vhl inhibitor vh298  (Tocris)

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

Journal: European journal of medicinal chemistry

Article Title: Design, synthesis, and biological evaluation of first-in-class indomethacin-based PROTACs degrading SARS-CoV-2 main protease and with broad-spectrum antiviral activity.

doi: 10.1016/j.ejmech.2024.116202

Figure Lengend Snippet: Fig. 5. Validation of the mechanism of PROTACs 5 and 6. A) Human 293T cells were pre-treated with 15 at 25 μM and then transfected to overexpress SARS- CoV-2 Mpro. Non-trasfected (NT) cells and cells pre-treated with either DMSO or INM were included as controls. B) Human 293T cells were pre-treated with 5 and 6 at 25 μM in the absence or in the presence of 50 μM VHL inhibitor VH298 and then transfected to overexpress SARS-CoV-2 Mpro. NT cells and cells pre- treated with either DMSO were included as controls. In both panels, whole cell lysates obtained from cells collected at 24 h post-transfection were analyzed by Western Blot with an antibody recognizing SARS-CoV-2 Mpro. β-actin was used as a loading control. Molecular masses in kDa are indicated on the left.

Article Snippet: BOC and the VHL inhibitor VH298 were purchased from Selleckchem and Abcam, respectively.

Techniques: Biomarker Discovery, Transfection, Western Blot, Control

Journal: Science Advances

Article Title: Glycolytic state of aortic endothelium favors hematopoietic transition during the emergence of definitive hematopoiesis

doi: 10.1126/sciadv.adh8478

Figure Lengend Snippet: ( A ) Mito stress test performed to examine the effect of VH298 on metabolic state of the aortic ECs. ( B ) Comparison of average OCR values in sorted aortic ECs before and after the addition of mitochondrial activity modulators. ( C ) Comparison of maximal respiration calculated from mito stress test. ( D ) Comparison of OCR examined by mito stress test performed using HCs sorted from control and VH298-treated E10.5 embryos. ( E ) Confocal microscopy–based whole-mount embryo imaging to compare the emergence of IAHCs in control and VH298-treated embryos ( n = 5 to 8 independent biological replicates). The left panel shows gross morphology of the embryos imaged on a stereozoom microscope. The middle and right panels show confocal images for embryos immunostained for CD31 and c-kit. Scale bars, 50 μm. ( F ) Aortic clusters and ( G ) the total number of cluster cells counted for the dorsal and ventral sides along the aorta and compared between control and VH298-treated embryos. ( H ) Number of c-kit + cells in the aortic clusters along the dorsal and ventral side of DA in control and VH298-treated embryos. ( I ) Comparison between the control and VH298-treated embryos for c-kit + cells per cluster. ( J ) Comparison of the colonies appeared in the explant cultures of E10.5 embryos maintained with or without VH298. ( K ) Number of BFU-Es, CFU-Gs, CFU-Ms, CFU-GMs, and CFU-GEMMs obtained per cultured embryo explant plotted for the two groups. ( L ) Confocal-based whole-mount immunofluorescence images of UA in control and ( L′ ) VH298-treated embryos stained for CD31 and c-kit. Scale bars, 50 μm. ( M ) Density of hematopoietic clusters represented as numbers normalized for a length of 500 μm of UA in control and NaOX-treated embryos. Data are represented as means ± SEM. ns indicates P > 0.05; * P < 0.05, ** P < 0.01, and *** P < 0.001 by two-tailed unpaired t test.

Article Snippet: Stabilization of HIF-1α in the developing embryos was achieved by intraperitoneal injection of VHL inhibitor VH298 (150 mg/kg; Sigma-Aldrich).

Techniques: Comparison, Activity Assay, Confocal Microscopy, Imaging, Microscopy, Cell Culture, Immunofluorescence, Staining, Two Tailed Test