Boucher et al NCOMMS B
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2 Supplementary Figure 1 (linked to Figure 1). mvegfr1 constitutively internalizes in endothelial cells. (a) Immunoblot of mflt1 from undifferentiated mouse embryonic stem (ES) cells with indicated genotypes; 2 replicates. (b) Quantification of VEGFR1 fluorescence intensity of HUVEC with indicated treatments via integrated density. (n=44/control; n=42/chx). CHX, cycloheximide; 2 replicates. Statistics: Shown are means + 95% CI, student s t-test with Tukey s post-hoc range test; ns, not significant. (c-d) Immunofluorescence of VEGFR1 from HUVEC internalization assay with indicated treatments. Strip: 0.2M acetic acid, 4min; 3 replicates. (e) Rab11a immunofluorescence in HUVEC with indicated treatments. DRAQ7 (nuclei); 2 replicates. All scale bars: 20µm. 2
3 Supplementary Figure 2 (linked to Figure 2). Destabilized mvegfr1 degrades via the lysosome. (a-b) (a) VEGFR1 immunofluorescence of d5 HUVEC angiogenic sprouts treated with 2-BH for 12h prior to CHQ for 5h, scale bar: 25µm. (b) Quantification of VEGFR1 fluorescence via integrated density. Shown are means + 95% CI (#sprouts; EtOH/H 2 O, n=6; EtOH/CHQ, n=6; 2-BH/H 2 O, n=8; 2-BH/CHQ, n=5); 2-BH, 2-bromohexadecadnoic acid; CHQ, chloroquine; 2 replicates. Statistics: one-way ANOVA and pairwise comparison with post-hoc Tukey s range test. **, p 0.01; ns, not significant. (c-d) Immunoblot of HUVEC with indicated treatments, 3 replicates. 3
4 Supplementary Figure 3 (linked to Figure 3). Palmitoylation regulates mvegfr1 localization with lysosomes. (a-b) (a) VEGFR1 and LAMP-1 (lysosome marker) immunofluorescence of HUVEC with indicated treatments and (b) Mander s Correlation Coefficient quantification of VEGFR1 overlap with LAMP-1. (#cells: EtOH, n=38; 2-BH, n=36); 3 replicates, scale bar: 10µm. Statistics: Shown are means +95% CI. Pairwise comparison with post-hoc Tukey s range test. **, p
5 Supplementary Figure 4 (linked to Figure 4). Knockdown of the palmitoyl acetyl transferase DHHC3 reduces mvegfr1 levels in endothelial cells. (a) Acyl-biotin exchange workflow. B, biotin; PA, palmitic acid; NEM, N-ethylmaleimide; HAM, hydroxylamine. (b) Acyl-biotin exchange on immunoprecipitated VEGFR1 from HUVEC with indicated treatments, then immunoblot for biotin, stripped and reprobed for VEGFR1. HAM, hydroxylamine; 2 replicates. (c) DHHC3 immunofluorescence of HUVEC with indicated treatments for 24h; 2 replicates, scale bar: 20µm. (d-e) Immunoblot for mvegfr1 in HUVEC with 24h knockdown of DHHC3 (d) or DHHC7 (e); 3 replicates. 5
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7 Supplementary Figure 5 (linked to Figure 5). Rab27a and DHHC3 but not VEGFR1 colocalize in endothelial cells. (a-b) (a) Immunofluorescence of DHHC3 and Rab27a in HUVEC with indicated treatments 24h post-knockdown, scale bar: 10µm. (b) Mander s Colocalization Coeffcient quantification of overlap. Shown are means +95% CI. (#cells: ntrna, n=31; sirab27a, n=20); 2 replicates. (c) Immunoblot of DHHC3 in HUVEC with indicated treatments. (d) Mander s Colocalization Coefficient quantification of DHHC3 and TGN46 (Golgi) overlap in HUVEC with indicated treatments. Shown are means + 95% CI. (#cells: ntrna, n=19; sirab11a, n=27); 2 replicates. (e) Subcellular fractionation and immunoblot of HUVEC from 3 different lots. Pyruvate kinase1/2 (PKM1/2), soluble marker; vascular-endothelial cadherin (VE-CAD), membrane and cytoskeletal marker; cis-golgi Marker 130 (GM130), membrane marker; pan actin, soluble (G-actin) and cytoskeletal (F-actin) marker. 2 replicates. (f) Immunofluorescence of VEGFR1 and Rab27a overlap quantified with Mander s Correlation Coefficient, +/- SD, scale bar 15µm. Statistics: Student s t-test with post-hoc Tukey s range test. ***, p 0.001; ns, not significant. 7
