percent of CD24 low /CD44 high /CD104 high cells is graphed for cells responding to TGFβ under 3D culture conditions. The data source is identical to that of panel E ( n = 3 biological replicates).

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Generated, Control, Expressing

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: 3D mammosphere growth and invasion under the influence of TGFβ. (A) Schematic drawing of the experiment as in Fig. . Mammospheres were placed on adherent conditions to measure cell migration. (B) Representative DIC and fluorescence microscopy overlay images for the indicated proteins (green), RFP (red) and nuclei (DAPI, blue) of E‐cadherin ‐RFP/Py2T mammospheres and migrating cells. Mammospheres were generated from 2D cells that were pre‐treated with 5 ng·mL −1 TGFβ1 for 7 days and then trypsinized and seeded in 3D cultures in the absence (3D control) or presence of 5 ng·mL −1 TGFβ1 (3D TGFβ) for 5 days, and finally transferred to 2D culture on glass chambers ( n = 3 biological replicates). Scale bar, 100 μm. (C) Violin plots quantifying RFP intensity of 2D control (epithelial) and 2D TGFβ‐treated (EMT) cells as calibration controls, and in migratory cells emanating from mammospheres. Each point represents a single cell. The total cell number was n = 60 and significance ( P ‐value) was assessed using two‐tailed paired Student’s t ‐test. (D) Representative DIC and fluorescence microscopy overlay images of migratory cells emanating from mammospheres under different TGFβ1 concentrations ( n = 2 biological replicates). Scale bar, 100 μm. (E) Mammosphere size after treatment with different TGFβ1 concentrations. Data show average values and SD from n = 6 biological replicates per condition and significance ( P ‐value) assessed using two‐tailed paired Student’s t ‐test. (F) Percentage of mammospheres exhibiting migratory cells emanating from spheres at the indicated time points and at different TGFβ1 concentrations. Data show average values from triplicate determinations. (G) Quantification of invasive area around the mammospheres after stimulation with the indicated TGFβ1 concentrations. DIC and fluorescence microscopy overlay images of two representative mammospheres (0 and 5 ng·mL −1 TGFβ1). Data show average values and SD from triplicate ( n = 3) determinations and associated significance ( P ‐value) assessed by two‐tailed paired Student’s t ‐test. (H) Percentage of migratory/non‐migratory cells emanating from mammospheres under the indicated conditions. The TGFβ type I receptor kinase inhibitor LY2157299 was used at 2.5 μ m with DMSO as vehicle. A representative from n = 3 independent experiments is shown. (I) TGFβ1 concentration was measured by ELISA in the conditioned medium of the indicated conditions. Data show average values with SD from triplicate ( n = 3) determinations and associated significance ( P ‐value) assessed by two‐tailed paired Student’s t ‐test.

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Migration, Fluorescence, Microscopy, Generated, Control, Two Tailed Test, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: EMTimage breast tumour analysis in mice. (A) Schematic drawing of the experiment as in Fig. , with mammospheres injected orthotopically in the mammary fat pads of mice. (B) Quantification of primary tumour volume 6 weeks after orthotopic injection of the indicated cells into 2 distinct sites of a mammary fat pad in each mouse. Data show average values and SD from n = 7 (2D control, 3D control and 3D TGFβ) or n = 8 (2D TGFβ) mice and associated significance ( P ‐value) assessed by two‐tailed paired Student’s t ‐test. (C) Primary tumour and lung metastasis incidence in the indicated conditions. (D) Representative images of lung metastases (out of n = 13 lungs analyzed). Upper, macroscopic images of whole lungs with metastatic nodules (yellow arrowheads) generated by the indicated conditions. Scale bar, 0.5 cm. Lower, H&E staining of single metastatic nodules. Scale bar, 100 μm. (E) Representative images of immunohistochemical staining of E‐cadherin and RFP in metastatic breast tumour in the lung, generated by injection of 3D control cells into mouse mammary fat pads ( n = 3 independent lungs analyzed). The tumour (T), nearby stromal area (demarcated by dotted lines and verified based on extracellular matrix content) and a blood vessel (V) are indicated. Scale bar, 20 μm. (F) The single lung metastasis in only one mouse (see panel C), observed 6 weeks after the injection of 3D TGFβ cells. H&E staining of the single lung metastatic nodule (orange arrowhead) along with immunohistochemistry for the indicated proteins in adjacent serial sections. Scale bar, 50 μm.

