Journal: International Journal of Molecular Sciences

Article Title: Derivation of Porcine Extra-Embryonic Endoderm Cell Lines Reveals Distinct Signaling Pathway and Multipotency States

doi: 10.3390/ijms222312918

Figure Lengend Snippet: Signaling dependence analysis of pXEN cells. ( A ) The morphology and JC1 staining of pXEN cells cultured in LCDM, LCDM+10μm SD1008, LCDM+10μm SU5402 and LCDM+10μm SB431542. ( B ) Quantitative RT-PCR analysis of FGF, TGFβ and LIF signaling related genes in pXEN cells, porcine nESCs and porcine embryo fibroblasts (PEF). Relative expression reflected as a fold difference in pXEN cells and porcine nESCs compared to PEF, PEF = 1. Data are depicted as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus Control ( t -test). ( C ) The morphology of pXEN cells cultured in LCDM and LCDM supplemented with 1, 5 and 10μM concentration of PD0325901. ( D ) Western blotting analysis of the phosphorylation status of ERK, STAT3 andSMAD2/3, and the expression of ERK, STAT3 andSMAD2/3 in pXEN cells and porcine nESCs. ( E ) The quantification of proteins after Western blotting. Scale bar, 100μm.

Article Snippet: The cells were cultured in medium supplemented with or without the JAK inhibitor SD1008 (Tocris), the FGF receptor (FGFR) inhibitor SU5402 (Tocris), Mek inhibitor PD0325901 (Sigma) and the TGFβ receptor inhibitor SB431542 (Tocris) for LIF, FGF and TGFβ signal pathway identification, respectively.

Techniques: Staining, Cell Culture, Quantitative RT-PCR, Expressing, Control, Concentration Assay, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: Differential effects of amnion and chorion membrane extracts on osteoblast-like cells due to the different growth factor composition of the extracts

doi: 10.1371/journal.pone.0182716

Figure Lengend Snippet: (A) MG-63 cells were cultured in OIM supplemented with DMSO (0.1% v/v) and were pretreated with various concentrations of SU5402 (0.1–1 μM) or SB505124 (0.5–1 μM) for 1 h, followed by treatment with 400 μg/mL CME. After 9 days, mineralization was determined by performing Alizarin red S staining. Alizarin red S stain was extracted using 10% cetylpyridinium chloride, and absorbance was measured at 570 nm. (B) For the calcium assay, the cells were pretreated with DMSO (0.1% v/v) and various concentrations of SU5402 (0.1–1 μM) or SB505124 (0.5–1 μM) alone or their combination for 1 h, followed by treatment with 200 μμg/mL CME. Calcium assay was performed after 9 days. (C) Expression of genes encoding ALP and IBSP was determined on day 4 by performing quantitative real-time RT-PCR under the experimental condition described in (A): SU5402: FGF specific inhibitor, SB505124: TGFβ-1 specific inhibitor, SU+SB: SU5402+SB505124. Data are presented as the mean ± SD of multiple repeated experiments; * p < 0.05, ** p < 0.01, and # p < 0.001 versus only CME, indicate statistical significance.

Article Snippet: Recombinant human bFGF, TGFβ-1, EGF, and BMP2 were purchased from Peprotech (Rocky Hill, NJ, USA), and inhibitors of FGF (SU5402), TGFβ-1 (SB505124), and EGF (PD153035) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Cell Culture, Staining, Calcium Assay, Expressing, Quantitative RT-PCR

Journal: Cancers

Article Title: Long-Pentraxin 3 Affects Primary Cilium in Zebrafish Embryo and Cancer Cells via the FGF System