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9 Supplementary Figure 6 (linked to Figure 5). Rab27a regulates steady-state mvegfr1 levels. (a-c) (a-b) Immunofluorescence of DHHC3 and VEGFR1 in HUVEC with indicated treatments, scale bars: 10µm (a) and 5µm (b). (c) Mander s Colocalization Coefficient quantification of fluorescence. (#cells: ntrna, n=33, sirab27a, n=40); 3 replicates. (d) Immunoblot of HUVEC with indicated treatments. Values are relative mvegr1 levels. (e-f) (e) VEGFR2 immunofluorescence and (f) quantification of fluorescence via integrated density of d4 HUVEC angiogenic sprouts with indicated treatments. Scale bar: 20µm, (#sprouts: ntrna, n=10; sirab27a, n=12; sirab3a, n=7); 3 replicates. (g-h) (g) VEGFR1 immunofluorescence of HUVEC 48h post KD and (h) quantification of fluorescence via integrated density, scale bar 15µm. (# cells: DMSO/ntRNA, n=14; MG132/ntRNA, n=18; DMSO/siRab27a, n=14; MG132/siRab27a, n=16); 2 replicates. (i) Immunoblot of HUVEC with indicated treatments. Values are relative mvegr1 levels; 2 replicates. Statistics: Shown are means +95% CI. One-way ANOVA and pairwise comparison with post-hoc Tukey s range test. *, p 0.05; **, p 0.01; ***, p 0.001; ns, not significant. 9
10 Supplementary Figure 7 (linked to Figures 6 and 7). Rab27a negatively regulates angiogenesis and increases HUVEC proliferation. (a-d) Quantification of sprouting from HUVEC with indicated treatments on indicated days. (n=10 beads/condition/time point); 3 replicates. (e) Immunoblot of HUVEC with indicated treatments and times; 2 replicates. (f) Immunoblot of HUVEC with indicated treatments and times; 3 replicates. (g) Quantification of phish3 (mitotic) nuclei of HUVEC 48h post-knockdown (#nuclei: ntrna, n=682; sirab27a, n=549); 2 replicates. Statistics: (a-d) Shown are means +95% CI. One-way ANOVA and pairwise comparison with post-hoc Tukey s range test. (g) Shown are means +SEM, Student s t- test. **, p 0.01; ns, not significant. 10
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12 Supplementary Figure 8 (linked to Figure 8). Loss of Rab27a protein in ash/ash mice leads to hyperactive retinal blood vessels. (a) Immunofluorescence of P6.5 mouse retinas for vessels (isolectin) and Rab27a of indicated genotypes; 3 replicates, scale bar: 15µm. (b) Immunoblot of Rab27a from P6.0 retinal lysates of indicated genotypes; 4 replicates. (c-e) (c) Immunofluorescence of P6.5 retinal vessels with indicated genotypes. Filopodia were quantified at the vascular front (d) and within the plexus (e). Red boxes, higher magnification to right. Arrows, filopodia. (#retinas: 2 independent litters: +/+, n=7; ash/ash, n=6). (f-h) (f) Immunofluorescence of P8.5 retinal vessels with indicated genotypes. Filopodia were quantified at the vascular front (g) and within the plexus (h). Red boxes, higher magnification to right. Arrows, filopodia. (#retinas: 2 independent litters: +/+, n=6; ash/ash, n=4). Statistics: Shown are means +95% CI. Students t-test. *, p 0.05; **, p 0.01; ***, p 0.001; ns, not significant. Scale bars, 25µm. 12