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Injection, Control, Two Tailed Test, Generated, Staining, Immunohistochemical staining, Immunohistochemistry

Journal: JCI Insight

Article Title: Quinolinic acid potentially links kidney injury to brain toxicity

doi: 10.1172/jci.insight.180229

Figure Lengend Snippet: Kidney cortex RNA and protein lysates were prepared from Mdm2 -cKO vs. control mice ( n = 4 per group). ( A ) Transformation related protein 53 ( Trp53 ) mRNA and ( B ) p53 protein levels. ( C ) Cyclin-dependent kinase inhibitor 1A ( Cdkn1a ) mRNA and ( D ) p21 protein levels. ( E ) Cytochrome b-245, beta polypeptide ( Cybb ), mRNA and ( F ) CYBB protein levels. ( G ) CXC chemokine ligand 1 ( Cxcl1 ) mRNA level. ( H ) CXCL1 protein concentration. ( I ) Interleukin-1β ( Il1b ) mRNA level. ( J ) IL-1β protein concentration. ( K ) Neutrophil-to-lymphocyte ratio (VetScan Analyzer). ( L ) Plasma interleukin-6 (IL-6) protein concentration. ( M ) Plasma soluble urokinase plasminogen activator receptor (suPAR) concentration. Quantitative PCR (qPCR) results (normalized to Gapdh expression) and protein levels measured via Western blot (normalized to actin) are presented as fold-change relative to control. The proteins analyzed via ELISA are reported as concentrations. Graphs display means ± SEM. Two-tailed t tests: * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Quantitative protein levels in plasma and tissue lysates were assessed using ELISA kits following manufacturer’s protocols: mouse uPAR DuoSet ELISA (R&D Systems, Bio-Techne, DY531), mouse CXCL1/KC Quantikine ELISA Kit (R&D Systems, Bio-Techne, MKC00B-1), mouse IL-6 Quantikine ELISA Kit (R&D Systems, Bio-Techne, M6000B-1), and mouse IL-1β/IL-1F2 Quantikine ELISA Kit (R&D Systems, Bio-Techne, MLB00C-1).

Techniques: Control, Transformation Assay, Protein Concentration, Clinical Proteomics, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: JCI Insight

Article Title: Quinolinic acid potentially links kidney injury to brain toxicity

doi: 10.1172/jci.insight.180229

Figure Lengend Snippet: Mice were fed regular chow (control) or 0.2% adenine-enriched chow for 1 month ( n = 4 per group). ( A and B ) Plasma suPAR and IL-6 levels measured by ELISA. Concentrations of Trp, KYN, 3HK, QA, and 5-HIAA in ( C – G ) plasma, kidney cortex ( H – L ), and brain cortex ( M – Q ). QA and 5-HIAA were measured using a different mass spectrometry method. Graphs display means ± SEM. Two-tailed t test; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Quantitative protein levels in plasma and tissue lysates were assessed using ELISA kits following manufacturer’s protocols: mouse uPAR DuoSet ELISA (R&D Systems, Bio-Techne, DY531), mouse CXCL1/KC Quantikine ELISA Kit (R&D Systems, Bio-Techne, MKC00B-1), mouse IL-6 Quantikine ELISA Kit (R&D Systems, Bio-Techne, M6000B-1), and mouse IL-1β/IL-1F2 Quantikine ELISA Kit (R&D Systems, Bio-Techne, MLB00C-1).

Techniques: Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Two Tailed Test

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Comparison between human ICOS full length and splice variant. A, Schematic representation of full length and spliced variant of human ICOS. The full length of ICOS consists of 5 exons. The gray boxes denote untranslated regions, and the black boxes denote coding sequences. The transmembrane domain (TM) is located in exon 3 and labeled with a green bar. The splicing region is shown in red. B, Comparison of cDNA nucleotide sequences, amino acid sequences, and 5′UTR sequences between ICOS-FL and the splice variant. Nucleotide sequences of cDNA for ICOS-FL (b1) and the splice variant of ICOS (b4) are represented; spliced region of ICOS is shown in red and delineated by red brackets; the nucleotides and amino acids of TM are shown in green. Asterisk indicates the stop codon. Amino acid sequences of full length of ICOS (b2) and the splice variant of ICOS (b5) are shown. The underline depicts additional differences in amino acid sequence compared with ICOS-FL. “Stop” represents a stop codon. 5′UTR sequences of ICOS-FL (b3) and the splice variant of ICOS (b6) are shown. The 5′UTR of the ICOS-SV lacks 32 nucleotides compared with the 5′UTR of ICOS-FL shown in red and delineated by red brackets. C, Schematic diagram of ICOS-FL and the ICOS-SV protein. Thirty-two out of 38 amino acids in the intracellular domain are spliced, as shown in red.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Comparison, Variant Assay, Labeling, Sequencing