doi: 10.3390/cancers12071756

Figure Lengend Snippet: Inhibition of FGF signaling increases the length of primary cilium in TRAMP-C2 cells. ( A – E ) Cilia were visualized in serum-starved cells by acetylated α-tubulin immunostaining and their length was measured using the ImageJ software. Black dots represent individual cilia; red bars show the mean values. Data were obtained from three independent experiments, *** p < 0.001, Student’s t -test; ( A ) primary cilium length in TRAMP-C2 cells overexpressing the C -terminal or the N -terminal fragment of human PTX3; ( B ) primary cilium length in mock_TRAMP-C2 and N -term- hPTX3 -TRAMP-C2 cells treated for 48 h with 30 ng/mL FGF2; ( C ) primary cilium length in TRAMP-C2 cells treated for 48 h with recombinant PTX3 protein (66 nM) or with anti-FGFR1 single-chain antibody fragment scFv-RR-C2 (300 nM); ( D ) primary cilium length in TRAMP-C2 cells treated for 48 h with the tyrosine kinase FGFR inhibitors PD173074 (100 nM), SU5402 (100 nM), or BGJ398 (100 nM); ( E ) primary cilium length in TRAMP-C2 treated for 48 h with the MAPK inhibitors PD98059 (10 µM) or U0126 (1.0 µM) or with the PI3K inhibitors LY294002 (10 µM) or perifosine (1.0 µM).

Article Snippet: PD173074, SU5402, PD98059, and LY294002 were from Sigma-Aldrich (St. Louis, MO, USA), BGJ393 was from Selleckchem (Houston, TX, USA), UO126 was from MedChem Express, (Monmouth Junction, NJ, USA) and perifosine was from Aeterna Zentaris (Frankfurt, Germany).

Techniques: Inhibition, Immunostaining, Software, Recombinant

Journal: Cancers

Article Title: Long-Pentraxin 3 Affects Primary Cilium in Zebrafish Embryo and Cancer Cells via the FGF System

doi: 10.3390/cancers12071756

Figure Lengend Snippet: Inhibition of the FGF/FGFR system affects cilium-related signaling. ( A ) Serum-starved mock and hPTX3 -TRAMP-C2 cell protein extracts were probed with an anti-trichoplein (TCHP) antibody. Uniform loading of the gel was assessed by probing the membrane with an anti-β actin antibody; ( B ) densitometric analysis of trichoplein levels normalized to β actin; ( C ) serum-starved mock-TRAMP-C2 cells were treated with the FGFR inhibitors BGJ398 (100 nM) or SU5402 (100 nM) for 48 h. After lysis, the extracts were probed with an anti-trichoplein antibody; ( D ) densitometric analysis of trichoplein levels normalized to β-actin; ( E ) Gli1 expression in serum-starved mock and hPTX3 _TRAMP-C2 cells; ( F ) serum-starved hPTX3 -TRAMP-C2 cells were incubated with recombinant FGF2 (30 ng/mL) for 1, 6, or 12 h. Then, Gli1 expression was evaluated by qRT-PCR and normalized to Gaphd mRNA levels. All data are the mean ± S.E.M. of 3–4 independent experiment, * p < 0.05, ** p < 0.01, Student’s t -test.

Article Snippet: PD173074, SU5402, PD98059, and LY294002 were from Sigma-Aldrich (St. Louis, MO, USA), BGJ393 was from Selleckchem (Houston, TX, USA), UO126 was from MedChem Express, (Monmouth Junction, NJ, USA) and perifosine was from Aeterna Zentaris (Frankfurt, Germany).

Techniques: Inhibition, Membrane, Lysis, Expressing, Incubation, Recombinant, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Reproducible production and image-based quality evaluation of retinal pigment epithelium sheets from human induced pluripotent stem cells

doi: 10.1038/s41598-020-70979-y

Figure Lengend Snippet: Small-molecule-based differentiation of RPE from hiPSC. ( A ) Timetable for stepwise treatment for RPE differentiation from hiPSC. Y27632 (10 µM), LDN (LDN193189, 100 nM), A83 (A83-01, 500 nM), IWR (IWR-1- endo , 1 µM), CHIR99021 (3 µM), and SU5402 (2 µM) were added to the culture medium for indicated periods. ( B ) Generation of RPE progenitors from hiPSC. Differentiated cells on day 12 were processed for immunostaining for PAX6 and MITF. ( C ) Generation of polygonal cells from hiPSC. Differentiated cells on day 32 were processed for phalloidin staining. ( D ) Generation of RPE from hiPSC. Macroscopic photographic images (left) and phase-contrast images (right) of differentiated cells on day 35. Note that most cells are pigmented and polygonal. ( E ) Co-induction of non-RPE cells from hiPSC. Dotted lines mark the non-pigmented cells (left). Arrow heads indicate neural process-like structure of the differentiated cells other than RPE on day 35 (right). ( F ) Minor populations of neural retinal progenitors. Representative immunostaining for MITF (a marker for RPE progenitors) and CHX10 (a marker for neural retinal progenitors) on day 35. Dotted lines mark the CHX10-positive cells. Scale bars: 20 μm ( B , C ), 50 μm ( D , F ), and 100 μm ( E ).

