Fig 1: TGFß and TLR4 pathway intermediates are upregulated in adult TNC KO lungs. Panel a: mRNA expression of TGFß pathway intermediates evaluated by RT-qPCR in whole lung lobe homogenates of adult TNC KO and WT mice. N = 3–4 animals/genotype. Panel b-e: Representative results of phosphorylated smad2 and smad3 protein detection in whole lung lysates from adult TNC KO and WT mice; 20 or 160ug of whole lung lobe lysates were loaded on 7.5% SDS-Page gels. N = 5 animals/genotype. Cropped blots are displayed. Full length blots are presented in Supplementary Figure S2. Panel f: mRNA expression of TLR4 pathway intermediates evaluated by RT-qPCR in whole lung lobe homogenates of TNC KO and WT adult mice. N = 3–4 animals/genotype. Results are expressed as mean ± SD. Statistical analyses were made by Student t test; statistical significance was set at p < 0.05; *p < 0.05, **p < 0.01.
Fig 2: a-SMA protein expression in lungs of adult TNC KO and WT mice. Panel a-f: representative images from lung sections of WT (a,c,d) and TNC KO (b,e,f) P5 (a,b) and P90 (c–f) animals immunostained with anti-a-SMA antibody (dark grey), and counter-stained with Nuclear Red. Magnification: 20x for c,e and 40x for a,b,d,f. N = 4–7 animals/genotype for newborns and N = 6 animals/genotype for adults. Panel g-j: representative results of a-SMA protein detection in whole lung lysates from newborn (g,i) and adult (h,j) TNC KO and WT animals by Western blots; quantification is shown in histogram; N = 3–4 animals/genotype for newborns and N = 5 animals/genotype for adults. Cropped blots are displayed. Full length blots are presented in Supplementary Figure S2. Panel k,m: representative results of phosphorylated form of smooth muscle myosin light chain (PMLC2) protein detection in whole lung lysates from adult TNC KO and WT animals by Western blots; quantification is shown in histogram; N = 5 animals/genotype. Cropped blots are displayed. Full length blots are presented in Supplementary Figure S2. Panel l,n: representative results of smooth muscle myosin heavy chain (MYH11) protein detection in whole lung lysates from adult TNC KO and WT animals by Western blots; quantification is shown in histogram; N = 5 animals/genotype. Cropped blots are displayed. Full length blots are presented in Supplementary Figure S2. Results are expressed as mean ± SD. Panel o-s: flow cytometry experiment done on adult TNC KO and WT lung samples using a-SMA, desmin and vimentin antibodies. Ki67 was used to measure proliferation. N = 3 animals/genotype. Statistical analyses were made by Student t test; statistical significance was set at p < 0.05; **p < 0.01.
Fig 3: Genetic knockdown of VEGFA suppresses the stimulatory effects of TDSC-derived exosomes on tenocyte growth, migration, and transition to a fibroblastic phenotype. (A) TDSCs with VEGFA knockdown induced by a lentivirus and control TDSCs. (B) Knockdown of VEGFA determined by western blotting and quantification. Tenocytes were treated with VEGFA or exosomes derived from shVEGFA-TDSCs or shNC-TDSCs. (C) An MTT assay to investigate the growth of tenocytes. (D, E) A transwell assay to monitor the migration of tenocytes. (F–H) The mRNA (F) and protein (G, H) levels of collagen I, a-SMA, collagen III, Scx, and TnC determined by RT-qPCR and western blotting. *P <0.05, **P <0.01 relative to the indicated group.
Fig 4: Optic nerve crush causes modifications in the proteome of adult visual targets.a Experimental design and timeline. 6 week-old mice underwent bilateral optic nerve crush and brains were dissected 28 days after the injury (28 dpc). b Cleared optic nerve labelled with CTB-Alexa555 in intact and injured conditions, allows to verify the efficiency of optic nerve crush. Red stars indicate the injury site. Scale bar: 100 µm. Data are representative of N = 4 biologically independent animals. c–g Volcano plots showing differentially expressed proteins in the optic chiasm, SCN, vLGN, dLGN and SCol in injured versus intact conditions. Samples from visual targets at 28 days post-crush (28 dpc) were compared with the intact condition by MS-based label-free quantitative proteomics, using four biological replicates for each condition. The injury leads to modifications of the proteome of the visual targets, with 311 proteins found differentially expressed in the optic chiasm, 45 in the SCN, 26 in the vLGN, 92 in the dLGN and 92 in the SCol. Statistical testing was conducted using limma test. Differentially-expressed proteins were sorted out using a log2(fold change) cut-off of 0.8 and a p-value cut-off allowing to reach a FDR inferior to 5% according to the Benjamini-Hochberg procedure. h GO terms (DAVID analysis) associated with lesion-modulated hits in the optic chiasm and in the vLGN. In red is represented the protein count of the corresponding GO for up-regulated proteins. In blue is represented the protein count of the corresponding GO for down-regulated proteins. In grey is represented the significance as -log10(FDR). i Western blot analysis of Tenascin-C expression on independent biological replicates for intact and 28dpc optic chiasm and SCN (left) and corresponding quantification (right). Each lane corresponds to tissue collected from one animal. For each sample, protein expression is quantified by pixel densitometry relative to actin and normalized to intact condition. N = 3 biologically independent animals. Data are presented as mean values + /-SEM. Two-tailed unpaired Student’s t-tests, ns: not significant, *p-value = 0.0464, ns: not significant. j Epifluorescence images of immunofluorescent labelling on coronal brain section showing the modulation of GFAP expression in the optic chiasm and not in the SCN in intact and injured conditions (left) with corresponding quantification (right). Scale bar: 50 µm. N = 3 biologically independent animals. Data are presented as mean values + /- SEM. Two-tailed unpaired Student’s t-tests, ns: not significant, ***p-value = 0.0009, ns: not significant. Source data are provided as a Source Data file.
Fig 5: Serum TNC levels are increased in IBD. (a) Serum TNC levels in patients with UC (n = 40), CD (n = 91), and NCs (n = 52). (b) Serum TNC levels in NCs (n = 52), CD remission (n = 40), and CD active (n = 51). *P < 0.05, ***P < 0.001, and ****P < 0.0001. UC, ulcerative colitis; CD, Crohn's disease; NCs, normal controls.
Supplier Page from Abcam for Anti-Tenascin C antibody [EPR4219]