catalog no 3447  (R&D Systems)

Journal: EMBO Molecular Medicine

Article Title: KRAS-dependent glycolytic reprogramming of endothelial cells in sporadic arteriovenous malformations

doi: 10.1038/s44321-026-00383-y

Figure Lengend Snippet: Experiments were conducted with IM-HUVEC KRAS WT and KRAS G12V cells (−/+ doxycycline [Dox] induction). ( A ) Representative immunofluorescence (IF) staining and quantification of VE-cadherin (Green). Mean ± standard deviations (SD). One-way ANOVA with Tukey’s post hoc tests. n (independent experiments) = 3 (3–4 fields of view per replicate). Scale bars = 100 μm. ( B ) Fluorescence measurements of 70 kDa FITC transwell leak assay. Two-tailed paired t test. n = 6. ( C ) Representative western blots and quantification of VE-cadherin. Mean ± SD. One-way ANOVA. n = 3. ( D ) Representative spheroid sprouting assay (24 h). Bar graph: mean ± SD. Box and whiskers: min to max. Kruskal–Wallis test with Dunn’s multiple comparisons. n = 5 (5–12 spheroids per condition per replicate). Scale bars = 200 μm. ( E ) Representative Angiogenesis Dot Blot profiling of cell culture media. Significantly changed proteins are highlighted using yellow dotted boxes. n = 3. ( F ) Representative image of wound migration assay (6 h). Yellow dotted lines highlight cell boundaries after migration. Mean ± SD. One-way ANOVA with Tukey’s post hoc tests. n = 4. Scale bars = 200 μm. ( G ) Representative IF staining and intensity quantification of hyaluronic acid binding protein (HABP). Mean ± SD. Two-tailed unpaired t tests (comparing Dox vs No Dox). n = 4. Scale bars = 100 μm. ( H ) Representative IF staining and intensity quantification of fibronectin (FN1). Mean ± SD. Two-tailed unpaired t tests (comparing Dox vs No Dox). n = 4. Scale bars = 50 μm. ( I ) Glycolysis stress test (left) and mitochondrial (mito) stress test (right). SL327 treatment = 2 μM. Mean ± standard error of means (SEM). Selected statistics shown represent significant changes between KRAS G12V +Dox condition compared to all other conditions. One-way ANOVA with Tukey’s post hoc tests. n = 6 (glycolysis test), n = 5 (mito test) and n = 4 for SL327 treatments. In this figure, * P value < 0.02 with exact P values shown in Appendix Table . .

Article Snippet: For each mL of collagen matrix mixture, the mixture contained: 550 μL of methylcellulose solution with 40% FBS + 150 μL of 15.6 mg/mL NaHCO 3 in water + 10 μL of 1 M NaOH + 300 μL of Cultrex 3-D Culture Matrix Rat Collagen I (R&D Systems, Cat #: 3447-020-01).

Techniques: Immunofluorescence, Staining, Fluorescence, Two Tailed Test, Western Blot, Dot Blot, Cell Culture, Migration, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: KRAS-dependent glycolytic reprogramming of endothelial cells in sporadic arteriovenous malformations

doi: 10.1038/s44321-026-00383-y

Figure Lengend Snippet: ( A ) Schematic of co-culturing IM-HUVEC KRAS and IM-HUVEC F-Tractin cells. Example images showing the difference between the F-Tractin and mScarlet-KRAS signals. ( B – G ) Representative IF staining of ( B ) pERK ( n = 3), ( C ) VE-cadherin ( n = 3), ( D ) Fibronectin (FN1) ( n = 3), ( E ) BrdU ( n = 3), ( F ) Ki67 ( n = 3), ( G ) TUNEL ( n = 3) within the co-culture system (−/+ Dox). Time of KRAS induction varies depending on the experiment: (i) proliferation assays (i.e., BrdU, Ki67) = 24 h; (ii) FN1 and TUNEL assays = 3 days; (iii) pERK and VE-cadherin assays = 4 days. White asterisks in ( C ) indicate examples of barrier gaps. Arrows in ( D ) indicate regions under WT ECs that have reduced FN1 levels. ( H ) Representative IF staining of pERK within the co-culture system showing changes to the areas occupied by IM-HUVEC F-Tractin cells after 4 days of co-culture with IM-HUVEC KRAS cells ( n = 3). In all images, “FT” marks regions of IM-HUVEC F-Tractin cells, while “K” marks regions of IM-HUVEC KRAS WT or G12V cells. White solid lines show the boundary between IM-HUVEC KRAS and F-Tractin cells. All scale bars = 100 μm. ( I , J ) Quantifications of cell area ( I ) and circularity ( J ) by tracing VE-cadherin staining images from the same experiments as ( C ). Violin plots with dotted lines show interquartile range. One-way ANOVA with Tukey’s post hoc tests. n = 3 (540 cells were quantified per condition). ( K , L ) Quantifications of proportions of ( K ) BrdU and ( L ) Ki67 positive IM-HUVEC KRAS cells from the same experiments as ( E , F ). Mean ± SD. One-way ANOVA tests. n = 3 (5 fields of view per condition per replicate). ( M ) Quantification of area occupied by IM-HUVEC F-Tractin cells after co-culture with IM-HUVEC KRAS cells (−/+ Dox). Two groups of experiments were conducted: (1) KRAS expression was induced right after seeding (prior to 100% confluency) (left); (2) KRAS expression was induced after the formation of confluent monolayers (right). Mean ± SD. Unpaired, two-tailed t tests. n = 3 (6 fields of view per condition per replicate).

Article Snippet: For each mL of collagen matrix mixture, the mixture contained: 550 μL of methylcellulose solution with 40% FBS + 150 μL of 15.6 mg/mL NaHCO 3 in water + 10 μL of 1 M NaOH + 300 μL of Cultrex 3-D Culture Matrix Rat Collagen I (R&D Systems, Cat #: 3447-020-01).

Techniques: Staining, TUNEL Assay, Co-Culture Assay, Expressing, Two Tailed Test