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Secretion of the ICOS-SV from ICOS-SV–overexpressing cells. A, RT-PCR to detect the ICOS-SV in human T cells. Total RNA was isolated from purified T cells in PBMCs of a healthy donor. Expression of the ICOS variant was examined by RT-PCR with specific primers. RT-PCR of ICOS-FL is shown as a control. B, DNA sequencing of ICOS-SV cDNA from human T cells. The cDNA of the PCR product was cloned into a TOPO TA vector. The ICOS-SV was confirmed by DNA sequencing using an M13 forward primer. Splicing point is shown by the red arrow. C, DNA sequencing of ICOS-FL cDNA is shown as a control. No splicing occurs in the region corresponding to the position in ( B ), as shown in blue. D, Expression of ICOS-SV and ICOS-FL in 293T cells determined by immunoblot. 293T cells were transduced with recombinant GFP retroviral vector encoding either ICOS-SV or ICOS-FL as a positive control. The parental cells and the GFP-empty vector–transduced cells were used as negative controls. Sample loading was normalized to actin. E, Cell culture supernatants were collected from the cells described in ( A ). Secreted ICOS-SV was examined by ELISA. Two-tailed unpaired t test was used to compare ICOS secretion between the two groups. ***, P < 0.001. Standard deviation of the mean (SD) is shown. F, To validate the secreted ICOS variant protein in the cell supernatant, immunoprecipitation, SDS-PAGE, and immunoblotting assays were performed. Sample loading was normalized to cell numbers.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Purification, Expressing, Variant Assay, Control, DNA Sequencing, Clone Assay, Plasmid Preparation, Western Blot, Transduction, Recombinant, Retroviral, Positive Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Standard Deviation, Immunoprecipitation, SDS Page

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Suppressive effects of sICOS-SV on the costimulation of T cells. A, CHO-K1 cells were transduced with either ICOSL-expressing lentiviral vector or GFP-empty vector (CHO-ICOSL − ) as a negative control. Expression of ICOSL on the cell surface of CHO-K1 was examined by flow cytometry using anti-ICOSL (open histogram with solid line) or lentivirus GFP-empty vector (open histogram with dotted line). Isotype antibody was used as a negative control (filled histogram). B, ICOSL-expressing CHO-K1 cells were stained with ICOS-SV Ig (red line), control IgG Ig (blue line), or PD-1 Ig (orange line) and analyzed by flow cytometry. Both soluble control Ig and PD-1 Ig were used as negative controls. GFP-empty vector-expression CHO-K1 cells stained with ICOS-SV Ig (green line) were used as another negative control for the binding assay. Isotype antibody control was used as a negative control (gray filled histogram). C, CD154 expression was determined by FACS analysis. Isotype control antibody is shown in gray. Activated T cells treated with a suboptimal dose of anti-CD3 were incubated with control Ig + CHO-ICOSL − cells (CHO cells transduced with GFP-empty vector, blue), control Ig + CHO-ICOSL + cells (green), PD-1 Ig + CHO-ICOSL + cells (red), or ICOS-SV Ig + CHO-ICOSL + cells (yellow). Both control Ig and PD-1 Ig were used as negative controls. D, Two-tailed unpaired t test was used to compare expression of CD154 between two groups. **, P < 0.01; ***, P < 0.001. Standard deviation of the mean (SD) is shown. E, CD69 expression was determined by FACS analysis. Isotype control antibody is shown in gray. Activated T cells treated with a suboptimal dose of anti-CD3 were incubated with control Ig + CHO-ICOSL − cells (CHO cells transduced with GFP-empty vector, blue), control Ig + CHO-ICOSL + cells (green), PD-1 Ig + CHO-ICOSL + cells (red), or ICOS-SV Ig + CHO-ICOSL + cells (yellow). Both control Ig and PD-1 Ig were used as negative controls. F, Two-tailed unpaired t test was used to compare expression of CD69 between the two groups. **, P < 0.01; ***, P < 0.001. Standard deviation of the mean (SD) is shown. G, CHO cells transduced with GFP-empty vector (CHO-ICOSL − ) were used as negative controls. Sample loading was normalized to total pan-Akt. H, T-cell proliferation was determined by a [ 3 H]-TdR thymidine incorporation assay. CHO cells transduced with empty vector (CHO-ICOSL − ) were used as negative controls. Two-tailed unpaired t test was used to compare 3 H uptake between the two groups, respectively. *, P < 0.1. Standard deviation of the mean (SD) is shown. I, Schematic diagram of ICOS/ICOSL costimulatory T-cell proliferation, as well as the blocking function of the sICOS-SV. Depicted are the cytoplasmic tail sequences of ICOS-FL and sICOS-SV isoforms. The YMFM Src Homology 2 (SH2) binding motif in the cytoplasmic tail of the ICOS-FL is highlighted in pink. Upon ICOS engagement by the ICOSL on CHO cells, the unique YMFM motif recruits a p85a and a p50a subunits of PI3K, resulting in the elevated phosphorylation of Akt, thereby inducing PI3K activity. In contrast, the ICOS-SV, a truncated isoform lacking the YMFM motif in its cytoplasmic tail, cannot elicit phosphorylation of Akt. Consequently, it fails to promote T-cell proliferation. The secreted ICOS-SV (red) competes with membrane-bound ICOS for binding to ICOSL, thereby blocking the interaction between ICOSL and membrane ICOS. As a result, the sICOS-SV suppresses phosphorylation of Akt and T-cell proliferation, leading to the inhibition of T-cell immunity. The diagram was created with BioRender.com. All data shown are representative of at least 2 independent experiments.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Transduction, Expressing, Plasmid Preparation, Negative Control, Flow Cytometry, Staining, Control, Binding Assay, Incubation, Two Tailed Test, Standard Deviation, Thymidine Incorporation Assay, Blocking Assay, Phospho-proteomics, Activity Assay, Membrane, Inhibition

