Journal: Scientific Reports

Article Title: SOCS domain targets ECM assembly in lung fibroblasts and experimental lung fibrosis

doi: 10.1038/s41598-024-83187-9

Figure Lengend Snippet: VHL protein inhibition does not decrease SMA intensity in myofibroblasts. ( A ) IMR90 lung fibroblasts were untreated or differentiated with 5 ng/mL TGFβ for 48 h. Differentiated myofibroblasts were then treated with 100 µM VH298 for 1 h. Cells were then lysed in SDS buffer and immunoblotted for HIF1-⍺. β-Tubulin was used as the loading control. ( B ) Fibroblasts, myofibroblasts and myofibroblasts treated as in ( A ) were immunostained for FN (red) or SMA (red) and nuclear stain, DAPI (blue). Scale bar = 10 µm.

Article Snippet: VH298 (Axon MedChem, #2810) was used at working concentrations of 100 μM for 1 h in serum media.

Techniques: Inhibition, Control, Staining

Journal: Nature Communications

Article Title: The urotensin II receptor triggers an early meningeal response and a delayed macrophage-dependent vasospasm after subarachnoid hemorrhage in male mice

doi: 10.1038/s41467-024-52654-2

Figure Lengend Snippet: a Immunolabeling of ERTR7 + (white) meningeal fibroblasts of leptomeninges showing UT (magenta) overexpression and DAPI (blue) in SAH compared with sham conditions. Quantification of UT intensity from D1 to D7 in ERTR7 + areas ( n = 5). Scale bar = 50 µm. b Immunolabeling of the dura mater showing UT (green) overexpression in SAH compared to sham conditions at D1 in ERTR7 + (magenta) meningeal fibroblasts bordering dural lectin + (gray) vessels ( n = 3/condition). Scale bar = 50 µm. c Immunolabeling of leptomeninges showing HIF-1α (cyan) overexpression at D1 in AKAP12 + (green) meningeal fibroblasts in SAH compared with sham conditions. Quantification of meningeal HIF-1α intensity in AKAP12 + areas ( n = 6). Scale bar = 50 µm. d Timeline of intracisternal injections of siRNA targeting HIF-1α gene expression one day before SAH surgery. e Immunolabeling of leptomeninges showing at D1 HIF-1α (green) and UT (magenta) expression in SAH compared to sham conditions in mice pretreated with siCTRL or siHIF-1α in laminin + (gray) meningeal fibroblasts. Quantification of HIF-1α (upper panel) and UT (lower panel) intensity in laminin + areas ( n = 4). Scale bar = 50 µm. f MCA delimited via lectin + (green) and DAPI (blue) staining at D7. MCA lumen area/wall thickness ratio in SAH compared to sham conditions in mice pretreated with siCTRL or siHIF-1α (lower panel). Quantification of the lumen area/wall thickness ratio ( n = 6). Scale bar = 50 µm. HIF-1α (magenta) expression in peri-MCA in SAH compared to sham UT +/+ and UT -/- mice (upper panel). Quantification of perivascular HIF-1α intensity in the MCA ( n = 6). Scale bar = 50 µm. g Timeline of intracisternal injection of aCSF or VH298 prior to intracisternal injection of aCSF or UII. h Immunolabeling of HIF-1α (red) or UT (cyan) within leptomeninges at D1 in sham- or VH298-pretreated mice and in the absence or presence of UII. Quantification of HIF-1α (upper panel) and UT (lower panel) intensity in meningeal cells. Scale bar = 50 µm. i MCA delineation with lectin (green) and DAPI (blue) staining at D7. Quantification of the lumen area/wall thickness ratio ( n = 3). Scale bar = 50 µm. a – i Values are expressed as the mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 ( a – g , i ) Two-sided two-way analysis of variance (ANOVA), Bonferroni’s correction). h two-sided two-way analysis of variance (ANOVA), Tukey’s correction). Source data are provided as a Source Data file. a , d , g created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: In another experimental set, HIF-1α stabilization was performed by intracisternal injection of the Von Hippel‒Lindau (VHL) VH298 inhibitor (10 μl, 5.2 μg/ml) (Tocris, #6156) or aCSF volume as a control 2 h before intracisternal injection of murine UII (50 μl, 2 μg/ml) (Phoenix Pharmaceuticals, #071-08) or aCSF volume solution for two consecutive days (D-1, D0).

