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goat anti spp1  (R&D Systems)


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    R&D Systems goat anti spp1
    a-b , Compared to control, Tyr-CreER T2 ;Braf V600E mice induced with tamoxifen at P2-4, showed prominent hair growth. Representative P56 skin samples are shown in ( a ). Anagen HFs are marked in (a) and quantified in ( b ). In b , at P44 ( n = 12 in control, n = 20 in mutant; P = 0.00218), at P56 ( n = 12 in control, n = 21 in mutant; P = 0.00000000804), at P69 ( n = 12 in control, n = 16 in mutant; P = 0.0000526), at P100 ( n = 12 in control, n = 16 in mutant; P = 0.00000662). c, d , Following tamoxifen-induction at P21-25, Tyr-CreER T2 ;Braf V600E mice developed nevi by P44 and started to display ectopic hair growth from P56 onward. Representative wholemount and histology samples at five time points between P44-100 are shown in ( c ), and anagen HF density quantification is shown in ( d ). In d , n = 9. e , On ELISA, <t>SPP1</t> levels became significantly higher in the supernatant from day 5 cultures of primary sorted Tyr-CreER T2 ;Braf V600E melanocytes at five indicated timepoints from P56 onward. Data from P69 cells is also shown in main Fig. . In e , at P44 ( n = 4; P = 0.2686), at P56 ( n = 4; P = 0.0000269), at P62 ( n = 4; P = 0.003095), at P69 ( n = 4; P = 0.00224), at P100 ( n = 4; P = 0.0035). f-i , On cytometry, SPP1 levels in permeabilized cells ( f, h ) as well as surface-bound SPP1 levels in non-permeabilized cells ( g, i ) were significantly higher in melanocytes from induced Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf) compared to wild type control mice (pWT, sWT) at indicated time points. Representative cytometry plots are shown in ( f, g ) and quantification is show in ( h, i ). Data from P69 cells is also shown in main Fig. . In h , at P44 ( n = 3 for pWT, n = 4 for pBraf; P = 0.000318), at P56 ( n = 3; P = 0.0000533), at P62 ( n = 3; P = 0.00426), at P69 ( n = 3; P = 0.001397), at P100 ( n = 3 for pWT, n = 4 for pBraf; P = 0.00000386). In i , at P44 ( n = 3; P = 0.0531), at P56 ( n = 3; P = 0.0912), at P62 ( n = 3; P = 0.2495), at P69 ( n = 3; P = 0.291), at P100 ( n = 3; P = 0.00399). j–k , Cytometry of permeabilized ( j ) and non-permeabilized melanocytes ( k ) showed significantly higher levels of SPP1 compared to isotype control both in Tyr-Nras Q61K mice (pNras, sNras) and Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf). Representative cytometry plots are shown on the left and quantification on the right of j and k . In j , for pNras ( n = 3; P = 0.00000175), for pBraf ( n = 3; P = 0.00000213). In k , for sNras ( n = 3; P = 0.00297), for sBraf ( n = 3; P = 0.000000536). l , Skin of P69 Tyr-CreER T2 ;Braf V600E mice contained Trp2 + /Spp1 + melanocytes in upper dermis adjacent to bulge regions of HFs. In b, d , n = biologically independent samples. In e, h, i, j, k , n = independent experiments. Data are mean ± s.d. P values are calculated using unpaired one-tailed (in b at P56) or two-tailed (in b at P44, P69, P100, e, h, i, j, k ) Student’s t-test. NS, P ≥ 0.05, * P ≤ 0.05, ** P ≤ 0.01. Scale bars, a (wholemount) – 1 mm; c (wholemount) – 300 μm; c (histology) – 200 μm; a (histology), l – 100 μm.
    Goat Anti Spp1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Signalling by senescent melanocytes hyperactivates hair growth"

