Journal: Nature

Article Title: Dynamic cytoskeletal regulation of cell shape supports resilience of lymphatic endothelium.

doi: 10.1038/s41586-025-08724-6

Figure Lengend Snippet: Fig. 1 | Junctional heterogeneity in capillary LECs. a, Whole-mount immunofluorescence of ear skin from a 25-week-old Cdh5-GFP mouse expressing VE-cadherin-GFP fusion protein (VE-cad). Boxed areas magnified below show unsegmented (arrows) and focal (arrowheads) VE-cadherin+ junctions in lymphatic capillary (left) and precollecting vessel (middle), and continuous zipper junctions in LYVE1− collecting vessel (right). b, Immunofluorescence in 12-week-old mouse ear skin showing VE-cadherin colocalization with CLDN5 at junctions. Line intensity profiles through lines 1 and 2 of respective stainings are depicted. c, Immunofluorescence at the indicated ages depicting junctional heterogeneity. Boxed areas are magnified. d,e, Quantification of lymphatic vessel sprouting (percentage of spiky ends of all lymphatic capillary ends, n = 7, 12 per respective stage, mean ± s.e.m.; d) and LEC proliferation (percentage Ki67+ of all LECs by flow cytometry, n = 3, 6, 8, 6 mice per respective stage, mean ± s.e.m.; e). f, Whole-mount silver nitrate (Ag) staining of ear dermis showing deposits around cell perimeter, including the lobe tips (arrow),

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used The following antibodies were used for whole mount immunofluorescence (dilution 1:100-1:500): chicken anti-GFP (ab13970, Abcam), goat anti-mouse VEGFR3 (AF743, R&D Systems), goat anti-mouse VE-cadherin (R&D Systems, AF1002), goat anti-mouse PECAM1 (R&D, Systems AF3628), mouse anti-HA tag, Alexa Fluor 647 (Cell Signalling Technology, 6E2), rabbit anti-alpha tubulin (Abcam, ab52866), rabbit anti-GFP (A11122, Thermo Fisher Scientific), rabbit anti-DsRed (Takara Bio, 632496), rabbit anti-mouse LYVE1 (Reliatech, 103-PA50AG), rabbit anti-mouse CLDN5 (Invitrogen, 34-1600), rat anti-mouse PECAM1 (553370, BD Pharmingen), rat anti-mouse LYVE1-Alexa FluorTM 488, Clone ALY7 (Invitrogen, 53-0443-82), rat anti-mouse LYVE1 (R&D Systems (MAB2125), rat Anti-Mouse CD29 Clone 9EG7 (BD Pharmingen, 553715) All secondary antibodies were conjugated to Cy3(JIR, 712-165-153),(JIR, 711-166-152), Dylight 405 (JIR, 712-475-153), Alexa Fluor 488 (JIR, 703-545-155), (JIR, 712-545-153), (JIR, 711-545-152), Alexa Fluor 594 (JIR, 705-585-147), Alexa Fluor 647(JIR, 712-605-153),(JIR, 705-605-147),(JIR, 711-605-152) or Alexa Fluor 680 (JIR, 705-625-147) were raised in donkey and obtained from Jackson ImmunoResearch(JIR) .

Techniques: Immunofluorescence, Expressing, Flow Cytometry, Staining

Journal: Nature

Article Title: Dynamic cytoskeletal regulation of cell shape supports resilience of lymphatic endothelium.

doi: 10.1038/s41586-025-08724-6

Figure Lengend Snippet: Fig. 2 | Morphology and remodelling of intercellular overlaps between capillary LECs. a, Constructs for mosaic multicolour labelling of LECs using membrane-localized fluorescent proteins. b, Whole-mount immunofluorescence of mosaically labelled dermal LECs in a 6-week-old iMb2-Mosaic;Vegfr3-creERT2 mouse after 4-OHT treatment at 3 weeks, showing lobate shape in LYVE1+ capillaries and elongated shape in LYVE1− collectors (arrowheads). c, Whole-mount immunofluorescence of embryonic back skin (E17) or ear skin at indicated postnatal stages in iMb2-Mosaic;Vegfr3-creERT2 mice. d–f, Dermal LEC and vessel parameters, represented as mean ± s.d.: cell size (d, n = 24, 17 and 20 cells per respective stage), lobe number (e, n = 24, 48 and 20 cells per respective stage) and average lymphatic capillary width (f, n = 7, 5 and 9 mice per respective stage). Ordinary one-way ANOVA. g, Immunofluorescence of ear skin of a 12-week-old iMb2-Mosaic;Vegfr3-creERT2 mouse showing LYVE1 at LEC overlaps, with corresponding intensity plot. h, Double staining for cell surface and total LYVE1 (left), or with intradermally

