Journal: bioRxiv

Article Title: Perivascular Macrophages Convert Physical Wound Signals Into Rapid Vascular Responses

doi: 10.1101/2024.12.09.627538

Figure Lengend Snippet: ( a ) Upper panel, cartoon scheme representing the experimental workflow of fluorescence microangiography, tailfin wounding, and ionic/osmotic bath treatment. HYPO, regular E3 solution. ISO (NaCl) , E3 adjusted to interstitial osmolarity by supplementing E3 with 135 mM NaCl. ISO*, E3 adjusted to isotonicity with other salts/osmolytes. Dotted box indicates time of solution switch (shift). ( b ) Cartoon schemes depicting the regions of interest for measuring the dextran permeability of vessels (Iv), wounds (Ib), as well as vessel dilation (Dv). Left panel, confocal maximal intensity projection (MIP) of wounded endothelial reporter larvae (Tg( kdrl :eGFP)) before (T= 270 s) and after (T= 3600 s) switch of bathing solutions. Green, kdrl :eGFP fluorescence. Magenta, pseudo-coloured 70 kDa dextran fluorescence. Right panel, kymographs of vessel diameter before and after switch of bathing solution. ( c ) Top panel, rate (dI dt −1 ) of vessel leakage (blue) and wound leakage (red) versus time upon switching from ISO (NaCl) to HYPO. Arrows indicate the maximal rate of change for vessel permeability (cyan) or wound permeability (black). Bottom panel, rate (dI dt −1 ) of vessel leakage versus time upon switching from ISO (NaCl) to HYPO (blue) or ISO (NaCl) to ISO (NaCl) . Shaded error bars, SD. Note, the blue curves represent the same, replotted dataset. ( d ) Quantification of normalized, integrated (between T= 0-3600 s) dextran leakage (Iv tot/norm ) or ( e ) or normalized dextran leakage from the wound (Ib norm ) measured at T= 3600 s. ( f ) Maximal, normalized endothelial diameter (max(Dv norm )), obtained from kymograph). If indicated, the raw data was normalized by the mean of the first 10 frames (T= 0-270 s, i.e., preceding solution switching). Note, ISO* contains pooled results from different isotonic salt/osmolyte treatments. Error bars, SD. White numbers, animals. Black numbers, mean of dataset. P values, one-way analysis of variance (ANOVA) with Dunn’s post-hoc test (in d, f, e ). All P values are listed in the Supplementary Excel File 1 (F1_T1). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Scale bars, 50 μm.

Article Snippet: Subsequently, the LM-agarose was enveloped with ∼2-3 mL of hypotonic (Π=10 mOsm) or ISO (NaCl) (Π=280 mOsm) E3 media for creating a submerging environment for the 25X Objective lens (N.A.=1.1 ꚙ/0-0.17 WD=2μm Water Dipping, Nikon) in Nikon Eclipse FN1 upright microscope.

Techniques: Fluorescence, Permeability

Journal: bioRxiv

Article Title: Perivascular Macrophages Convert Physical Wound Signals Into Rapid Vascular Responses

doi: 10.1101/2024.12.09.627538

Figure Lengend Snippet: ( a ) simplified scheme of enzymatic derivatives of AA, the pathways enzymes tested in the study are highlighted in red. Representative confocal MIP images of pseudo-coloured 70 kDa dextran fluorescence before (T=270 s, left panel) and after (T-3600 s, right panel) ISO (NaCl) ◊ HYPO shift in wild-type animals (Casper), ( b ) alox12 mk215/mk215 mutants, ( c ) alox5a mk211/mk211 mutants, ( d ) and lta 4 h mk216/mk216 mutants. 3 dpf larvae were compared to their matched wild-type siblings. ( e-g ) Quantification of integrated (T= 0-3600 s), normalized vessel leakage and wound permeability (T= 3600 s). The P values in ( e ) and ( g ) were calculated using a non-paired, two-tailed student’s t-test. The data in ( f ) were not normally distributed, and a two-sided Mann–Whitney U test was used instead. ( h ) Integrated vessel leakage after pretreatment of wild-type (Casper) larvae with 130 nM Diclofenac, 50 μM Licofelone and 10 μM MK886, or DMSO (vehicle) and ISO (NaCl) ◊ HYPO shifting. P values, one-way ANOVA with Dunn’s post-hoc test. White numbers, animals. Black numbers, mean of dataset. Error bars, SD. All figure source data and numerical P values are listed in the Supplementary Excel File 1. **P ≤ 0.01, ****P ≤ 0.0001. ns, not significant (P > 0.05). Timestamp, hh: mm: ss. Scale bars, 50 μm.