Article Snippet: For RPE induction, cells were treated with 100 nM LDN193189 (Sigma, St. Louis, MO), 500 nM A-83-01 (Wako), 1 μM IWR-1- endo (Wako), and 10 μM Y-27632 were added to IMDM/Ham’s F12 (1:1, both from Sigma) supplemented with 10% KnockOut Serum Replacement (Thermo Fisher Scientific), 0.5 mM Monothioglycerol Solution (Wako), 1% Chemically Defined Lipid Concentrate (Wako), and 2 mM l -glutamine (Wako) for the initial 6 days, and then with 3 μM CHIR99021 (Wako), 2 μM SU5402 (Wako), and 10 μM Y-27632 in IMDM/F12 for another 12 days.

Techniques: Immunostaining, Staining, Marker

Journal: Scientific Reports

Article Title: Reproducible production and image-based quality evaluation of retinal pigment epithelium sheets from human induced pluripotent stem cells

doi: 10.1038/s41598-020-70979-y

Figure Lengend Snippet: Efficient generation of pure RPE by RPE6iN method. ( A ) Timetable for the new differentiation method RPE6iN. Dissociated hiPSC were cultured on iMatrix 511-coated dishes in the presence of Y27632 (10 µM), LDN (LDN193189, 100 nM), A83 (A83-01, 500 nM), IWR (IWR-1-endo, 1 µM), CHIR99021 (3 µM), and SU5402 (2 µM) for indicated periods. NIC (Nicotinamide, 10 mM) was added into the culture medium from day 12 to day 24. ( B ) Promotion of RPE differentiation by NIC treatment. The percentage of MITF-positive cells were determined by immunostaining for MITF on days 12, 18, 24, and 35. ** P < 0.01, *** P < 0.001, compared with control. ( C ) Phase-contrast images of induced cells with or without NIC treatment on day 35. Dotted lines mark the non-pigmented cells. Note that NIC treatment decreased the non-pigmented area. ( D ) Representative photomicrographs showing RPE6iN-treated hiPSC. HiPSC were differentiated in the presence or absence of NIC, fixed on day 35, and then subjected to immunostaining for MITF and CHX10. ( E ) The decrease in the percentage of CHX10-positive cells by NIC treatment on day 35. *** P < 0.001, compared with control. ( F ) Promotion of RPE maturation by NIC treatment. Gene expression of tyrosinase was quantified by RT-qPCR on day 24. *** P < 0.001, compared with control. ( G ) Generation of polygonal cells from RPE6iN-treated hiPSC. Differentiated cells were processed for phalloidin staining on day 31. ( H ) Formation of tight junctions in RPE6iN-treated hiPSC. RPE6iN-indued cells were immunostained for the tight junction marker ZO-1 on day 35. ( I ) Macroscopic photographic images and phase-contrast images of RPE6iN-indued pigmented cells on day 35 in culture. RPE6iN-treated cells were cultured in RPE maintenance medium. Scale bars: 20 μm ( G , H ), 50 μm ( D , I ), and 100 μm ( C ).

Article Snippet: For RPE induction, cells were treated with 100 nM LDN193189 (Sigma, St. Louis, MO), 500 nM A-83-01 (Wako), 1 μM IWR-1- endo (Wako), and 10 μM Y-27632 were added to IMDM/Ham’s F12 (1:1, both from Sigma) supplemented with 10% KnockOut Serum Replacement (Thermo Fisher Scientific), 0.5 mM Monothioglycerol Solution (Wako), 1% Chemically Defined Lipid Concentrate (Wako), and 2 mM l -glutamine (Wako) for the initial 6 days, and then with 3 μM CHIR99021 (Wako), 2 μM SU5402 (Wako), and 10 μM Y-27632 in IMDM/F12 for another 12 days.