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: sICOS from melanoma patients inhibits T-cell activation and proliferation induced by GM-CSF–driven DCs in MLRs. A, CD4 + T cells (responders) were stimulated by allogeneic GM-CSF–driven MoDCs (stimulators: negative control DCs, DCs generated by GM-CSF/IL4 + anti-IgG, or DCs generated by GM-CSF/IL4 + anti-CD116) in MLRs. T cells were assessed for activation via flow cytometry using anti-ICOS (blue), anti-GITR (green), or anti-CD25 (gray). Isotype control antibodies were used as negative controls. B, Statistical analysis of the percentage of CD4 + ICOS + , CD4 + GITR + , and CD4 + CD25 + T-cell populations from different groups as described above. C, Soluble ICOS levels were determined by ELISA using supernatants from the MLRs described above. D – E, Serum from sICOS-high and sICOS-negative patients were added to the MLRs, and T-cell proliferation ( D ) and CD69 expression ( E ) were evaluated. Additionally, sICOS was depleted from sICOS-high serum to assess its effects on T cells. F, Statistical analysis of the percentage of CD4 + CD69 + T-cell populations from different groups as described above. All data shown are representative of at least 2 independent experiments. Two-tailed unpaired t test was used between the two groups. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant ( P > 0.05). Standard deviation of the mean (SD) is shown.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Activation Assay, Negative Control, Generated, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test, Standard Deviation