    Article Title: Signalling by senescent melanocytes hyperactivates hair growth

    Journal: Nature

    doi: 10.1038/s41586-023-06172-8

    a-b , Compared to control, Tyr-CreER T2 ;Braf V600E mice induced with tamoxifen at P2-4, showed prominent hair growth. Representative P56 skin samples are shown in ( a ). Anagen HFs are marked in (a) and quantified in ( b ). In b , at P44 ( n = 12 in control, n = 20 in mutant; P = 0.00218), at P56 ( n = 12 in control, n = 21 in mutant; P = 0.00000000804), at P69 ( n = 12 in control, n = 16 in mutant; P = 0.0000526), at P100 ( n = 12 in control, n = 16 in mutant; P = 0.00000662). c, d , Following tamoxifen-induction at P21-25, Tyr-CreER T2 ;Braf V600E mice developed nevi by P44 and started to display ectopic hair growth from P56 onward. Representative wholemount and histology samples at five time points between P44-100 are shown in ( c ), and anagen HF density quantification is shown in ( d ). In d , n = 9. e , On ELISA, SPP1 levels became significantly higher in the supernatant from day 5 cultures of primary sorted Tyr-CreER T2 ;Braf V600E melanocytes at five indicated timepoints from P56 onward. Data from P69 cells is also shown in main Fig. . In e , at P44 ( n = 4; P = 0.2686), at P56 ( n = 4; P = 0.0000269), at P62 ( n = 4; P = 0.003095), at P69 ( n = 4; P = 0.00224), at P100 ( n = 4; P = 0.0035). f-i , On cytometry, SPP1 levels in permeabilized cells ( f, h ) as well as surface-bound SPP1 levels in non-permeabilized cells ( g, i ) were significantly higher in melanocytes from induced Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf) compared to wild type control mice (pWT, sWT) at indicated time points. Representative cytometry plots are shown in ( f, g ) and quantification is show in ( h, i ). Data from P69 cells is also shown in main Fig. . In h , at P44 ( n = 3 for pWT, n = 4 for pBraf; P = 0.000318), at P56 ( n = 3; P = 0.0000533), at P62 ( n = 3; P = 0.00426), at P69 ( n = 3; P = 0.001397), at P100 ( n = 3 for pWT, n = 4 for pBraf; P = 0.00000386). In i , at P44 ( n = 3; P = 0.0531), at P56 ( n = 3; P = 0.0912), at P62 ( n = 3; P = 0.2495), at P69 ( n = 3; P = 0.291), at P100 ( n = 3; P = 0.00399). j–k , Cytometry of permeabilized ( j ) and non-permeabilized melanocytes ( k ) showed significantly higher levels of SPP1 compared to isotype control both in Tyr-Nras Q61K mice (pNras, sNras) and Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf). Representative cytometry plots are shown on the left and quantification on the right of j and k . In j , for pNras ( n = 3; P = 0.00000175), for pBraf ( n = 3; P = 0.00000213). In k , for sNras ( n = 3; P = 0.00297), for sBraf ( n = 3; P = 0.000000536). l , Skin of P69 Tyr-CreER T2 ;Braf V600E mice contained Trp2 + /Spp1 + melanocytes in upper dermis adjacent to bulge regions of HFs. In b, d , n = biologically independent samples. In e, h, i, j, k , n = independent experiments. Data are mean ± s.d. P values are calculated using unpaired one-tailed (in b at P56) or two-tailed (in b at P44, P69, P100, e, h, i, j, k ) Student’s t-test. NS, P ≥ 0.05, * P ≤ 0.05, ** P ≤ 0.01. Scale bars, a (wholemount) – 1 mm; c (wholemount) – 300 μm; c (histology) – 200 μm; a (histology), l – 100 μm.