Techniques: Cell Culture, Immunostaining, Expressing, Quantitative RT-PCR, Staining, Marker

Journal: eLife

Article Title: Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

doi: 10.7554/eLife.62196

Figure Lengend Snippet: ( A ) Schematic representation of the fli1aep:ERK-KTR-Clover (EKC) construct, and endothelial cells (ECs) with nuclear enriched EKC (bottom left, inactive Erk-signalling) and nuclear depleted EKC localisation (bottom right, active Erk-signalling). ( B–E ) Lateral confocal images of the EC-EKC ( B,D ) and Tg(fli1a:EGFP) ( C,E ) embryos/larvae at 24 hours post-fertilisation (hpf) ( B,C ) and 5 days post-fertilisation (dpf) ( D,E ). Blood vessel development is not altered in EC-EKC embryos/larvae. ( F–H’’ ) Lateral spinning disc confocal images of ISV ECs in 28 hpf EC-EKC embryos treated for 1 hr with either 0.5% dimethyl sulfoxide (DMSO) ( F–F’’ ), with active EC Erk-signalling, or 15 μM SL327 ( G–G’’ ) or 4 μM SU5416 ( H–H’’ ), all of which with inactive EC Erk-signalling. Images ( F-H ) show fli1aep:EKC expression, while images ( F’-H’) show both fli1aep:EKC and fli1a:H2B-mCherry expression. Images ( F’’-H’’ ) show the nuclear fli1aep:EKC expression with intensity difference represented in 16 colour LUT (Fiji). The fli1a:H2B-mCherry signal was used to mask the nucleus. ( I ) Quantification of nucleus/cytoplasm EKC intensity in ISV tip ECs of 28 hpf embryos treated with either 0.5% DMSO (0.881, 93 ISV tip ECs, n = 20 embryos), 15 μM SL327 (1.419, 114 ISV tip ECs, n = 27 embryos), or 4 μM SU5416 (1.591, 118 ISV tip ECs, n = 27 embryos). ISV: intersegmental vessel. Statistical test: Kruskal-Wallis test was conducted for graph ( I ). Error bars represent standard deviation. Scale bars: 200 μm for images ( B ) and ( D ), 25 μm for image ( F ). Figure 1—source data 1. Nuclear/cytoplasm EKC measurements in leading ISV ECs of DMSO-, SL327-, and SU5416-treated 28 hpf embryos.

Article Snippet: Chemical compound, drug , SU5416 , Merck, Darmstadt, Germany , S8442 , Diluted in DMSO.