13 Supplementary Figure 9 (linked to Figure 8). Retinal vessels from ash/ash mice have longer plexus filopodia and model for regulation of mvegfr1 stability. (a-c) (a) Immunofluorescence of filopodia in P8.5 retinal vessels of indicated genotypes (left, low magnification plexus, scale bar, 25µm left column; middle, high magnification plexus; right, high magnification tip cells, scale bars 10µm, middle and right columns). (b-c) Quantification of filopodia length from vascular plexus (b) and vascular front (c). (#filopodia: plexus: +/+, n=396; ash/ash, n=827; front: +/+, n=175; ash/ash, n=220); 2 independent replicates. Statistics: Shown are means +95% CI. One-way ANOVA and pairwise comparison with post-hoc Tukey s range test. **, p 0.01; ns, not significant. 13
14 Supplementary Figure 10 (linked to all Figures). Uncropped Western blots. 14
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28 Supplementary Table 1. List of antibodies used for this study. Antibody Company IB Titer IF Titer PRIMARY α-tubulin Sigma 1: β-actin CST 1: DHHC3 Abcam 1: DHHC3 SCBT 1:2000 1:750 Erk1/2 (4695) CST 1: p-erk1/2 (4370) CST 1:1000 1:500 GM130 (ab-52649) Abcam 1: HA CST 1: P-Histone H3 (9701) CST 1:1000 1:1000 LAMP1 Abcam - 1:2000 LYPLA1 (APT1) Abcam 1: PKM1/2 (3186S) CST 1: P-VEGFR2 Y1175 (2478) CST 1:500 - PECAM Abcam - 1:500 Rab3a (MN1250) Thermo Scientific 1:750 - Rab4/14 (sc ) SCBT 1: Rab5 SCBT 1:500 - Rab11 (sc ) SCBT 1:2000 1:750 Rab27a (sc-81914) SCBT 1:1000 1:500 Streptavidin HRP (N100) Life Technologies 1: TGN46 Abcam - 1:2000 VE-Cadherin (sc-28644) SCBT 1: VEGFR1 (ab-32152) Abcam 1:2000 1:500* Alexa-fluor 488-VEGFR1 (ab ) Abcam - 1:50 VEGFR2 (2479) CST 1:5000 1:1000 ZDHHC21 (ab ) Abcam 1:750 - SECONDARY Anti-mouse IgG, HRP-linked (secondary) CST 1: Anti-rabbit IgG, HRP-linked (secondary) CST 1: Goat anti-mouse 488 Life Technologies - 1:1000 Goat anti-mouse 594 Life Technologies - 1:1000 Goat anti-rabbit 488 Life Technologies - 1:1000 Goat anti-rabbit 594 Life Technologies - 1:1000 *A titer of 1:200 was used for Flt-1 staining in the retina Abbreviations: CST= Cell Signaling Technologies SCBT= Santa Cruz Biotechnology 28
29 Supplementary Table 2. List of Inhibitors used for this study. Inhibitors Concentration Company Target 2-Bromohexadecanoic Acid (2-BH) 20 µm in EtOH Sigma Palmitoyl-acetyl transferases Chloroquine (CHQ) 10 µg/ml in H2O Sigma Lysosome Cycloheximide (CHX) 500 µg/ml in H2O Sigma Ribosome Dynasore 1µM in DMSO Santa Cruz Dynamin MG132 5µM in DMSO Calbiochem Proteasome Palmostatin B (Pal-B) 10 µm in DMSO Calbiochem Acyl-protein thioesterases 29
30 Supplementary Table 3. List of small interfering RNA knockdown probes used for this study. sirnas Catalogue # Company Pmol/10 6 Target species cells APT1 S20410 ThermoFisher 100 Human DHHC3 S27899 ThermoFisher 125 Human DHHC3 #2 S27900 ThermoFisher 125 Human DHHC7 S31109 ThermoFisher 125 Human Flt-1 SI Qiagen 75 Human Non-targeting SI Qiagen varies none Rab4a S11677 ThermoFisher 100 Human Rab11a S16702 ThermoFisher 100 Human Rab27a#1* SI Qiagen 100 Human Rab27a#2** S11695 ThermoFisher - Human/mouse Rab27a # 3 S11494 ThermoFisher 100 Human/mouse *Used for all human cell work in 2D and 3D. **Used for mouse aortic ring assay (200pmol) and to validate effects of Rab27a-1 probe. Used at 100pmol/10 6 cell for Rab27a knockdown validation in Supplementary Figure 6d. 30
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p = formed with HCI-001 p = Relative # of blood vessels that formed with HCI-002 Control Bevacizumab + 17AAG Bevacizumab 17AAG
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a. b. c. d. e. f. g. h. i. j. k. l. m. n. o. p.
