Fig 1: Representative immunohistochemistry of SCF or c-Kit on the same area of interest around CD31+ blood vessels in the normal skin (A, E, I), compound nevi (B, F, J), dysplastic nevi (C, G, K), and melanomas (D, H, L) tissue sections. The micrographs show a significative gradual increased CD31, SCF, and c-Kit expression in malignant melanoma lesions compared to premalignant ones and normal skin (A–L). The SCF- or c-Kit-positive mast cells appear mostly with a strong red-granulous membranous and moderate cytoplasmic staining in all the samples (see the inserts in the red rectangles; scale bar: 10 μm). The morphometric analysis performed on the same region of interest for all three markers confirmed an increased SCF+/c-Kit+ mast cell infiltrate around blood vessels in melanoma compared to normal skin, compound nevi, and dysplastic ones, both in percentages of immunolabeling positivity and number of positive cells (M, N). Linear regression analysis shows positive relationships between SCF or c-Kit and CD31(O), and between SCF and c-Kit (P) [(O) For SCF: y=3.698x+0.008438; R2 = 0.7048; p ≤ 0.0001. (O) For c-Kit: y=1.435x+0.004774; R2 = 0.792; p ≤ 0.0001. (P) y=2.61x-0.004682; R2 = 0.9123; p ≤ 0.0001]. Data are reported as means ± SD, and Tukey post-test was used to compare all groups after one-way ANOVA. Statistical significance: ns, not significative; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Scale bar: 200 μm.
Fig 2: Localization of pre-HSCs in the AGM region.(a) E10.5 AoV, AoD and UGRs were co-aggregated with OP9 and cultured for 5 days, and the formation of dHSCs was then tested by transplantation into irradiated mice (2 e.e. per recipient; AoV: six independent experiments; AoD: four independent experiments; UGRs: two independent experiments). Dashed line indicates the cutoff for high-level engraftment (>70% donor chimaerism). (b) E11.5 aortas and UGRs were transplanted after reaggregate culture (Ao: 0.2 e.e. per recipient and UGRs: 1 e.e. per recipient; two independent experiments). (c,d) Pre-HSCs type I (VC+CD45−) (c) or type II (VC+CD45+) (d) sorted from E11.5 AoV and AoD were co-aggregated with OP9 cells and transplanted after culture (1 e.e. per recipient; two independent experiments). (a–d) Levels of engraftment are plotted, and number of repopulated versus total number of transplanted mice are shown in brackets. Number of embryo equivalents (ee) injected in each experiment are indicated on the graphs. (*P<0.05; ***P<0.005; Mann–Whitney U-test). In all these experiments, tissues were cultured with three growth factors (Flt3I, Il3 and SCF). AGM, aorta–gonad–mesonephros region; Ao, dorsal Aorta; AoV, ventral domain of the dorsal aorta; AoD, dorsal domain of the dorsal aorta; UGRs, urogenital ridges.
Fig 3: Sertoli cell-specific deletion of Scf remodels the spermatogonial hierarchy. (A) Schematic overview of the workflow for sample preparation for scRNA-seq analysis. FACS, fluorescence-activated cells sorting. (B) Clustering analysis of single-cell transcriptome data from combined testicular germ cells, in which control testicular germ cells were visualized in the UMAP space. Diff, differentiating; Undiff, undifferentiated. (C) Heat map of the top 120 differentially expressed markers among five germ cell types. (D) Gene expression patterns of distinct stage-specific markers in control testicular germ cells visualized in the UMAP space. (E) UMAP plot of combined testicular germ cells from testes of 6-week-old Scffl/fl and Amh-cre; Scffl/fl mice. (F) UMAP plot of testicular germ cells from testes of 6-week-old Amh-cre; Scffl/fl mice. (G) Cell numbers of testicular cells from distinct sources in each cell cluster. (H) Summary schematic depicting percentages of undifferentiated spermatogonia (Undiff SPG), differentiating spermatogonia (Diff SPG), spermatocytes (SPC), round spermatids (RS) and elongating spermatids (ES) in testes of 6-week-old Scffl/fl and Amh-cre; Scffl/fl mice. cKO, conditional knockout; CT, control.
Fig 4: LD alters the number of ICCs and protein expression levels of C-kit and SCF in the gallbladders of guinea pigs. (A) Immunofluorescence staining of ICCs in the gallbladders of guinea pigs fed a SD or LD. ICCs were labelled using a primary antibody targeting C-kit (green), and nuclei were labelled using DAPI (blue). White arrowheads indicate C-kit-positive gallbladder ICCs, which are shown in the inset. Magnification, x200. (B) The mean number of ICCs in a field of view of guinea pig gallbladder. (C and D) Western blotting of C-kit and SCF protein expression levels in guinea pig gallbladders of the SD and LD groups. *P<0.05, **P<0.01 and ***P<0.001 vs. SD/2 weeks or SD/8 weeks. SD, standard diet; LD, lithogenic diet; ICCs, interstitial cells of Cajal; SCF, stem cell factor.
Fig 5: RNA-seq Revealed the Involvement of Key Genes for SCF Treatment. (A) Volcano plot of differentially expressed genes (DEGs) in SCF group compared with controls after 1 week treatment. (B) Gene Ontology (GO) analysis showing the enriched gene functions of SCF group versus control group. (C) Heatmap analysis showing DEGs in indicated groups. The relative abundance of each genus was indicated by a gradient of color from green (low abundance) to red (high abundance). (D) Crystallins family members were significantly up-regulated in SCF treated retinas. (E) Among top10 DEGs in SCF exposed mice, several neuro-protective factors were up-regulated, including Wnt7 related genes, Foxe3/Pitx3, and gap junction protein coding genes Gja8/Gja3. (F) Real-time qPCR analysis showing relative mRNA expression for the Crystallins family members among WT, NMDA +PBS and NMDA + SCF treated mice after 1 week. (G) Real-time qPCR analysis confirming the expression for the genes highly expressed in RNA-seq analysis. Data were shown as mean ± SD (n = 5 for each time point). *p < 0.05, **p < 0.01, compared with WT mice, #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the NMDA + PBS group.
Supplier Page from Abcam for Anti-SCF antibody