EMBO/SourceData huggingface dataset:
Figure 1Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP, and FLAG-TCHP at N- and C- terminal. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP-FLAG C-terminal or constructs with deletions of the coiled-coil domain 1 (TCHP ∆1) and 2 (TCHP ∆2), as indicated in the scheme. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.Anti-PCM1 immunoprecipitation from HUVECs cells and TCHP and PCM1 immunoblot.Co-localization of TCHP-V5 and centriolar satellite proteins. HUVECs were transduced with TCHP-V5 lentivirus and stained for anti-PCM1, anti-CEP290, anti-CEP72 and anti-V5 antibodies. Scale bars, 25 µm and 5 µm in the inset. Right panel: the two-channel intensity correlation of pixels corresponding to regions identified with TCHP-V5 and satellite markers (n=90 cells; Pearson colocalization coefficient).TCHP knock-down or control cells were stained for anti-PCM1 antibody. Panel below: quantification (n=50 cells; unpaired t-test; **p=0.004 vs control). Scale bars, 50 µm and 5 µm in the inset.
Figure 2Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. PCM1 levels were analysed in the insoluble fraction. Laminin has been used as a loading control. Below panel: Quantification of PCM1 degradation. (one-way ANOVA; *p=0.0180 vs control time 0).Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. Condition as in (A), Western blot for anti-GABARAP and anti-ACTIN antibodies. Below panel: Quantification of GABARAP degradation. (one-way ANOVA; *p =0.0215 vs control time 0).Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-PCM1 and anti-V5 antibodies. Laminin has been used as a loading control.Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-V5, anti-GABARAP and anti-ACTIN antibodies. Below panels: Quantification of C (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0003 vs control CHX) and D (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0056 vs control CHX).
Figure 3Immunofluorescent staining for LC3 and p62 in TCHP knock-down and control cells. Scale bars, 25μm. Lower panel: quantification. (n=90 cells, unpaired t-test; LC3: **p<0.0001 vs control; p62: **p<0.0001 vs control).Western blot of p62 and LC3 during under normal culture condition or starved condition (HBSS) or the presence of BafA1 (2 hours) in TCHP knock-down or control cells. Lower panels: p62 quantification. (one-way ANOVA; **p=0.0003 vs control, ##p=0.0093 vs shTCHP); LC3 quantification. (one-way ANOVA; **p=0.0008 vs control, ##p=0.0011 vs shTCHP).HUVECs were transduced with the tandem mCherry-EGFP-LC3 and with shRNA TCHP or control vectors. Left panels: Representative picture of mCherrry-EGFP-LC3 reporters. Scale bars, 25μm. Right panel: Quantification of the number of mCherry-only (red bars, autolysosomes) or double-positive (mCherry+/EGFP+; yellow bars, autophagosomes). (n=80 cells, one-way ANOVA; *p=0.0447 vs control; ##p=0.0007 vs shTCHP).Ratiometric flow cytometric analysis of mCherrry-EGFP-LC3 reporters as in C. (one-way ANOVA; *p=0.0247 vs control; ##p=0.0088 vs shTCHP).Quantification of long-lived protein degradation assay in TCHP knock-down and control cells by flow cytometry (one-way ANOVA; **p=0.0018 vs control, ##p<0.0001 vs shTCHP).HUVECs were transduced with TCHP-V5 and control vectors. Western blot for anti-V5 and anti-p62 antibody during under normal culture condition or HBSS or BafA1 or Torin-1 treatment. Lower panel: quantification. (one-way ANOVA; **p=0.0044; #p=0.0117 vs TCHP-V5).
Figure 4The postnuclear fraction (PNS) from TCHP knock-down and control HUVECs in the presence or absence of BafA1 was separated into (low-speed pellet) LSP and (high-speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies. The sub-fractions were treated with proteinase K (Prot. K) with or without Triton X- 100 (TX-100). Quantification of the Western blot: the p62, LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non-treated samples.Representative TEM images of autophagosomes in control and TCHP knock-down HUVECs in the complete medium after 3h incubation with BafA1 (Scale bars, 600nm): i, quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and ii, distribution of the cross-section areas of the analysed vesicles expressed in percentage. (n=7 cells; >100 vesicles per sample; AVs: unpaired t-test; **p<0.0001 vs control; APs: unpaired t-test **p<0.0001 vs control).Left panels: Representative single-channel and merged images of HUVECs expressing GFP-LC3 and immunostained for STX17. Scale bars, 25 µm and 2 µm in the inset. Right panels: Quantification of LC3/STX17 positive cells in the presence or absence of BafA1 (one-way ANOVA; **p=0.0060 vs control; #p=0.0136 vs control BafA1) and quantification LC3/STX17 positive puncta per cells (n=50 cells; one-way ANOVA; **p<0.0001 vs shTCHP BafA1).
