1 recombinant human gdnf  (R&D Systems)

Journal: bioRxiv

Article Title: Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling

doi: 10.64898/2026.03.31.715459

Figure Lengend Snippet: GDNF-RET signaling tunes branching in mouse and human ureteric bud tissues. A. GDNF-RET signaling interactions involved in kidney branching. B. Immunofluorescence of cleared E14 mouse kidney. Top left , ECAD and SIX2. Bottom left, RET and GFRA1. Top right , isolated RET. Bottom right , isolated GFRA1 signal. C. Air-liquid interface (ALI) culture of mouse embryonic kidney explants. D. Design of GDNF-RET signaling perturbation experiments in ALI culture. E. Immunofluorescence of E13 kidneys grown in ALI culture for 4 days in 100 nM Selpercatinib (+RETi), 100 ng ml -1 GDNF (+GDNF), or control media. Inset , close-up of tip domains. Explants are immunostained for EpCAM (epithelium), RET (tip cells), and JAG1 (early nephrons). F. Tip number on days 1-4 across all conditions, N = 4 kidneys per condition. P -values by one-way Kruskal-Wallis test with Dunn’s post hoc test. G. Differentiation of iUB organoids from hiPSCs H. Immunofluorescence of epithelial (ECAD) and tip (RET) markers in a day 12 iUB organoid. I. Design of GDNF-RET signaling perturbations for iUB organoids. J. Merged brightfield and GATA3:mCherry images of iUB organoids at days 9 and 12. Organoids were cultured in control (-GDNF) or complete branching medium (+GDNF, 50 ng ml -1 ), excess GDNF (++GDNF, 250 ng ml -1 ), or complete branching medium with 100 nM Selpercatinib (+RETi). See: Fig. S4F . K. Projected area (x10 4 µm 2 ) on days 9 and 12 for all conditions, n = 47, 56, 40, 46 iUB organoids (-GDNF, +RETi, +GDNF, ++GDNF) from 2 independent biological replicates. L. Circularity (a.u.) on days 9 and 12. M. Bud number on days 9 and 12. P -values in panels K, L, M by one-way ANOVA with Dunnett’s post hoc test using +GDNF as reference group.

Article Snippet: MDCK cells were not starved and were treated with 50 ng ml -1 GDNF (#212-GD-050, R&D Systems) and 100 ng ml -1 recombinant human Gfrɑ1 (#714-GR-100, R&D Systems) at defined time points.

Techniques: Immunofluorescence, Isolation, Control, Cell Culture

Journal: bioRxiv

Article Title: Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling

doi: 10.64898/2026.03.31.715459

Figure Lengend Snippet: Blue light stimulation of optoRET drives ERK signaling and ligand-independent budding in iUB organoids. C. Schematic of hiPSC-optoRET generation using piggyBac transposase and differentiation into iUB-optoRET organoids. D. Immunofluorescence of iUB-optoRET monolayers stimulated for 2 hr under the indicated conditions (±light). Blue light stimulation was provided by an optoPlate-96 (470 nm, 50 mW cm -2 , 1 s every 10 s) for 2 hrs. Top , optoRET (detected by anti-GFP) and nuclei (DAPI). Bottom , intensity-coded ppERK (a.u.). E. Violin plot of ppERK (a.u.) for iUB-optoRET cells under -light and +light conditions, n = 2458, 2030 cells (control, +light) pooled from 2 independent biological replicates. P -value by Welch’s t-test. F. Immunofluorescence density plot of ppERK (a.u.) as a function of optoRET(eGFP) intensity (a.u.) for cells shown in B. n = 2458, 2030 cells (control, +light) pooled from two independent biological replicates. G. Stimulation conditions for iUB-optoRET organoids: control (−light/−GDNF), +light, +GDNF, and combined (+light/+GDNF) treatments. H. Live fluorescence images of GATA3 mCherry iUB-optoRET organoids under optogenetic and ligand-based stimulation conditions. Blue light stimulation was provided by an optoPlate-96 (320 mW cm -2 , 0.5 s every 4 min) between days 7-11 and the +GDNF and +light/+GDNF groups received 50 ng ml -1 GDNF. I. Bud number on day 11 across all conditions, n = 50, 50, 51, 50 organoids (control, +light, +GDNF, +light/+GDNF) from 2 independent biological replicates. P -values by one-way Kruskal-Wallis test with Dunn’s post hoc test. J. Differential expression analysis from bulk RNA-seq of iUB-optoRET organoids grown in ±light stimulation conditions (see also: Fig. S14 ). Top , relative density of tip and trunk marker genes. Bottom , volcano plot of all genes organized by significance (-log 10 ( p )) and log 2 (fold-change) with tip and trunk-specific markers highlighted. Data are derived from 3 biological replicates. K. Gene Set Enrichment Analysis (GSEA) as a function of normalized enrichment score (NES). Hallmark gene sets are color coded by false discovery rate (FDR), size coded by the size of the gene set.

