Fig 1: Immunofluorescence characterization of cultured buffalo spermatogonial stem-like cells colonies. Overlapping images after double-fluorescence staining of 9 days cultured cells with Hochest33342 and antibodies. Hochest33342 positive cells stained blue and cells positive for antibodies appear green. PGP9.5 (A); THY-1 (B); c-kit (C); Oct-4 (D). Scale bar, 100 µm.
Fig 2: Immunohistochemical double staining of the rat bladder. (A) Dystrophin (green) with smooth muscle marker aSMA (red), ×200, Hoechst (blue). Dystrophin was expressed at the sarcolemma, and aSMA was expressed in the cytoplasm of smooth muscle cells. (B) Dystrophin (green) with interstitial cell marker VIM (red), ×200. (C) Dystrophin (green) with urothelial cell marker PGP 9.5 (red), ×400, Hoechst (blue). (D) Dystrophin (green) with neuronal cell marker CGRP (red), ×400, Hoechst (blue). Dystrophin is always shown in green; in all images, there is a weak green signal present in the structure, which, histologically, looks like the urothelium. In close inspection of (C), structures (asterisks) can be seen in the suburothelium, which stain positive for dystrophin but do not stain for PGP 9.5. These structures are most likely CGRP+ neurons, of which some colocalized (asterisk, D) with dystrophin in the LP. aSMA, a-smooth muscle actin; CGRP, calcitonin gene-related peptide; LP, lamina propria; ML, smooth muscular layer; PGP 9.5, protein gene product 9.5; UL, urothelial layer; VIM, vimentin. [Color figure can be viewed at wileyonlinelibrary.com]
Fig 3: Z-depth reduction (ZDR) and tissue morphology(A) Axillary subcutaneous white adipose tissue (axi-scWAT) and inguinal (ing-scWAT) depots were Z-depth reduced and mounted on slides.(B) Tissue area was measured before ZDR (control), after ZDR, and after mounting. N = 3, paired two-tailed Student’s t-test, alpha level 0.05, error bars are SEMs.(C) Tissue thickness in the z-axis was measured at the thickest point before and after ZDR and mounting. Mounted tissues were measured by 3D projecting tissue autofluorescence captured on a Nikon A1R with a 20X objective. N = 3, paired two-tailed Student’s t-test, alpha level 0.05, error bars are SEMs.(D) Ing-scWAT was either whole mount processed (Z-depth reduced) or left unaltered (control) and placed on a slide and imaged with transmitted light. Four representative 10X micrographs were captured for each tissue and the area for 30–120 cells was measured per micrograph and analyzed using a two-tailed Student’s t-test, Alpha level 0.05, error bars are SEMs.(F) Hematoxylin staining of 7 µm ing-scWAT sections that either received ZDR before paraffin embedding or did not (control). Both ing-scWAT depots were excised from a PGP9.5+/- (green) direct reporter mouse (C57BL/6-Tg(Uchl1-EGFP)G1Phoz/J) and co-stained with IB4 (vasculature, red) and DAPI (nuclei, blue). One depot was Z-depth reduced and mounted whereas the other was left uncompressed to demonstrate morphological changes introduced by compressing the tissue; demonstrated through 3D reconstructions of similar structures.Other details: Transmitted light micrographs were captured on a Nikon E400 upright microscope (D and E). Confocal micrographs captured on Nikon A1R (C and F). Scale bars are 186 µm and 174 µm (C), 200 µm (D and F) and 50 µm (E).
Fig 4: Nociceptor neurons affect allergic skin inflammation.(A) Timeline for induction of allergic skin inflammation using calcipotriol (vitamin D analog). Calcipotriol or vehicle (EtOH) was applied once daily on mouse dorsal skin for 8 days to littermate control or genetically ablated (TRPV1cre/wt DTAfl/wt) mice. Groups of WT mice were additionally treated once daily with QX-314 (10 mg/mL, topical) from days 3 to 8. Animals were sacrificed on day 17. (B) Skin histology of day 5 control and calcipotriol-treated mice showed skin thickening, ECM deposits, loss of dendritic epidermal CD3+ T cells, recruitment of CD3+ T cells to inflamed tissue, and loss of PGP9.5 hair follicle innervation after sensitization. Scale bar represents 100 µm. Calcipotriol treatment increased numbers of (C) CD45+ cells, (D) dendritic cells, (E) CD3+ T cells, (F) CD4+ T cells, (G) regulatory T cells, and (H) CD8+ memory T cells in skin tissue. Nociceptor ablation (TRPV1Cre/wt DTAfl/wt) reversed calcipotriol-mediated skin inflammation and significantly decreased (J) serum IgE levels. (I) Serum IgG1 levels were not affected by nociceptor ablation. Graphs show range, median, and “+” as mean. P values determined using 1-way ANOVA and Tukey’s multiple comparisons test. * denotes comparison with vehicle-exposed, nociceptor-intact mice and + comparison with calcipotriol-exposed, nociceptor-intact mice. P < 0.05 is indicated by +; P < 0.01 is indicated by ** or ++; P < 0.001 is indicated by +++; P < 0.0001 is indicated by **** or ++++. n = 3–5/group. Experiments were replicated at least 2 independent times.
Fig 5: UCHL1 methylation in breast cancer cell lines and reactivation of UCHL1 by demethylation.(A) UCHL1 is frequently methylated in breast cancer cell lines. (B) Pharmacological demethylation restores UCHL1 expression. A+T: Aza and TSA treatment. M, methylated; U, unmethylated.
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