Figure 5Expression of TCHP, p62, IL-1β, IL-8, and IL-6 in ECs from vessels wall from patients with CAD (n=8 patients per group, unpaired t-test; TCHP **p <0.0001; p62 **p = 0.0007; IL-1β **p <0.0001; IL-8 **p <0.0001; and IL-6 **p <0.0001 vs. Healthy subject).Staining, and quantification of p62 in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. Scale bars, 50 µm; (n=3 patients per group; n=80 cells; one-way ANOVA; **p<0.0001 vs healthy DMSO; ##p=0.003 vs patients DMSO).Expression of IL-6, IL-8 and IL-1β (n=3 patients per group; one-way ANOVA; IL-6: *p = 0.0135 vs healthy DMSO; #p = 0.0180 vs patients DMSO; IL-8: *p = 0.0139 vs healthy DMSO; ##p = 0.0024 vs patients DMSO; IL-1β: *p = 0.0104 vs healthy DMSO; ##p = 0.0069 vs patients DMSO).migration speed, in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. (n=3 patients per group; one-way ANOVA; *p=0.0325 vs healthy DMSO; #p=0.0336 vs patients DMSO).Left panels: representative images of the heart of wild-type and Tchp knock-out mice stained for CD31 (green) and p62 (magenta). Scale bars, 100µm, and 50µm for the inset. Right panels: Quantification of the percentage of cardiac area (n=4 mice per group; unpaired t-test; **p=0.083 vs wild-type) or vessels positive for p62 (n=4 mice per group; unpaired t-test; **p=0.058 vs wild-type) and vessel density (n=4 mice per group; unpaired t-test; **p=0.0097 vs wild-type).
Figure 1Electron micrograph of fibrils formed by a synthetic peptide encompassing the M45 RHIM. Representative of three samples examined. Scale bar 100 nmHT29 cell survival 18h after treatment with TNF+BV-6+z-VAD-fmk to induce necroptosis. Cells expressed lentivirus constructs encoding 1-90 or 1-277 fragments of M45 and were cultured +/- the addition of coumermycin A1 to induce dimerisation. N=2 for M45-90 gyrase +/- coumermycin A1 and 4 for M45-277 gyrase and for empty vector, +/- coumermycin A1
Figure 2Thioflavin T fluorescence as a function of time, following dilution of WT or AAAA His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 from 8 M to 200 mM urea-containing buffer. Buffer sample contains ThT but no protein. Samples were tested in triplicate and solid line is the average of the triplicates and the error bars (in grey) represent +/- s.dAbsorbance spectra from solutions containing Congo red with WT (black lines) or AAAA mutant (dark grey lines) His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 that have been dialysed against Assembly Buffer. Congo red sample alone with no protein (light grey line) served as a control for the experiment. Experiment conducted onceElectron micrograph of material eluted in peak 1 from the three MBP fusion proteins, as labelled. These are representative micrographs of the material observed. Scale bar 100 nm in all panelsRepresentative micrographs of insoluble material generated after treatment with thrombin removed the MBP. Experiment repeated three times. Scale bar 100 nm in all panel
Figure 3Fluorescence polarisation profiles from incorporation of FITC-M451‑90 into excess unlabeled M451‑90 (green), RIPK1497‑583 (red), RIPK3387‑518 (blue) or ZBP1170-355 (purple). Average polarisation profile from smoothed triplicate samples plotted, with s.d. indicated. Experiment performed twice and representative results shown hereRepresentative fluorescence intensity trace showing homomeric assembly over 40 s in mixtures of (C) mCHERRY-RIPK1497‑583 and YPet-RIPK1497‑583 and (D) mCHERRY-RIPK3387‑518 and YPet-RIPK3387‑518. E, F Representative fluorescence intensity traces showing heteromeric assembly over 40 s in mixtures of (E) mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 (inset is expansion of region indicated by *) and (F) mCHERRY-RIPK1497‑583-AAAA and YPet-RIPK3387‑518. All mixtures examined in at least three different experiments on three separate occasions
Figure 4Representative fluorescence intensity traces collected over 50 s for mixtures of (A) mCHERRY-M451-90 and YPet‑only; (B) mCHERRY-M451-90 and YPet‑RIPK1497‑583; (C) mCHERRY-M451-90 and YPet-RIPK3387‑518; (D) mCHERRY-M451-90 and YPet-ZBP1170-355; (E) mCHERRY-M451-90AAAA and YPet‑only; (F) mCHERRY-M451-90AAAA and YPet-RIPK1497-583; (G) mCHERRY-M451-90AAAA and YPet-RIPK3387‑518 and (H) mCHERRY-M451-90 and YPet-ZBP1170-355. Independent replicates performed at least twice