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MANUSCRIPT TITLE: Protein kinase C δ signaling is required for dietary prebiotic-induced strengthening of intestinal epithelial barrier function Authors: Richard Y. Wu 1,2, Majd Abdullah 1, Pekka Määttänen
Nature Medicine: doi: /nm.4322
1 2 3 4 5 6 7 8 9 10 11 Supplementary Figure 1. Predicted RNA structure of 3 UTR and sequence alignment of deleted nucleotides. (a) Predicted RNA secondary structure of ZIKV 3 UTR. The stem-loop structure
Supplementary Figure S1
Supplementary Figure S1 Supplementary Figure S1. PARP localization patterns using GFP-PARP and PARP-specific antibody libraries GFP-PARP localization in non-fixed (A) and formaldehyde fixed (B) GFP-PARPx
Supplemental Information. Inhibition of the Proteasome b2 Site Sensitizes. Triple-Negative Breast Cancer Cells
Cell Chemical Biology, Volume 24 Supplemental Information Inhibition of the Proteasome b2 Site Sensitizes Triple-Negative Breast Cancer Cells to b5 Inhibitors and Suppresses Nrf1 Activation Emily S. Weyburne,
p47 negatively regulates IKK activation by inducing the lysosomal degradation of polyubiquitinated NEMO
Supplementary Information p47 negatively regulates IKK activation by inducing the lysosomal degradation of polyubiquitinated NEMO Yuri Shibata, Masaaki Oyama, Hiroko Kozuka-Hata, Xiao Han, Yuetsu Tanaka,
Exosomes function in antigen presentation during an in vivo Mycobacterium tuberculosis infection
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Supplementary Figure 1: Neuregulin 1 increases the growth of mammary organoids compared to EGF. (a) Mammary epithelial cells were freshly isolated,
1 2 3 4 5 6 7 8 9 10 Supplementary Figure 1: Neuregulin 1 increases the growth of mammary organoids compared to EGF. (a) Mammary epithelial cells were freshly isolated, embedded in matrigel and exposed
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk -/- mice were stained for expression of CD4 and CD8.
Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsrna-induced retinal degeneration
Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsrna-induced retinal degeneration The Harvard community has made this article openly available. Please
Supplementary Figure 1 Induction of cellular senescence and isolation of exosome. a to c, Pre-senescent primary normal human diploid fibroblasts
Supplementary Figure 1 Induction of cellular senescence and isolation of exosome. a to c, Pre-senescent primary normal human diploid fibroblasts (TIG-3 cells) were rendered senescent by either serial passage
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/8/389/ra79/dc1 Supplementary Materials for HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P 1 to limit vascular
Supplementary Figure 1. Expression of CUGBP1 in non-parenchymal liver cells treated with TGF-β
Supplementary Figures Supplementary Figure 1. Expression of CUGBP1 in non-parenchymal liver cells treated with TGF-β and LPS. Non-parenchymal liver cells were isolated and treated with or without TGF-β
Supplemental Information. Autophagy in Oncogenic K-Ras. Promotes Basal Extrusion. of Epithelial Cells by Degrading S1P. Current Biology, Volume 24
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Figure S1: Effects on haptotaxis are independent of effects on cell velocity A)
Supplemental Figures Figure S1: Effects on haptotaxis are independent of effects on cell velocity A) Velocity of MV D7 fibroblasts expressing different GFP-tagged Ena/VASP family proteins in the haptotaxis
Supplementary Figure 1. The CagA-dependent wound healing or transwell migration of gastric cancer cell. AGS cells transfected with vector control or
Supplementary Figure 1. The CagA-dependent wound healing or transwell migration of gastric cancer cell. AGS cells transfected with vector control or 3xflag-CagA expression vector were wounded using a pipette
Cesarini et al., http ://www.jcb.org /cgi /content /full /jcb /DC1
Supplemental material JCB Cesarini et al., http ://www.jcb.org /cgi /content /full /jcb.201504035 /DC1 THE JOU RNAL OF CELL BIO LOGY Figure S1. Lamin A/C depletion generates two distinct phenotypes in
AAV-TBGp-Cre treatment resulted in hepatocyte-specific GH receptor gene recombination
AAV-TBGp-Cre treatment resulted in hepatocyte-specific GH receptor gene recombination Supplementary Figure 1. Generation of the adult-onset, liver-specific GH receptor knock-down (alivghrkd, Kd) mouse
The subcortical maternal complex controls symmetric division of mouse zygotes by
The subcortical maternal complex controls symmetric division of mouse zygotes by regulating F-actin dynamics Xing-Jiang Yu 1,2, Zhaohong Yi 1, Zheng Gao 1,2, Dan-dan Qin 1,2, Yanhua Zhai 1, Xue Chen 1,
Protein tyrosine phosphatase 1B targets PITX1/p120RasGAP. thus showing therapeutic potential in colorectal carcinoma
Protein tyrosine phosphatase 1B targets PITX1/p120RasGAP thus showing therapeutic potential in colorectal carcinoma Hao-Wei Teng, Man-Hsin Hung, Li-Ju Chen, Mao-Ju Chang, Feng-Shu Hsieh, Ming-Hsien Tsai,
Supplementary Figure 1 Cell line TRIB2 status. Supplementary Figure 2 TRIB2 status has no impact on the cell cycle after PI3K inhibition. a. b.
Supplementary Figure 1 Cell line TRIB2 status. TRIB2 protein expression to determine endogenous expression and to determine the effectiveness of each of our TRIB2 knockdown constructs. Supplementary Figure
Supplementary Figure 1 hlrrk2 promotes CAP dependent protein translation.
` Supplementary Figure 1 hlrrk2 promotes CAP dependent protein translation. (a) Overexpression of hlrrk2 in HeLa cells enhances total protein synthesis in [35S] methionine/cysteine incorporation assays.
SUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb3021 Supplementary figure 1 Characterisation of TIMPless fibroblasts. a) Relative gene expression of TIMPs1-4 by real time quantitative PCR (RT-qPCR) in WT or ΔTimp fibroblasts (mean ±
Supplementary information. The Light Intermediate Chain 2 Subpopulation of Dynein Regulates Mitotic. Spindle Orientation
Supplementary information The Light Intermediate Chain 2 Subpopulation of Dynein Regulates Mitotic Spindle Orientation Running title: Dynein LICs distribute mitotic functions. Sagar Mahale a, d, *, Megha
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/8/366/ra25/dc1 Supplementary Materials for Viral entry route determines how human plasmacytoid dendritic cells produce type I interferons Daniela Bruni, Maxime
Erzsebet Kokovay, Susan Goderie, Yue Wang, Steve Lotz, Gang Lin, Yu Sun, Badrinath Roysam, Qin Shen,
Cell Stem Cell, Volume 7 Supplemental Information Adult SVZ Lineage Cells Home to and Leave the Vascular Niche via Differential Responses to SDF1/CXCR4 Signaling Erzsebet Kokovay, Susan Goderie, Yue Wang,
T H E J O U R N A L O F C E L L B I O L O G Y
T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Krenn et al., http://www.jcb.org/cgi/content/full/jcb.201110013/dc1 Figure S1. Levels of expressed proteins and demonstration that C-terminal
Control GST GST-RAP. α2-mg. 170 kda. b-actin. 42 kda LRP-1
% of max Supplementary Figure 1 Control GST GST-RP 17 kda α2-mg 42 kda b-actin Gate: CD11c+ (DCs) Gate: F4/8+ (Mfs) IgG Cd11cCre + Lrp1 fl/fl LRP-1 Supplementary figure 1. () MDCs were pretreated with
Supplementary Figure (OH) 22 nanoparticles did not affect cell viability and apoposis. MDA-MB-231, MCF-7, MCF-10A and BT549 cells were
Supplementary Figure 1. Gd@C 82 (OH) 22 nanoparticles did not affect cell viability and apoposis. MDA-MB-231, MCF-7, MCF-10A and BT549 cells were treated with PBS, Gd@C 82 (OH) 22, C 60 (OH) 22 or GdCl
Santulli G. et al. A microrna-based strategy to suppress restenosis while preserving endothelial function
ONLINE DATA SUPPLEMENTS Santulli G. et al. A microrna-based strategy to suppress restenosis while preserving endothelial function Supplementary Figures Figure S1 Effect of Ad-p27-126TS on the expression
SUPPLEMENTARY INFORMATION. Supplementary Figures S1-S9. Supplementary Methods
SUPPLEMENTARY INFORMATION SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane Jian Huang 1,2#, Jie Yan 1,2#, Jian Zhang 3#, Shiguo Zhu 1, Yanli Wang
supplementary information
DOI: 10.1038/ncb1875 Figure S1 (a) The 79 surgical specimens from NSCLC patients were analysed by immunohistochemistry with an anti-p53 antibody and control serum (data not shown). The normal bronchi served
(a-r) Whole mount X-gal staining on a developmental time-course of hearts from
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Supplementary Figure 1 (a-r) Whole mount X-gal staining on a developmental time-course of hearts from Sema3d +/- ;Ephb4 LacZ/+ and Sema3d -/- ;Ephb4 LacZ/+ embryos.
SUPPLEMENTARY DATA. Supplementary Table 2. Antibodies used for Immunofluoresence. Supplementary Table 3. Real-time PCR primer sequences.
Supplementary Table 2. Antibodies used for Immunofluoresence. Antibody Dilution Source Goat anti-pdx1 1:100 R&D Systems Rabbit anti-hnf6 1:100 Santa Cruz Biotechnology Mouse anti-nkx6.1 1:200 Developmental
Supplementary Figure 1
Supplementary Figure 1 The average sigmoid parametric curves of capillary dilation time courses and average time to 50% peak capillary diameter dilation computed from individual capillary responses averaged
Supplementary Figure S1. Effect of Glucose on Energy Balance in WT and KHK A/C KO
Supplementary Figure S1. Effect of Glucose on Energy Balance in WT and KHK A/C KO Mice. WT mice and KHK-A/C KO mice were provided drinking water containing 10% glucose or tap water with normal chow ad
Supplementary Figure 1 IMQ-Induced Mouse Model of Psoriasis. IMQ cream was
Supplementary Figure 1 IMQ-Induced Mouse Model of Psoriasis. IMQ cream was painted on the shaved back skin of CBL/J and BALB/c mice for consecutive days. (a, b) Phenotypic presentation of mouse back skin
Nature Immunology: doi: /ni eee Supplementary Figure 1
eee Supplementary Figure 1 Hyphae induce NET release, but yeast do not. (a) NET release by human peripheral neutrophils stimulated with a hgc1 yeast-locked C. albicans mutant (yeast) or pre-formed WT C.