Fig 1: Hypothetical model of the possible mechanisms used by M. leprae to modulate the expression of the Notch signaling pathway in the epidermis and their consequences. This figure shows how M. leprae could be related to the basement lamina (laminin receptor–dystroglycan complex) of the keratinocytes. The proliferation of M. leprae inside of these cells could induce a chronic inflammatory process that would reduce the glycosylation of the Notch receptor and its interaction with the ligand Jagged-1. This fact could induce aberrant signaling and reduced expression of Hes-1, affecting the activation of the protein P-63, the differentiation of the keratinocytes, and the innate and adaptative immune response against M. leprae.
Fig 2: Notch activity promotes microglial development in vivo and in vitro.a, b Immunofluorescence (a) and fluorescent intensity emission values (b) of RBPJ (red) in F4/80+ (green) cells in the sagittal sections of E14.5 Rbpjfl/fl and Cx3cr1cre/+Rbpjfl/fl mice (n = 3 mice in each group). Each dot in (b) denotes one cell. Ten cells per mouse were quantified. The white arrow indicates the co-localized signals of RBPJ and F4/80, whereas white arrowhead point to the limited signals of RBPJ. c, d Immunofluorescence (c) and quantified cell density (d) of IBA1+ cells in the sagittal sections of E14.5 Rbpjfl/fl, Cx3cr1cre/+ and Cx3cr1cre/+Rbpjfl/fl mice (n = 6 mice in each group). Each dot denotes one mouse. Three slices per mouse were quantified. e Transcriptional levels of Sall1, Tgfb1, Fcrls, Slc2a5, and Gpr34 in CD11bhiCD45lo cells from E14.5 Rbpjfl/fl and Cx3cr1cre/+Rbpjfl/fl mice mesencephalon. The data are from three independent experiments. Each dot represents an independent experiment. f Immunofluorescence of IBA1 and Hes1 in the embryonic microglia in vitro furnished with MCSF or additional DLL3 for 5 days. g Quantification of IBA1+ cell density in (f). Data were pooled from three independent experiments. Cultured IBA1+ cells in each experiment were from twenty E12.5 mice mesencephalon. Each dot represents individual experiment. h The percentage of Hes1-IBA1+ or Hes1+IBA1+ cells after cultured 5 days in (f). The number in each histogram indicates the average percentage. Data are pooled from three independent experiments. i Transcriptional levels of Hes1, Mcm5, Dab2, Pros1, Tmem119, Gpr34, and Sall1 in embryonic microglia after cultured for 5 days. The data were from three independent experiments. Each dot represents an independent experiment. j Schematic diagram of Notch activation in mouse microglial differentiation, created with BioRender.com. Error bars, mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns no significant, Unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.
Fig 3: ?Np63a transcriptionally regulates the effect of the Notch pathway on TS-induced lung CSC-like properties. (A and C) THBE sphere-forming cells were transfected with ?Np63a plasmids, a control vector, siRNA-?Np63a, or control- siRNA for 4 days and the protein levels of NICD and Hes1 were determined by western blotting. (B and D) Densitometry results are shown as the fold-change compared to the vector or control siRNA after ß-actin normalization. (E and F) THBE sphere-forming cells were co-transfected with wt-Notch1 promoter-luciferase, ?Np63a (E), or ?Np63a-siRNA (F), and luciferase activity was measured after incubating for 3 days. (G and H) THBE sphere-forming cells were co-transfected with mut-Notch1 promoter-luciferase, ?Np63a (G), or ?Np63a siRNA (H), and their luciferase activity was measured. Three independent experiments were performed. Data are expressed as the mean ± SD. Significance was assessed by unpaired two-tailed Student's t tests. * P < 0.05, compared to the vector group. # P < 0.05, compared to the control siRNA group.
Fig 4: Baxtm1Sjk mutant mice exhibit compromised microglial maturation and Notch signaling.a Immunofluorescence of BAX (red) with SOX2 (gray) and F4/80 (green) in the sagittal sections of E14.5 mice brains. b Quantification of IBA1+ and F4/80+ cell density in E14.5 WT and Baxtm1Sjk mice midbrains. Each dot denotes one mouse (n = 5 mice in each group). Three sections per mouse were quantified. c, d 3D reconstructions of confocal z-stacks (c) and measured volume (d) of IBA1+ cells in E14.5 WT and Baxtm1Sjk mice mesencephalon (n = 3 mice in each group). Each dot in (d) denotes one cell. Ten cells per mouse were quantified. e Transcriptional levels of P2ry12, Hexb, C1qa, Slc2a5, and Fcrls in CD11bhiCD45lo cells from E14.5 WT and Baxtm1Sjk mice mesencephalon. The data are from three independent experiments. Each dot represents an independent experiment. f Immunofluorescence of p-CaMKII (left) and p-CREB (right) in brain sagittal sections of E14.5 WT and Baxtm1Sjk mice. g WB of p-CaMKII, p-CREB and GAPDH in the brain cells from E14.5 WT and Baxtm1Sjk mice. The experiments were triply repeated. The results were similar. h Immunofluorescence of HuC/HuD (green) and DLL3 (red) in the brain sagittal sections of E14.5 WT and Baxtm1Sjk mice. i, j WB of DLL3 and ß-catenin (i), NICD1 and Hes1 (j) in the brain cells from E14.5 WT and Baxtm1Sjk mice. The experiments were triply repeated. The results were similar. k Immunofluorescence of F4/80 (green) and Hes1 (red) in the brain sagittal sections of E14.5 WT and Baxtm1Sjk mice. White arrows indicated the co-stained signals. White arrowheads presented the F4/80+ signals only. l The percentage of F4/80+ microglia with or without Hes1+ signals in (k) (n = 4 mice in each group). Three sections per mouse were quantified. The number in each histogram indicates the average percentage. m A diagram of the Bax-Notch axis in regulating microglial maturation, created with BioRender.com. The small images in the right corners of (a, h, and k) are the enlargement regions (white dotted box). Error bars, mean ± SEM. **P < 0.01; ****P < 0.0001, Unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.
Fig 5: Suppression of cognitive deficits induced by Pb exposure are prevented by inhibition of Notch signaling. (a) Western blot analysis and shading analysis of Notch1, RBP-J, Hes1, and Hes2 in DG in Hippocampus in the three groups. (b) Western blot analysis and shading analysis of Notch1, RBP-J, Hes1, and Hes2 in olfactory bulb in the three groups. (c) Western blot analysis and shading analysis of Notch1, RBP-J, Hes1, and Hes2 in DG in Control, high-dose Pb exposure, DAPT, and high-dose Pb exposure+DAPT groups. (d) Freeze time during the cue and contextual fear conditioning test in the four groups. (e) Swimming speed, Platform+Edge frequency, and Lantency period during Morris water maze test in the four groups. *P < 0.05,**P < 0.01.
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