Techniques: Construct, Expressing, Standard Deviation

Journal: eLife

Article Title: Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

doi: 10.7554/eLife.62196

Figure Lengend Snippet: ( A,B ) Lateral confocal images of a 4 days post-fertilisation (dpf) Tg(kdrl:EGFP) larva following vessel wounding (post-ablation). Image ( A ) shows the kdrl:EGFP expression and image ( B ) shows the trans-light image of image ( A ). Ablated ISV, adjacent ISVs, and the wounded site are indicated with white arrows. ( C ) Schematic representation of imaging schedule for larvae in images ( D-G ) and – . ( D–G’ ) Still images from ( D–E’ ) and ( F–G’ ) showing ISV endothelial cells (ECs) before (pre-ablation) and after vessel wounding. Ablated and adjacent ISV ECs rapidly activate Erk-signalling. ( D-G ) fli1aep:EKC expression; ( D’-G’ ) nuclear intensity. ( H,I ) Quantification of post-/pre-ablation nuclear EKC intensity of ECs in non-ablated control ISVs (black, 24 ECs, n = 8 larvae), ablated ISVs (red, 27 ECs, n = 9 larvae), and adjacent ISVs (light blue, 27 ECs, n = 9 larvae). ( H ) shows quantification of individual ECs and ( I ) shows the average of all biological replicates. Green dotted line indicates 15 min post-ablation (mpa). ( J ) Quantification of post-/pre-ablation nuclear EKC intensity 15 mpa in ECs of non-ablated control ISVs (103 ECs, n = 34 larvae), ablated venous ISVs (75 ECs, n = 25 larvae), and ablated arterial ISVs (57 ECs, n = 19 larvae). Both venous and arterial ISV ECs activate Erk-signalling. ( K ) Quantification of post-/pre-ablation nuclear EKC intensity 15 mpa in ECs of non-ablated uninjected control ISVs (45 ECs, n = 15 larvae), non-ablated spi1 / csf3r morphant ISVs (42 ECs, n = 14 larvae), uninjected control ISVs (45 ablated/adjacent ISV ECs, n = 15 larvae), and spi1 / csf3r morphant ISVs (56 ablated ISV ECs and 57 adjacent ISV ECs, n = 19 larvae). Macrophages are not required to rapidly activate Erk-signalling in ablated or adjacent ISV ECs. ( L ) Quantification of post-/pre-ablation nuclear EKC intensity 15 mpa in ECs of 0.5% dimethyl sulfoxide (DMSO)-treated non-ablated control ISVs (33 ECs, n = 11 larvae) and ISVs of larvae treated with either 0.5% DMSO (42 ablated/adjacent ISV ECs, n = 14 larvae), 15 μM SL327 (39 ablated/adjacent ISV ECs, n = 13 larvae), 4 μM SU5416 (36 ablated/adjacent ISV ECs, n = 12 larvae), 10 μM SU5416 (42 ablated/adjacent ISV ECs, n = 14 larvae), or 500 nM AV951 (42 ablated/adjacent ISV ECs, n = 14 larvae). Vegfr-signalling is not required to rapidly activate Erk-signalling in ablated or adjacent ISV ECs. ISV: intersegmental vessel. Statistics: permutation test was conducted for graph ( H ). Kruskal-Wallis test was conducted for graphs ( J-L ). Error bars represent standard deviation. White dotted lines/circle shows the wounded sites of each larva. Scale bar: 100 μm for image ( A ), 20 μm for image ( D ). Figure 3—source data 1. Post-/pre-ablation nuclear EKC measurements in control, ablated, and adjacent ISV ECs.

Article Snippet: Chemical compound, drug , SU5416 , Merck, Darmstadt, Germany , S8442 , Diluted in DMSO.

Techniques: Expressing, Imaging, Control, Standard Deviation

Journal: eLife

Article Title: Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

doi: 10.7554/eLife.62196

Figure Lengend Snippet: ( A–B’’ ) Lateral spinning disc confocal images of ISV endothelial cells (ECs) in 28 hours post-fertilisation (hpf) EC-EKC embryos treated for an hour with either 0.5% dimethyl sulfoxide (DMSO) ( A–A’’ ), with active EC Erk-signalling, or 500 nM AV951 ( B–B’’ ), with inactive EC Erk-signalling. Images ( A ) and ( B ) show the fli1aep:EKC expression, while images ( A’ ) and ( B’ ) show both the fli1aep:EKC and the fli1a:H2B-mCherry expression. Images ( A’’ ) and ( B’’ ) show the nuclear fli1aep:EKC intensity. ( C ) Quantification of nucleus/cytoplasm EKC intensity in ISV tip ECs of 28 hpf embryos treated with either 0.5% DMSO (0.849, 65 ECs, n = 14 embryos) or 500 nM AV951 (1.423, 53 ECs, n = 12 embryos). ( D–Z’ ) Vegfr-signalling inhibitors do not block rapid Erk-signalling activation in ablated and adjacent ISVs following vessel wounding. Lateral spinning disc confocal images of ISV ECs in 4 days post-fertilisation (dpf) EC-EKC larvae treated with either 0.5% DMSO ( D–I’ ), 15 μM SL327 ( J–M’ ), 4 μM SU5416 ( O–R’ ), 10 μM SU5416 ( S–V’ ), or 500 nM AV951 ( W–Z’ ). Images ( D-E’ ) show non-ablated control ISV ECs. Images ( F-G’ ), ( J-K’ ), ( O-P’ ), ( S-T’ ), and ( W-X’ ) show ablated ISV ECs. Images ( H-I’ ), ( L-M’ ), ( Q-R’ ), ( U-V’ ), and ( Y-Z’ ) show adjacent ISV ECs. Images ( F, H, J, L, O, Q, S, U, W, Y ) were taken pre-ablation and images ( G, I, K, M, P, R, T, V, X, Z ) were taken 15 min post-ablation (mpa). Images ( D-Z ) show the fli1aep:EKC expression and images ( D’-Z’ ) show the nuclear fli1aep:EKC intensity. White dotted lines show the wounded sites of each larva. ISV: intersegmental vessel. Statistical test: Mann-Whitney test was conducted for graph ( C ). Error bars represent standard deviation. Scale bars: 25 μm for image ( A ), 15 μm for image ( D ). Figure 3—figure supplement 3—source data 1. Nuclear/cytoplasm EKC measurements in leading ISV ECs of DMSO- and AV951-treated 28 hpf embryos.

