Fig 2: Fourteen-day orthotopic transplantation.(a) GFP-positive cells after 3 weeks’ culture on tubular scaffold (scale bar—50 µm). (b) Orthotopic replacement of the entire cervical oesophagus with a reseeded tissue-engineered graft (g), the trachea (t) was partially opened (*) to facilitate breathing, the lower part of the larynx is observed (l). (c) Postoperative weight curves. The animals receiving tissue-engineered grafts lost weight in lesser extent to control surgery and weight gain was observed at some time points (* indicate significance levels between the groups). (d) The neo-epithelium stained positively for pankeratin (scale bar—50 µm). (e) Proliferative cells were found in the basal part of the epithelium (scale bar—20 µm). (f) High power magnification showed a seven- to nine-layer thick epithelium with a superficial keratin layer (f, k; scale bar—50 µm). (g) High power magnification of regenerated muscle fibres (scale bar—50 µm). (h) Sagittal section of the oesophageal wall, the whole graft (marked with dashed lines) was re-epithelized, several blood vessels (black arrows), a few accumulations of inflammatory cells (red arrow) were found. (scale bar—500 µm. m indicates mucosa, sm submucosa, mu muscular layer, o and g indicate oral and gastric sides of the explant). (i–n) Repopulation of the scaffold of different cell types was proven by immunohistochemistry (scale bars—50 µm, for ZO 2–20 µm); desmin-positive cells were found throughout the graft (i), Gap43 and Beta-III-tubulin-positive staining suggested in-growth of neurons (j,k). Zona occludens-II, a tight junction protein, showed a pattern consistent with a healthy epithelium (l). Potential vasculature stained positively for von willebrand factor (m) out of which some stained positively for desmin (n), suggesting arterioles. *P<0.05, ***P<0.001 by ANOVA followed by Bonferroni post tests for (c).

Fig 3: GAP43 expression is downregulated in CGRP-positive neurons upon loss of adipose mTORC2. (A) Immunoblot analysis of inguinal WAT (iWAT) tissue from control and iAdRiKO mice two weeks after tamoxifen treatment. (n = 6; 6). (B) Immunoblot analysis of iWAT tissue from control and iAdRiKO mice four weeks after tamoxifen treatment. (n = 6; 6). (C) Immunoblot analysis of surgically denervated iWAT depot (denervation) compared to iWAT depot from sham-operated mice (sham). Neurofilament heavy polypeptide (NFH). (n = 5; 5). (D) Representative image of a large nerve bundle in iWAT of control mice immunostained with growth-associated protein 43 (GAP43)-pS41 and calcitonin gene-related peptide (CGRP). (N = 11; 9). (E) Representative image of a large nerve bundle in iWAT of control mice immunostained with GAP43-pS41 and tyrosine hydroxylase (TH). (N = 19; 11).

Fig 4: Ischemia stimulates cell apoptosis and alters gap junction and neural remodeling levels in vivo. (a, b) Western blotting and densitometric analysis of the protein levels of bcl2, bax, and caspase-3 in myocardial infarction tissues compared with control tissues. *p < 0.05; n = 5 per group (Student's t-test). (c, d) Western blotting and densitometric analysis of the protein levels of CX43, TH, and GAP43 in myocardial infarction tissues compared with control tissues. *p < 0.05; n = 5 per group (Student's t-test).

Fig 5: GSLCs in tumor organoids.(A) Images of C2 tumor organoids at 2 and 13 days of growth using Pln-Apo 10×/0.45 objective of inverted confocal LSM700. The resulting images represent a max intensity projection of 5 and 31 sections (step size: 7 and 3.12 µm), respectively, stained for anti-aTubulin (microtubules, white), Phalloidin (actin in red) and nuclei (blue). Scale bars are 200 (top) and 500 µm (bottom). (B) Expression of differentiation and progenitor/stem cells markers in C1 and C2 organoids, respectively in green and orange. The relative gene expressions were quantified by RT-qPCR after RNA extraction from 23-days-old organoids, normalized over the expression of HPRT. Note the 12-fold increased expression of GAP43 in C2 tumor organoids, and GFAP and CHI3L1 show no expression in both conditions and are not represented on the graph. The graphs represent means with SD of three and four independent experiments for C1 and C2 respectively, each point performed in triplicate. Holm–Sidak method was applied to determine statistical significance between cells and organoids for each gene. P value <0.05 (*), P values >0.05 are not significant and not indicated on the figure. (C) C1 and C2 tumor organoids at 9 and 6 days, respectively, stained for anti-aTubulin (microtubules, green), Phalloidin (actin filaments, red), and nuclei (blue). Confocal images were acquired with 40× objective. Regions of interest show either aTubulin-devoid connections, defined as TNT-like (<1 µm), or thick aTubulin-positive connections (>1 µm), named TM-like. Dashed arrowheads indicate absence of fluorescent signal at the connection level, white-filled arrowhead show positiveness to the signal. Both images are max intensity projections of 12 slices (step size: 0.38 µm). Scale bar = 10 µm.