    Figure Legend Snippet: a-b , Compared to control, Tyr-CreER T2 ;Braf V600E mice induced with tamoxifen at P2-4, showed prominent hair growth. Representative P56 skin samples are shown in ( a ). Anagen HFs are marked in (a) and quantified in ( b ). In b , at P44 ( n = 12 in control, n = 20 in mutant; P = 0.00218), at P56 ( n = 12 in control, n = 21 in mutant; P = 0.00000000804), at P69 ( n = 12 in control, n = 16 in mutant; P = 0.0000526), at P100 ( n = 12 in control, n = 16 in mutant; P = 0.00000662). c, d , Following tamoxifen-induction at P21-25, Tyr-CreER T2 ;Braf V600E mice developed nevi by P44 and started to display ectopic hair growth from P56 onward. Representative wholemount and histology samples at five time points between P44-100 are shown in ( c ), and anagen HF density quantification is shown in ( d ). In d , n = 9. e , On ELISA, SPP1 levels became significantly higher in the supernatant from day 5 cultures of primary sorted Tyr-CreER T2 ;Braf V600E melanocytes at five indicated timepoints from P56 onward. Data from P69 cells is also shown in main Fig. . In e , at P44 ( n = 4; P = 0.2686), at P56 ( n = 4; P = 0.0000269), at P62 ( n = 4; P = 0.003095), at P69 ( n = 4; P = 0.00224), at P100 ( n = 4; P = 0.0035). f-i , On cytometry, SPP1 levels in permeabilized cells ( f, h ) as well as surface-bound SPP1 levels in non-permeabilized cells ( g, i ) were significantly higher in melanocytes from induced Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf) compared to wild type control mice (pWT, sWT) at indicated time points. Representative cytometry plots are shown in ( f, g ) and quantification is show in ( h, i ). Data from P69 cells is also shown in main Fig. . In h , at P44 ( n = 3 for pWT, n = 4 for pBraf; P = 0.000318), at P56 ( n = 3; P = 0.0000533), at P62 ( n = 3; P = 0.00426), at P69 ( n = 3; P = 0.001397), at P100 ( n = 3 for pWT, n = 4 for pBraf; P = 0.00000386). In i , at P44 ( n = 3; P = 0.0531), at P56 ( n = 3; P = 0.0912), at P62 ( n = 3; P = 0.2495), at P69 ( n = 3; P = 0.291), at P100 ( n = 3; P = 0.00399). j–k , Cytometry of permeabilized ( j ) and non-permeabilized melanocytes ( k ) showed significantly higher levels of SPP1 compared to isotype control both in Tyr-Nras Q61K mice (pNras, sNras) and Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf). Representative cytometry plots are shown on the left and quantification on the right of j and k . In j , for pNras ( n = 3; P = 0.00000175), for pBraf ( n = 3; P = 0.00000213). In k , for sNras ( n = 3; P = 0.00297), for sBraf ( n = 3; P = 0.000000536). l , Skin of P69 Tyr-CreER T2 ;Braf V600E mice contained Trp2 + /Spp1 + melanocytes in upper dermis adjacent to bulge regions of HFs. In b, d , n = biologically independent samples. In e, h, i, j, k , n = independent experiments. Data are mean ± s.d. P values are calculated using unpaired one-tailed (in b at P56) or two-tailed (in b at P44, P69, P100, e, h, i, j, k ) Student’s t-test. NS, P ≥ 0.05, * P ≤ 0.05, ** P ≤ 0.01. Scale bars, a (wholemount) – 1 mm; c (wholemount) – 300 μm; c (histology) – 200 μm; a (histology), l – 100 μm.