Article Snippet: Chemical compound, drug , SU5416 , Merck, Darmstadt, Germany , S8442 , Diluted in DMSO.

Techniques: Expressing, Blocking Assay, Activation Assay, Control, MANN-WHITNEY, Standard Deviation

Journal: eLife

Article Title: Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

doi: 10.7554/eLife.62196

Figure Lengend Snippet: ( A ) Ongoing Erk-signalling requires Vegfr and Mek activity. Quantification of post-/pre-ablation nuclear EKC intensity 3 hours post-ablation (hpa) in endothelial cells (ECs) of 0.5% dimethyl sulfoxide (DMSO)-treated non-ablated control ISVs (33 ECs, n = 11 larvae) and ablated ISVs of larvae treated with either 0.5% DMSO (51 ECs, n = 17 larvae), 15 μM SL327 (42 ECs, n = 14 larvae), 4 μM SU5416 (47 ECs, n = 16 larvae), or 10 μM SU5416 (32 ECs, n = 11 larvae). ( B ) Kdrl is required for full induction of Erk activity in ablated ISV ECs. Quantification of post-/pre-ablation nuclear EKC intensity 3 hpa in non-ablated control ISV ECs of uninjected control (27 ECs, n = 9 larvae) and kdrl crispants (26 ECs, n = 9 larvae), and ablated ISV ECs of uninjected control (22 ECs, n = 8 larvae) and kdrl crispants (27 ECs, n = 9 larvae). ( C ) Quantification of ISV horizontal length (as percentage of control) for ablated ISVs in 24 hpa, 5 days post-fertilisation (dpf), EC-EKC larvae treated with either 0.5% DMSO (n = 18 larvae), 4 μM SU5416 (n = 12 larvae), 15 μM SL327 (n = 15 larvae), or 1 μM Trametinib (n = 13 larvae). ( D ) Macrophages are not required for maintaining Erk activity in ablated ISV ECs. Quantification of post-/pre-ablation nuclear EKC intensity 3 hpa in non-ablated control ISV ECs of uninjected control (24 ECs, n = 8 larvae) and spi1/csf3r morphants (21 ECs, n = 7 larvae), and ablated ISV ECs of uninjected control (29 ECs, n = 10 larvae) and spi1/csf3r morphants (31 ECs, n = 11 larvae). ( E–G ) Lateral spinning disc confocal images of ablated ISV ECs in 4 dpf, 3 hpa, EC-EKC larvae treated with either 0.5% DMSO ( E ), 4 μM SU5416 ( F ), or 10 μM SU5416 ( G ). EC Erk activity was consistently higher and more Vegfr-dependent closer to the wound. Arrows indicate first (white), second (yellow), and third (green) ECs from the wounded site. Full images: . ( H ) Quantification of post-/pre-ablation nuclear EKC intensity at 3 hpa in first (dark grey), second (red), and third (light blue) ECs from wound. Treatments were 0.5% DMSO-treated non-ablated control ISVs (11 first, second, and third ECs, n = 11 larvae), and ablated ISVs of larvae treated with either 0.5% DMSO (17 first, second, and third ECs, n = 17 larvae), 4 μM SU5416 (16 first and second ECs, and 15 third ECs, n = 16 larvae), or 10 μM SU5416 (11 first and second ECs, and 10 third ECs, n = 11 larvae). The same embryos were used in ( A ). ( I ) Quantification of post-/pre-ablation nuclear EKC intensity at 3 hpa in first (14 ECs, n = 14 larvae), second (14 ECs, n = 14 larvae), third (14 ECs, n = 14 larvae), forth (11 ECs, n = 11 larvae), and fifth (8 ECs, n = 8 larvae) ECs from the wounded site of ablated ISVs in 4 dpf EC-EKC larvae. Data for the first, second, and third ECs were taken from . ISV: intersegmental vessel; DA: dorsal aorta. Statistical test: Kruskal-Wallis test was conducted for graphs ( A, C, D, H, I ). Ordinary one-way ANOVA test was conducted for graph ( B ). Error bars represent standard deviation. 15 μm for image ( E ). Figure 5—source data 1. Post-/pre-ablation nuclear EKC measurements in control, ablated, and adjacent ISV ECs at 3 hpa.