Article Snippet: MDCK cells were not starved and were treated with 50 ng ml -1 GDNF (#212-GD-050, R&D Systems) and 100 ng ml -1 recombinant human Gfrɑ1 (#714-GR-100, R&D Systems) at defined time points.

Techniques: Immunofluorescence, Control, Fluorescence, Quantitative Proteomics, RNA Sequencing, Marker, Derivative Assay

Journal: bioRxiv

Article Title: Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling

doi: 10.64898/2026.03.31.715459

Figure Lengend Snippet: Spatially patterned optoRET stimulation drives asymmetric budding in iUB organoids. A. Strategy for targeted optogenetic stimulation of iUB-optoRET organoids. B. Average projections of GATA3:mCherry for small ( top panels ) and large ( bottom panels ) iUB-optoRET organoids under control, +GDNF, +light whole, and +light half conditions. Light stimulation was provided by 488 nm DMD-based light projection (0.5 s every 4 mins) for 4 days. Organoids in the +GDNF group received 50 ng ml -1 GDNF. Top row , average projections across individual organoids at day 7. Middle row , average projections across individual organoids at day 11. Bottom row , normalized average projections for individual organoids at day 11. C. Representative average projections of individual small ( top ) and large ( bottom ) iUB-optoRET organoids on day 11. The cyan dashed line divides the non-illuminated side ( left , -light half) from the illuminated side ( right , +light half). Magenta dots indicate bud locations. D. Confocal immunofluorescence images of optoRET+ and RET+ cells within iUB-optoRET bud tips in +light whole condition. Endogenous RET was visualized with an antibody against the extracellular domain (ECD). Left , ECAD, optoRET, and RET(ECD). Middle , optoRET and tip outline. Right , RET(ECD) and tip outline. E. Radial histograms of bud orientation angle (°) for small organoids across all conditions, n = 19, 20, 19, 20 organoids (control, +GDNF, +light whole, and +light half) pooled from 2 biological replicates. P -values by Kolmogorov-Smirnov test against a uniform reference distribution.

Article Snippet: MDCK cells were not starved and were treated with 50 ng ml -1 GDNF (#212-GD-050, R&D Systems) and 100 ng ml -1 recombinant human Gfrɑ1 (#714-GR-100, R&D Systems) at defined time points.

Techniques: Control, Immunofluorescence

Journal: bioRxiv

Article Title: Measurement of adhesion and traction of cells at high yield (MATCHY) reveals an energetic ratchet driving nephron condensation

doi: 10.1101/2024.02.07.579368

Figure Lengend Snippet: ( A ) Model schematic of ureteric bud tubule branching in the embryonic kidney. Right, detail of nephrogenic niche anatomy at ureteric bud tips and the role of RET in MAPK activation via ERK. ( B ) Phase contrast micrographs of RET-expressing MDCK cell line as a reductionist model, with and without activation using GDNF and the co-receptor GFRA1. ( C ) Montage of phase and immunofluorescence micrographs and traction force microscopy output (displacement and stress fields) for representative RET-MDCK cells after the indicated treatments on adhesive polyacrylamide substrate. ( D ) Violin plots of traction force distribution for indicated treatments showing means and quartiles (gray lines, n = 8 replicate wells per condition; red points are means of replicates). One-way ANOVA, Tukey’s test, **** p < 0.0001. ( E ) Plots of RET expression and pERK intensity by immunofluorescence upon GDNF/GFRA1 activation vs. untreated control (>1,700 cells combined across n = 8 replicate wells). Traction force is represented by point color and by running average plots (window size of 25 cells).

Article Snippet: To stimulate pERK overexpression, 100 ng/ml of soluble, recombinant human GFRA1 (R&D Systems, AF714-SP) along with 50 ng/ml recombinant human GDNF (R&D Systems, 212-GD-010) were administered for 12 hr.

Techniques: Activation Assay, Expressing, Immunofluorescence, Microscopy, Adhesive, Control