Techniques: Immunolabeling, Over Expression, Gene Expression, Expressing, Staining, Injection

Journal: Nature Communications

Article Title: The urotensin II receptor triggers an early meningeal response and a delayed macrophage-dependent vasospasm after subarachnoid hemorrhage in male mice

doi: 10.1038/s41467-024-52654-2

Figure Lengend Snippet: a Kinetics of F4/80 + (green) pial MΦ recruitment and UT (magenta) overexpression in leptomeninges from D1 to D7 in SAH compared to sham UT +/+ mice. Quantification of the number of F4/80 + MΦs (left panel, n = 5) and UT intensity in F4/80 + cells (right panel, n = 3) from D1 to D7. Scale bar = 50 µm. Values are expressed as the mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 (normally distributed, comparison of two groups, unpaired two sided t -test). b FACS analysis and gating strategy (upper right quadrant F4/80 + UT + ) for bone marrow-derived (in vitro, see Material and Methods) F4/80 + MΦs (BMDMs) obtained from UT +/+ or UT -/- mice and treated or not treated with VH298 (24 h). Quantification of the percentage of UT-expressing cells among total F4/80 + BMDMs ( n = 3). Values are expressed as the mean ± SEM. *** P < 0.001 (two-sided two-way analysis of variance (ANOVA), Tuckey’s correction). c Immunolabeling of leptomeninges of UT (magenta) and F4/80 + (green) MΦs at D1 in aCSF- or VH298-pretreated mice and in the absence or presence of UII in the subarachnoid space ( n = 3). Scale bar = 50 µm. Values are expressed as the mean ± SEM. * P < 0.05 (two-sided two-way analysis of variance (ANOVA), Tuckey’s correction). d Experimental timeline of leptomeningeal and PVMΦ depletion by intracisternal injection of clodronate-liposomes (CLO-lip) before SAH with blood from mice depleted of peripheral MΦs prior to behavioral testing and brain analyses. Values are expressed as the mean ± SEM. * P < 0.05; *** P < 0.001 (two-sided two-way analysis of variance (ANOVA), Tuckey’s correction). e Immunolabeling of F4/80 + (magenta) MΦs around lectin + (white) leptomeningeal and perivascular vessels. Histograms of quantification of the total number of F4/80 + cells at D1 in sham and SAH conditions. f Immunolabeling of lectin + (green) and nuclei by DAPI (blue) in MCA at D7 in PBS-lip and CLO pretreated mice. Quantification of lumen area/wall thickness ( n = 6). Scale bar = 50 µm. g Exploration and locomotion in OFT. Sensorimotor functions in the BWT and preference index in the NORT ( n = 9/condition). Radar plots illustrating the relative effect of SAH in CLO pretreated mice on performance in OFT, BWT, and NOR test. Each item of the radar represents the mean normalized to CLO pretreated PBS mice. e – g Values are expressed as the mean ± SEM. ns=non-significant, * P < 0.05; ** P < 0.01; *** P < 0.001. (two-sided two-way analysis of variance (ANOVA), Bonferroni’s correction). Source data are provided as a Source Data file. b , d – f created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: In another experimental set, HIF-1α stabilization was performed by intracisternal injection of the Von Hippel‒Lindau (VHL) VH298 inhibitor (10 μl, 5.2 μg/ml) (Tocris, #6156) or aCSF volume as a control 2 h before intracisternal injection of murine UII (50 μl, 2 μg/ml) (Phoenix Pharmaceuticals, #071-08) or aCSF volume solution for two consecutive days (D-1, D0).

Techniques: Over Expression, Comparison, Derivative Assay, In Vitro, Expressing, Immunolabeling, Injection, Liposomes