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 1. Vitamin D3 increases legumain expression, activity, and secretion in pre-osteoblastic cells. (A) The nucleotide sequence of the LGMN gene promoter region with annotations of potential vitamin D-responsive elements (VDRE; red) relative to the transcription start site (TSS). (B–F) Human BMSC- TERT cells (20,000 cells/cm2) were incubated with 1,25(OH)2D3 (B–F; 10, 50 or 100 nM), 25(OH)D3 (C–F; 100, 250, 500 or 1000 nM) or an equal volume of ethanol (control, 0 nM) in osteoblast induction medium for seven days before harvesting. (B) Legumain mRNA expression relative to housekeeping control (GAPDH) (2−∆∆CT; n = 3). (C) One representative immunoblot of legumain (proform 56 kDa, mature form 36 kDa) and GAPDH (housekeeping) in cell lysates (n = 3). (D) Quantification of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in C (n = 3). (E) Legumain activity (dF/s) in cell lysates adjusted for the total protein concentration (µg/mL) (n = 6–9). (F) Secreted legumain (pg/mL) in conditioned media measured by ELISA and adjusted for the total protein concentration in the corresponding cell lysates (n = 3–5). (B,D–F) Data represent mean ± SEM. (B,D) Kruskal–Wallis test. (E,F) One-way ANOVA. * p < 0.05 vs. 0 nM 1,25(OH)2D3 or 25(OH)D3. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Sequencing, Incubation, Control, Western Blot, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 2. Treatment with 25(OH)D3 increases legumain levels and activity in wild-type mice. Wild-type mice (Lgmn+/+) were treated with 50 µg/kg 25(OH)D3 (n = 7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (A) Legumain mRNA expression relative to the geometric mean of CT values of four housekeeping controls in kidney, liver, and spleen (2−∆∆CT; n = 5). (B) One representative immunoblot of legumain and GAPDH in kidney, liver, and spleen (n = 3). (C) Quantifi- cation of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH (housekeeping) in kidney, liver, and spleen from immunoblots represented in (C) (n = 3). (D) Legumain activity (dF/s) in kidney, liver, and spleen adjusted for total protein concentration (µg/mL, n = 5). (E) Legumain plasma concentration (ng/mL) measured by ELISA (n = 5). (F) Cor- relation between legumain (ng/mL and 1,25(OH)2D3 (pmol/L) concentrations in plasma (n = 5). (A,C,E) Two-tailed unpaired Student’s t-test. (D) Mann–Whitney test. Data represent mean ± SEM. * p < 0.05. (F) Simple linear regression. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Activity Assay, Control, Injection, Expressing, Western Blot, Protein Concentration, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 3. Legumain is required for VDBP processing and regulation. (A) Purified VDBP from human plasma (1.9 µM) was incubated in legumain assay buffer (pH 5.8) at 37 ◦C with or without purified active bovine legumain (2 µM) for 5 h before gel electrophoresis and immunoblotting of VDBP (n = 1). (B–H) Wild-type (Lgmn+/+) and legumain-deficient (Lgmn−/−) mice were treated with 50 µg/kg 25(OH)D3 (n = 6–7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (B) One representative immunoblot of VDBP and GAPDH (housekeeping) in kidney and liver (n = 4). (C–F) Quantification of VDBP immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in (B) (n = 4). (C) Hepatic VDBP 45 kDa immunoband. (D) Renal VDBP 45 kDa immunoband. (E) Hepatic VDBP 55 kDa immunoband. (F) Renal VDBP 55 kDa immunoband. (G) Plasma VDBP concentration (µg/mL) was measured by ELISA (n = 6–7). (H) Hepatic VDBP mRNA expression relative to the geometric mean of CT values of four house- keeping controls (2−∆∆CT, n = 5). (C–H) Data represent mean ± SEM. Two-way ANOVA. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. different genotype, same treatment. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Purification, Clinical Proteomics, Incubation, Nucleic Acid Electrophoresis, Western Blot, Control, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 5. Graphical representation of the suggested interplay between vitamin D and legumain. Left panel: Vitamin D (VD3) promotes legumain expression and activity through transcriptional upregulation of the legumain gene (LGMN). The free fraction of circulating VD3 metabolites diffuse through plasma membranes. 25-hydroxyvitamin D (25(OH)D3) is hydroxylated by 1α-hydroxylase (CYP27B1), forming the active metabolite 1α,25-dihydroxyvitamin D (1,25(OH)2D3). 1,25(OH)2D3 binds to the nuclear vitamin D receptor (VDR) and promotes transcription of legumain (LGMN). Synthesized prolegumain is either sorted and activated in the endolysosomal system or released to the extracellular environment. Right panel: In the proximal tubular epithelium, 25(OH)D3 bound to vitamin D binding protein (VDBP) is internalized from the tubular lumen through a megalin/cubilin- mediated process. The vitamin D metabolite is released, enabling subsequent hydroxylation by 1α-hydroxylase (CYP27B1) or 24-hydroxylase (CYP24A1), and VDBP is cleaved by legumain in the endolysosomal system. VDBP cleavage by legumain might be important in controlling the systemic level of vitamin D metabolites. Created with BioRender.com (accessed on 11 December 2023).

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Clinical Proteomics, Synthesized, Binding Assay