Article Snippet: Subsequently, the LM-agarose was enveloped with ∼2-3 mL of hypotonic (Π=10 mOsm) or ISO (NaCl) (Π=280 mOsm) E3 media for creating a submerging environment for the 25X Objective lens (N.A.=1.1 ꚙ/0-0.17 WD=2μm Water Dipping, Nikon) in Nikon Eclipse FN1 upright microscope.

Techniques: Fluorescence, Permeability, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Perivascular Macrophages Convert Physical Wound Signals Into Rapid Vascular Responses

doi: 10.1101/2024.12.09.627538

Figure Lengend Snippet: ( a ) Experimental timeline for metronidazole-induced depletion of macrophages or neutrophils in 3 dpf Tg( mpeg1.1 : YFP-NTR2.0) or Tg( lyz : YFP-NTR2.0) larvae, respectively. Blue arrow, start of the vehicle treatment (0.15% DMSO). Black arrow, start of the metronidazole (150 μM MTZ) treatment. Magenta arrow, dextran injection. Red arrow, start of imaging. ( b ) Representative confocal MIPs of leukocytes and tissue dextran fluorescence before (T= 270 s) and after (T= 3600 s) ISO (NaCl) ◊ HYPO shifting. Green, pseudo-coloured lyz: YFP-NTR2.0 (upper panel) or mpeg1.1 : YFP-NTR2.0 (lower panel) fluorescence. Magenta, pseudo-coloured 70 kDa dextran fluorescence. ( c , d ) Quantification of vessel and wound leakage in neutrophil-or macrophage-depleted animals. P values, non-paired, two-tailed Student’s t-test. ( e ) Quantification of vessel leakage after ISO (NaCl) ◊ HYPO shifting in macrophage-containing (DMSO + DMSO, DMSO + Licofelone) or –depleted (MTZ + Licofelone) larvae ± 50 μM Licofelone or 150 μM MTZ, respectively. P value, one-way ANOVA with Dunn’s post-hoc test. White numbers, animals. Black numbers, mean of dataset. Error bars, SD. The figure source data and numerical P values are listed in the Supplementary Excel File 1. ****P ≤ 0.0001. ns, not significant (P > 0.05) ( c-e ). Timestamp, hh:mm:ss. Scale bars, 50 μm.

Article Snippet: Subsequently, the LM-agarose was enveloped with ∼2-3 mL of hypotonic (Π=10 mOsm) or ISO (NaCl) (Π=280 mOsm) E3 media for creating a submerging environment for the 25X Objective lens (N.A.=1.1 ꚙ/0-0.17 WD=2μm Water Dipping, Nikon) in Nikon Eclipse FN1 upright microscope.

Techniques: Injection, Imaging, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Perivascular Macrophages Convert Physical Wound Signals Into Rapid Vascular Responses

doi: 10.1101/2024.12.09.627538

Figure Lengend Snippet: ( a ) Quantification of vessel and wound leakage in homozygous ( pla2g4aa mk217/mk217 ) and heterozygous ( pla2g4aa mk217/wt as control) cPla 2 mutant animals. ( b-c ) Representative MIPs of Tg( mpeg1.1 : cPla 2 -mKate2) larvae before and after laser injury (dashed, red circle) in HYPO (10 mOsm, upper panel) or ISO (280 mOsm, lower panel) bathing solution. Magenta pseudo-colour, mpeg1.1 : cPla 2 -mKate2. Green dashed line, vessel outline. Insets, correspond to the cells within the numbered ROIs depicted in the middle panel. Time stamp, hh:mm:ss. Scale bars, 50 μm ( d ) Line profiles of cPla 2 -mKate2 fluorescence in the ROI-labelled cells show translocation after laser injury in HYPO but not in ( e ) ISO bathing solution. ( f ) Left panel, cPla 2 -mKate2-INM binding dynamics. UV laser injury is induced at T= 40 s. INM-binding of cPla 2 -mKate2 is quantified as a ratio of nucleoplasmic to perinuclear fluorescence signal and normalized to its initial (T= 0 s) value. Right panel, comparison of peak translocation. N, number of analysed nuclei from 53 and 59 perivascular macrophages for ISO (NaCl) and HYPO laser wounds, respectively. Note, the panel depicts two different representations of the same dataset. ( g ) Simplified cartoon scheme. Perivascular macrophage nuclei are reversibly stretched by osmotic wound signals. In the presence of Ca 2+ , nuclear membrane tension causes arachidonic acid (AA) release by cPla 2 . AA is metabolized into a vessel permeabilizing lipid mediator. P values, non-paired two-tailed Student’s t-test ( a ) and non-paired two-tailed Mann Whitney test ( f ). White numbers, number of analysed nuclei pooled from 16-17 animals. Black numbers, mean of dataset. Error bars, SD. The figure source data and numerical P values are listed in the Supplementary Excel File 1. ****P ≤ 0.0001. ns, not significant (P > 0.05).

