Journal: eLife

Article Title: R-spondins engage heparan sulfate proteoglycans to potentiate WNT signaling

doi: 10.7554/eLife.54469

Figure Lengend Snippet: ( A ) Binding of HS20 to GPC3 or GPC4. An ELISA plate was coated with GPC3, GPC4, or mutant versions lacking HS chains (GPC3ΔHS or GPC4ΔHS). HS20 scFv was added at increasing concentrations (x-axis), followed by a goat anti-human IgG horseradish peroxidase conjugate. Bound HS20 was quantified with 3,3′,5,5′-tetramethylbenzidine detection reagent by measuring absorbance at 450 nm (y-axis). An irrelevant Fc-fusion protein (CD276-hFc, labeled ‘Control’) was used as an antigen control. ( B–C ) Fold-change in the EC 50 of WT RSPO3 ( B ) or RSPO3 ΔTSP/BR HS20 ( C ) in LGR4/5/6 KO cells and clonal derivatives in which different HSPGs were depleted through the indicated gene disruptions (see Results and Materials and methods for details). Since deletion of some GPCs in HAP1 cells leads to impaired WNT reception ( ; ), we first determined a concentration of WNT3A CM that yielded similar levels of WNT reporter activity in all the cell lines and then titrated RSPO3 variants to determine their EC 50 values. Error bars denote the standard error of the curve fits used to calculate the EC 50 . The statistical significance of differences between the measured EC 50 values in LGR4/5/6 KO cells and clonal derivatives thereof was determined by a two-tailed, unpaired t-test, and is indicated as **** ( p <0.0001), *** ( p <0.001), ** ( p <0.01), * ( p <0.05) or ns (non-significant).

Article Snippet: Increasing concentrations of HS20 (serial 2-fold dilutions starting from 1 μg/ml) were added into a 96-well ELISA plate coated with 5 μg/ml of glycosylated GPC3, GPC4 (R and D Systems Cat. # 2119-GP and 9195-GP, respectively), GPC3ΔHS, GPC4ΔHS, or an irrelevant human Fc-fusion protein (CD276-hFc, labeled ‘Control’ in ) and incubated for 1 hr at RT.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Labeling, Control, Concentration Assay, Activity Assay, Two Tailed Test

Journal: Nature Immunology

Article Title: Spatial patterning of fibroblast TGFβ signaling underlies treatment resistance in rheumatoid arthritis

doi: 10.1038/s41590-025-02386-2

Figure Lengend Snippet: a , UMAP plot of isolated single cells from the indicated 3D co-culture conditions . Fibroblast monoculture: organoid containing fibroblasts only; Fibroblasts + ECs: organoids containing fibroblasts and ECs; Fibroblasts + ECs + DAPT: organoids containing fibroblasts and ECs treated with a Notch inhibitor. b , UMAP plot of cells from organoid culture shaded by level of fibrogenic gene signature score calculated with UCell. c , Dot plots representing the expression of selected genes by condition. The asterisk indicates genes and conditions with P < 1 × 10 −100 between monoculture and co-culture. d , e , RT–qPCR analysis of TGFB isoform ( d ) and receptor gene expression ( e ) on unstimulated or DLL4-stimulated fibroblasts treated with or without DAPT (10 µM) for 72 h. f , g , Immunoblots of TGFβ isoforms ( f ) and receptors ( g ) with lysates from unstimulated or DLL4-stimulated fibroblasts (72 h). Data are representative of three independent experiments. h , RT–qPCR analysis of COL1A1 , COL3A1 and COL6A1 gene expression on unstimulated or DLL4-stimulated fibroblasts treated with or without TGFβ inhibitor (SB431542; 10 µM) or DAPT (10 µM) for 72 h. i , ELISA quantification of fibroblast production of pro-collagen I alpha 1 in 3 cell lines over 24 h after 3 days of treatment with siRNA (20 nM) with or without DLL4 stimulation. j , k , ELISA quantification of fibroblast production of POSTN ( j ) and COMP ( k ) over 24 h after 3 days of treatment with siRNA (20 nM) with or without DLL4 stimulation. For d , e and h , each data point represents an independent cell line ( n = 6); for i – k , each data point represents biological replicates ( n = 3) from a single-cell line and are representative of at least two independent experiments. Data are shown as the mean ± s.d. Statistical analysis was performed by a two-sided ratio paired t -test for d , e and h , two-sided Bonferroni-corrected Wilcoxon test for c and unpaired two-sided t -tests for i – k .

Article Snippet: For measurement of pro-collagen 1 alpha 1 (R&D Systems, DY6220-05), COMP (R&D Systems, DY3134) or POSTN (R&D Systems, DY3548B), TGFβ1 (R&D Systems, DY240-05), TGFβ2 (R&D Systems, DY302) and TGFβ3 (DY243) plates were incubated overnight at 4 °C with diluted capture antibody solution, washed with PBS-T and blocked for 1 h at 20 °C in blocking buffer (1% BSA in PBS).

Techniques: Isolation, Co-Culture Assay, Expressing, Quantitative RT-PCR, Gene Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Single Cell