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used The following antibodies were used for whole mount immunofluorescence (dilution 1:100-1:500): chicken anti-GFP (ab13970, Abcam), goat anti-mouse VEGFR3 (AF743, R&D Systems), goat anti-mouse VE-cadherin (R&D Systems, AF1002), goat anti-mouse PECAM1 (R&D, Systems AF3628), mouse anti-HA tag, Alexa Fluor 647 (Cell Signalling Technology, 6E2), rabbit anti-alpha tubulin (Abcam, ab52866), rabbit anti-GFP (A11122, Thermo Fisher Scientific), rabbit anti-DsRed (Takara Bio, 632496), rabbit anti-mouse LYVE1 (Reliatech, 103-PA50AG), rabbit anti-mouse CLDN5 (Invitrogen, 34-1600), rat anti-mouse PECAM1 (553370, BD Pharmingen), rat anti-mouse LYVE1-Alexa FluorTM 488, Clone ALY7 (Invitrogen, 53-0443-82), rat anti-mouse LYVE1 (R&D Systems (MAB2125), rat Anti-Mouse CD29 Clone 9EG7 (BD Pharmingen, 553715) All secondary antibodies were conjugated to Cy3(JIR, 712-165-153),(JIR, 711-166-152), Dylight 405 (JIR, 712-475-153), Alexa Fluor 488 (JIR, 703-545-155), (JIR, 712-545-153), (JIR, 711-545-152), Alexa Fluor 594 (JIR, 705-585-147), Alexa Fluor 647(JIR, 712-605-153),(JIR, 705-605-147),(JIR, 711-605-152) or Alexa Fluor 680 (JIR, 705-625-147) were raised in donkey and obtained from Jackson ImmunoResearch(JIR) .

Techniques: Construct, Membrane, Immunofluorescence, Double Staining

Journal: Nature

Article Title: Dynamic cytoskeletal regulation of cell shape supports resilience of lymphatic endothelium.

doi: 10.1038/s41586-025-08724-6

Figure Lengend Snippet: Fig. 3 | Cytoskeletal organization in lobate capillary LECs. a, Dot plot showing differential expression of cytoskeletal genes between capillary and collecting vessel LECs. Dot size illustrates percentage of cells with transcript counts, colour illustrates average expression (log2-fold difference). Cap, lymphatic capillary; Col, collecting vessel. b, Whole-mount immunofluorescence of adult ear skin showing microtubule network in dermal capillary LECs. Cell outline, based on VE-cadherin and LYVE1 staining, in red. c, Quantification of microtubule (MT) anchoring and density in capillary LECs in 9–12-week-old mice. Cell outline from c in green, with MT endpoints shown by yellow (concave) and purple (convex) dots. Data represent the percentage of MT anchoring (left; n = 5 LECs from five mice, 20–53 MT per cell), or MTs per µm of cortex in concave (right; n = 156 MTs, 5 LECs from five mice) versus convex (n = 56 MTs, 5 LECs

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used The following antibodies were used for whole mount immunofluorescence (dilution 1:100-1:500): chicken anti-GFP (ab13970, Abcam), goat anti-mouse VEGFR3 (AF743, R&D Systems), goat anti-mouse VE-cadherin (R&D Systems, AF1002), goat anti-mouse PECAM1 (R&D, Systems AF3628), mouse anti-HA tag, Alexa Fluor 647 (Cell Signalling Technology, 6E2), rabbit anti-alpha tubulin (Abcam, ab52866), rabbit anti-GFP (A11122, Thermo Fisher Scientific), rabbit anti-DsRed (Takara Bio, 632496), rabbit anti-mouse LYVE1 (Reliatech, 103-PA50AG), rabbit anti-mouse CLDN5 (Invitrogen, 34-1600), rat anti-mouse PECAM1 (553370, BD Pharmingen), rat anti-mouse LYVE1-Alexa FluorTM 488, Clone ALY7 (Invitrogen, 53-0443-82), rat anti-mouse LYVE1 (R&D Systems (MAB2125), rat Anti-Mouse CD29 Clone 9EG7 (BD Pharmingen, 553715) All secondary antibodies were conjugated to Cy3(JIR, 712-165-153),(JIR, 711-166-152), Dylight 405 (JIR, 712-475-153), Alexa Fluor 488 (JIR, 703-545-155), (JIR, 712-545-153), (JIR, 711-545-152), Alexa Fluor 594 (JIR, 705-585-147), Alexa Fluor 647(JIR, 712-605-153),(JIR, 705-605-147),(JIR, 711-605-152) or Alexa Fluor 680 (JIR, 705-625-147) were raised in donkey and obtained from Jackson ImmunoResearch(JIR) .

Techniques: Quantitative Proteomics, Expressing, Immunofluorescence, Staining