Article Snippet: Subsequently, the LM-agarose was enveloped with ∼2-3 mL of hypotonic (Π=10 mOsm) or ISO (NaCl) (Π=280 mOsm) E3 media for creating a submerging environment for the 25X Objective lens (N.A.=1.1 ꚙ/0-0.17 WD=2μm Water Dipping, Nikon) in Nikon Eclipse FN1 upright microscope.

Techniques: Control, Mutagenesis, Fluorescence, Translocation Assay, Binding Assay, Comparison, Membrane, Two Tailed Test, MANN-WHITNEY

Journal: Cell death and differentiation

Article Title: ANKRD13a controls early cell-death checkpoint by interacting with RIP1 independent of NF-κB.

doi: 10.1038/s41418-021-00906-9

Figure Lengend Snippet: Fig. 5 Poly-ubiquitination of Lys115, 377 residues of RIP1 by cIAP1/2 is required for the negative regulatory role of ANKRD13a on the Complex-II formation. a Schematic depiction of predicted lysine residues within the kinase and intermediate domains of RIP1. b RIP1-KO HEK293 cells were co-transfected with indicated combination of plasmids with CrmA. After 24 h, cells were treated with TNF (30 ng/ml) for 1 h. The ubiquitination status of RIP1 bound to ANKRD13a was determined by using IP with anti-flag antibody and followed by immunoblotting with indicated antibodies. c HEK293 cells were co-transfected with the indicated combination of plasmids. After IP with anti-HA antibody, co- immunoprecipitated proteins were detected by immunoblotting with indicated antibodies. d In vitro ubiquitination assay was performed with recombinant flag-RIP1, His-cIAP1 or His-cIAP2 and indicated E1/E2 enzymes. RIP1 ubiquitination was monitored by using IP with anti-flag antibody and followed by immunoblotting with anti-Ub antibody. e Purified GST-ANKRD13a was incubated with recombinant RIP1 and in vitro binding assay between ANKRD13a and RIP1 was performed, as described in Methods. f Purified GST-ANKRD13a was incubated with the indicated recombinant RIP1 and E1/E2/E3 enzymes and in vitro interaction between ANKRD13a and each types of RIP1 was examined, as in e. g RIP1-KO HEK293 cells reconstituted with the indicated RIP1 constructs and IκBαSR were treated for 6 h with TNF as indicated. Cell viability was measured as described in Fig. 1c. Data are presented as the mean ± S.E. (n = 3). *P < 0.05, compared with TNF-treated RIP1-KO cells expressing WT-RIP1. #P < 0.05, compared with TNF-treated RIP1-KO cells expressing RIP1K115R. h RIP1-KO HEK293 cells reconstituted with indicated constructs were treated with TNF for 1 h. Endogenous association of RIP1-FADD-caspase-8 complex and the recruitment of ANKRD13a was examined, as in Fig. 3b.

Article Snippet: In vitro ubiquitination assay was performed with E1 (UBE1, 100 nM), E2 (UbcH5a, 1μM), E3 (1μM of cIAP1 and cIAP2) with recombinant RIP1 protein-bound beads in E3 ligation buffer (50mM Tris, pH 7.5, 2.5 mM MgCl2, 2 mM DTT, 2 mM ATP) at 37 °C for 1 h. After samples were washed with 1x PBS and treated with Laemmli sample buffer (Bio-Rad) and supernatants separated by 6–8% SDS–PAGE.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Immunoprecipitation, In Vitro, Recombinant, Incubation, Binding Assay, Construct, Expressing