Article Snippet: Chemical compound, drug , SU5416 , Merck, Darmstadt, Germany , S8442 , Diluted in DMSO.

Techniques: Activity Assay, Control, Standard Deviation

Journal: eLife

Article Title: Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

doi: 10.7554/eLife.62196

Figure Lengend Snippet: ( A–J’ ) Lateral spinning disc confocal images of ISV endothelial cells (ECs) in 4 days post-fertilisation (dpf) EC-EKC larvae treated with either 0.5% dimethyl sulfoxide (DMSO) ( A–D’ ), 15 μM SL327 ( E–F’ ), 4 μM SU5416 ( G–H’ ), or 10 μM SU5416 ( I–J’ ). A higher concentration of SU5416 (10 μM) is required to block the Erk activity in ablated ISV ECs 3 hours post-ablation (hpa) immediately adjacent to the wound. Images ( A-B’ ) show non-ablated control ISV ECs. Images ( C, E, G, I ) were taken pre-ablation and images ( D, F, H, J ) were taken 3 hpa. Images ( A-J ) show fli1aep:EKC expression, and images ( A’-J’ ) show the nuclear fli1aep:EKC intensity. ( K,L ) Lateral confocal images of 4 dpf EC-EKC uninjected control (n = 100/100) ( K ) and kdrl crispant (n = 98/103 larvae displayed, phenotype indicated) ( L ). kdrl crispants phenocopy previously published kdrl mutant/morphant vascular phenotypes. ( M–T’ ) High Erk activity is not maintained in kdrl crispants 3 hpa. Lateral confocal images of ISV ECs in 4 dpf EC-EKC uninjected control ( M–N’, Q–R’ ) and kdrl crispants ( O–P’, S–T’ ). Images ( M-P’ ) show non-ablated control ISV ECs, and images ( Q-T’ ) show ablated ISV ECs. Images ( Q ) and ( S ) were taken pre-ablation, and images ( R ) and ( T ) were taken 3 hpa. Images ( M-T ) show fli1aep:EKC expression, and images ( M’-T’ ) show the nuclear fli1aep:EKC intensity. ( U–X ) Erk-signalling is required for vessel regeneration. Lateral spinning disc confocal images of 24 hpa, 5 dpf, EC-EKC larvae treated with either 0.5% DMSO ( U ), showing a regenerated ISV, or 4 μM SU5416 ( V ), 15 μM SL327 ( W ), or 1 μM Trametinib ( X ), all of which blocked ISV regeneration. DA, dorsal aorta; ISV: intersegmental vessel. White dotted lines/circles show the wounded site of each larva. Scale bars: 15 μm for image ( A ), 100 μm for image ( K ), 20 μm for image ( A ), and 50 μm for image ( U ).

Article Snippet: Chemical compound, drug , SU5416 , Merck, Darmstadt, Germany , S8442 , Diluted in DMSO.

Techniques: Concentration Assay, Blocking Assay, Activity Assay, Control, Expressing, Mutagenesis

Journal: eLife

Article Title: Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

doi: 10.7554/eLife.62196

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , SU5416 , Merck, Darmstadt, Germany , S8442 , Diluted in DMSO.

Techniques: Sequencing, Software