Journal: Nature Communications
Article Title: Macrophages induce AKT/β-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF
Figure Lengend Snippet: TNF-α is sufficient to induce HF TAT and is crucial for WIHN. ( a , b ) Intracutaneous injection of TNF-α can induce the HF telogen–anagen transition at the injection site in 7-week (W)-old mice (refractory phase) and 9-week-old mice (competent phase) ( a ), and the number of TNF-α-induced anagen hair follicles in the two different group was quantified ( b ). Seven-week-old mice, n =6 for each group; 9-week-old mice, n =8 for each group. ( c , d ) Wounding to the skin induced more anagen HFs in 9-week-old mice than in 7-week-old mice ( c ), and the number of anagen HFs in the two different groups was quantified ( d ). n =7 for each age group. ( e , f ) WIH-A analysis in TNFR1 −/− mice, TNFR2 −/− mice and WT mice ( e ), and the number of anagen HFs in different groups was quantified ( f ). Wild-type (C57BL/6) mice, n =7; TNFR1 −/− mice, n =6; TNFR2 −/− mice, n =9. ( g , h ) Wound-induced anagen HFs in mice constitutively expressing TNF-α (Tg-TNF-α) were significantly increased compared with WT mice, and the number of anagen HFs in the two different groups was quantified ( h ). Control mice (WT), n =6; Tg-TNF-α mice, n =5. ( i ) Bioluminescent imaging of Tnf-Luc-eGFP mice at different days (D) after wounding showed changes in the TNF-α level in the wound. Areas with high TNF-α levels (green/red) shifted from the wound (W) periphery to the wound centre with the progression of wound healing. n =15 for Tnf-Luc-eGFP mice, and n =10 for wild-type (WT) mice. ( j ) IF analysis of sections from the PWD-14 wound showed the presence of F4/80 + macrophages in the wound, which largely co-localized with TNF-α. Scale bar, 50 μm. ( k , l ) WIHN analysis in WT, TNFA −/− and Tg-TNF-α mice at PWD-30 ( k ), and the number of neogenic HFs in wounds was quantified ( l ). n =6 for both wild-type and TNFA −/− mice; n =12 for Tg-TNF-α mice. Scale bars, 2 mm. Data are expressed as the mean±s.e.m. * P
Article Snippet: Immunostaining Frozen skin tissue sections were incubated with different primary antibodies at 4 °C overnight; antibodies included anti-CD49f-biotin (GoH3, 1:150, BioLegend), anti-Ki67 (20Raj1, 1:100, eBioscience), anti-Akt (GTX28932, phospho Ser473, 1:150, GeneTex), anti-Akt (Akt 1+2+3) (GTX121937, 1:150, GeneTex), anti-CD34-biotin (RAM34, 1:150, eBioscience), anti-Lgr-5 (ab137484, 1:200, Abcam), anti-CD45 (30-F11, 1:150, BioLegend), anti-CD45-Biotin (30-F11, 1:150, BioLegend), anti-F4/80-Biotin (BM8, 1:150, BioLegend), anti-F4/80 (BM8, 1:150, BioLegend), anti-Ly6C-Biotin (RB6-8C5, 1:200, BioLegend), anti-Ly6C (HK1.4, 1:200, BioLegend), anti-MHC II (14-4-4S, 1:200, eBioscience), anti-TNF-α (1F3F3D4, 1:100, eBioscience), anti-TNFR1-Biotin (55R-170, 1:100, BioLegend), anti-β-catenin (C2206, 1:150, Sigma) and anti-Phospho-β-Catenin (Ser552) (5651S, 1:100, CST Signaling).
Techniques: Injection, Mouse Assay, Expressing, Imaging