Figure 5Representative electron micrographs of homofibrils formed by M451-90, RIPK1497‑583, His-Ub-RIPK3387‑518 or ZBP1170-355 individually, and heterofibrils formed by indicated mixtures.Samples prepared twice from independent protein preparationsHeterofibrils containing mCHERRY-M451-90 and YPet‑RIPK1497‑583 C Heterofibrils containing mCHERRY-M451-90 and YPet-RIPK3387‑518 D Heterofibrils containing mCHERRY-M451-90 and YPet‑ZBP1170‑355 E Heterofibrils containing mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑51Heterofibrils containing mCHERRY- RIPK3387‑518 and YPet‑ZBP1170‑355 Pearson's correlation coefficients measured from two confocal images of the indicated protein mixtures.Heterofibrils containing ECFP-M451-90 with mCHERRY- RIPK1497‑583 and YPet-RIPK3387‑518STED images of heteromeric fibrils formed by co-assembly of mCHERRY-M451-90 and YPet-RIPK3387‑518
Figure 6SDS AGE analysis of monomeric or assembled forms of YPet-RIPK3387‑518 and mCHERRY-M451-90WT or mCHERRY-M451-90AAAA. Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis, as indicated. Assembled samples incubated with 0 or 2% SDS at RT for 10 min before electrophoresis. Protein components as indicated above each lane.SDS AGE analysis of assemblies of mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 with or without four times molar equivalents of His-Ub-M45.SDS AGE analysis of assemblies of YPet-RIPK3387‑518 and mCHERRY-ZBP1170-355 with or without four times molar equivalents of His-Ub-M45.
Figure 7Confocal microscopy image of a single fibril composed of YPet-M451-90
Figure 1A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown.A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.G-I Splenicdendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenicdendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenicdendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.
Figure 2Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4-catalyzed oxidation of PAPC for 2 and 24 h. Mean ± SEM of duplicate determinations are shown.Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL-12 secretion of thioglycollate-elicited macrophages. Dots represent data of individual OxPAPC preparations.
Figure 3Selected candidate lipids were tested for their inhibitory activity on R837-induced (5 μg/ml; 18 h) cytokine secretion in BMDCs. Concentrations of indicated lipids: PECPC (10 μM), PEIPC (10 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), and KOdiAPC (40 μM). Representative data (mean ± SD of triplicate determinations) from one of three independent experiments are shown. ****P < 0.0001; ns, not significant; as determined by one-way ANOVA adjusted by Sidak's multiple comparisons test.Dose-response curves of IL-12 secretion in BMDCs pulsed for 60 min with the indicated lipids, followed by stimulation with R837 (5 μg/ml) for 18 h. Toxic concentrations of lipids were excluded from analysis. Mean ± SEM of triplicate determinations are shown.EC50 values of anti-inflammatory lipid products normalized to the capacity of 15d-PGJ2 to suppress IL-12 production in BMDCs (left panel) and their respective bioactivities (right panel) depicted as the fold increase relative to 15d-PGJ2. Mean ± SEM of triplicate determinations are shown.IL-12 production of BMDC stimulated via TLR 4 (LPS; 100 ng/ml), TLR 9 (CpG; 100 nM), and TLR 7 (R837; 5 μg/ml) after pretreatment with the indicated free and esterified versions of EC and 15d-PGJ2. Lipids were used at 1 μM (EC), 10 μM (PECPC), or 20 μM (15d-PGJ2 and 15d-PGJ2PC). Data (mean ± SEM) are representative of 3 independent experiments. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; determined by one-way ANOVA adjusted by Dunnett's multiple comparisons test.Analysis of the capacity of various OxPL-derived species to license splenicdendritic cells to polarize naïve CD4T cells toward the Th2 subset. Concentrations of indicated lipids: EC (1 μM), PECPC (10 μM), 15d-PGJ2 (20 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), KOdiAPC (40 μM).
Figure 4A IL-12 production of BMDC from wild-type, Nrf2−/−, Pparg−/−, and Pparg litter mate control mice normalized to medium control (open bars). Cells were treated with the indicated lipids (filled bars) for 60 min prior TLR 7 ligation with R837 (5 μg/ml) for 18 h. Lipids were used at starting concentrations of 40 μM (POVPC, PGPC, and KOdiAPC), 40 μg/ml (OxPAPC), 20 μM (15d-PGJ2), and 1.25 μM (EC), depicted as black bars, and 2-fold serial dilutions thereof (gray bars). Data represent mean ± SEM of triplicates from one of three independent experiments.B Expression of Nrf2 target genes Hmox1 and Nqo1 in wild-type and Nrf2−/−BMDM stimulated with EC (2 μM) for 60 min. Gene expression levels are presented relative to that of untreated cells after normalization to G6pdx. Data (mean ± SEM) are representative of two independent experiments.C, D mRNA expression levels of the Nrf2 targets Gclc and Gsta3 (C) and of the pro-inflammatory cytokines IL-6 and IL-12 (D) in wild-type, PPAR-γ-deficient, and Nrf2-deficient BMDM after treatment with EC or 15d-PGJ2 for 60 min followed by LPS treatment for 3 h. Expression levels are normalized to G6pdx. Data represent mean ± SEM of triplicate cultures from one of two independent experiments.E mRNA expression of the indicated chemokines as determined by qPCR. Wild-type BMDCs were treated with EC (1 μM) or 15d-PGJ2 (20 μM) for 60 min followed by R837 stimulation (5 μg/ml) for 3 h. Expression levels are shown normalized to G6pdx. Data (mean ± SEM, n = 2) are representative of three independent experiments.
Figure 5A C57BL/6mice were treated (i.v.) with 500 μg EC or DPPC control 2 h prior to i.p injection of 150 ng/g LPS together with 800 μg/g D-galactosamine. 4 h after LPS application, lungs were perfused with PBS and embedded in paraffin. Tissue sections were hematoxylin-stained to visualize adherent cells. Bars represent 100 µm.B, C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in (B) and as averages of single mice in (C). Pooled data of two independent experiments are shown (n = 10 for EC, n = 14 for DPPC). Unpaired two-tailed t-test.D, E C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils (D). Unpaired two-tailed t-test. Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.F, G The concentrations of IL-6 (F) and IL-12 (G) in the BAL of mice treated as in (D) were quantified by ELISA. Data (mean ± SEM) are representative of two independent experiments with 3 mice per group. ****P < 0.0001; unpaired two-tailed t-test.C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.
Figure 6Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h.Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in (A), presented relative to EC.
Figure 7Quantification of IL-6 and IL-12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 (5 μg/ml; 18 h). Bars represent mean ± SEM from one of three independent experiments. **P < 0.01; ***P < 0.001; one-way ANOVA adjusted by Dunnett's multiple comparisons test.mRNA expression of the Nrf2 targets Hmox1 and Nqo1 normalized to G6pdx expression. BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.ΔEC50 values of the indicated lipids and OxPL shown relative to that of EC as determined in (E).Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion in BMDCs by prior treatment with the indicated OxPL derivatives for 1 h.mRNA quantification of the indicated chemokines relative to G6pdx expression. BMDC were pretreated for 60 min with 500 nM cEC or the variant BisRed prior to R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.Quantification and characterization of cellular infiltrates in BAL. Groups of six C57BL/6mice were pretreated i.t. with 50 μg cEC, EC, or BisRed 24 and 2 h before challenge with 150 ng/g LPS in the presence of 800 μg/g D-galactosamine. BAL was harvested 4 h after LPS injection, and inflammatory cells were characterized by FACS analysis. **P < 0.01; ****P < 0.0001; one-way ANOVA with Sidak's multiple comparisons test.Comparison of the capacity of cEC, EC, and PECPC (1 μM) to license splenicdendritic cells to polarize naïve CD4T cells into IFN-γ-producing (Th1) and IL-4-producing (Th2) effector cells. Data (mean ± SD) are representative of two independent experiments. One-way ANOVA adjusted by Dunnett's multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 1(C) Representative confocal image of long propriospinal neurons expressing FGF22 (magenta) in cervical spinal cord.(E) Schematic illustration of the hCST collateral into the cervical cord and confocal images of hCST collaterals (green), stained for vGlut (red) and vGAT (blue) 21 days following spinal cord injury. Insets represent 3D views generated in Imaris of the deconvolved confocal image. (F) Quantification of bouton density and characterization of bouton between control and FGF22 overexpressing mice (n = 4-5 mice per group). ((G) Schematic illustration and representative confocal image of LPSN viral labeling (magenta) and CST collaterals (green). Insets represent 3D views generated in Imaris of the deconvolved confocal image. (H) Quantification of hCST contacts onto LPSNs and characterization of vGlut intensity onto individual LPSNs (n = 4-5 mice per group).
Figure 2(C) Schematic illustration and representative confocal image for vGlut2-Cre neuronal labeling (cyan) and hCST collaterals (white). Insets represent 3D views generated in Imaris of the confocal image. Quantification of hCST contacts onto vGlut neurons (n = 4-6 mice per group).