    Techniques Used: Mutagenesis, Enzyme-linked Immunosorbent Assay, Cytometry, One-tailed Test, Two Tailed Test

    a , d , On cytometry, SPP1 was increased in P56 Tyr-Nras Q61K ( a ) and P69 Tyr-CreER T2 ; Braf V600E ( d ) melanocytes. In a , for the permeabilized condition, n = 3 in WT and n = 5 in Tyr-Nras Q61K ( P = 0.000000115); for the surface-bound condition, n = 3 in WT and n = 5 in Tyr-Nras Q61K ( P = 0.0257). In d , for the permeabilized condition, n = 3 ( P = 0.001397); for the surface-bound condition, n = 3 ( P = 0.2888). See Extended Data Fig. . c , f , On western blot, SPP1 levels were increased in P56 Tyr-Nras Q61K ( c ) and P69 Tyr-CreER T2 ; Braf V600E ( f ) melanocytes. In c , n = 3; P = 0.00784. In f , n = 3; P = 0.0109. Uncropped gels are shown in Supplementary Fig. . b , e , On ELISA, SPP1 levels increased in day 5 cultures of P56 Tyr-Nras Q61K ( b ) and P69 Tyr-CreER T2 ;Braf V600E ( e ) melanocytes. In b , n = 3 in WT and n = 4 in Tyr-Nras Q61K ; P = 0.00072. In e , n = 4; P = 0.00224. See Extended Data Fig. . g , Unlike WT, Tyr-Nras Q61K skin contained Trp2 + Spp1 + melanocytes adjacent to HF bulges. h , Anagen HF quantification in Tyr-Nras Q61K ; Spp1 −/− versus Tyr-Nras Q61K ; Spp1 +/− control mice. At P44, n = 12 in control and n = 14 in Tyr-Nras Q61K ; Spp1 −/− ( P = 0.0000000191); at P56, n = 12 in control and n = 15 in Tyr-Nras Q61K ; Spp1 −/− ( P = 0.0000195). i , Tyr-CreER T2 ; Braf V600E ; Spp1 fl/fl mice showed hair cycle quiescence rescue. Representative samples (left) and quantification (right) are displayed. n = 9; P = 0.000731. Arrowheads mark anagen HFs. j , On ELISA, SPP1 levels were reduced in day 5 cultures of Tyr-CreER T2 ; Braf V600E ; Spp1 fl/fl versus Tyr-CreER T2 ; Braf V600E melanocytes. n = 4; P = 0.00242. k , Spp1 −/− mice showed reduced wound-induced hair growth. Representative samples (left) and quantification (right) are displayed. n = 8 in WT and n = 7 in Spp1 −/− ; P = 0.0000575. l , Unlike BSA-soaked beads (blue), SPP1-soaked beads induced anagen in WT skin 12 days after injection. Representative samples (left) and quantification (right) are displayed. n = 5; P = 0.00562. m , n , Unlike control, doxycycline (dox)-treated P54 Tyr-rtTA ; tetO-Spp1 mice displayed premature anagen. Representative mice ( m ) and quantification ( n ) are displayed. In n , n = 9; P = 0.000000377. In b , c , e , f , j , n refers to independent experiments. In a , d , h , i , k , l , n , n refers to biologically independent samples. Data are mean ± s.d. P values were calculated using unpaired two-tailed Student’s t -test. NS, P ≥ 0.05, * P ≤ 0.05 and ** P ≤ 0.01. Scale bars, 100 μm ( g ), 200 μm (histology; i , m ) and 500 μm (wholemount; i , k , l , m ).
    Figure Legend Snippet: a , d , On cytometry, SPP1 was increased in P56 Tyr-Nras Q61K ( a ) and P69 Tyr-CreER T2 ; Braf V600E ( d ) melanocytes. In a , for the permeabilized condition, n = 3 in WT and n = 5 in Tyr-Nras Q61K ( P = 0.000000115); for the surface-bound condition, n = 3 in WT and n = 5 in Tyr-Nras Q61K ( P = 0.0257). In d , for the permeabilized condition, n = 3 ( P = 0.001397); for the surface-bound condition, n = 3 ( P = 0.2888). See Extended Data Fig. . c , f , On western blot, SPP1 levels were increased in P56 Tyr-Nras Q61K ( c ) and P69 Tyr-CreER T2 ; Braf V600E ( f ) melanocytes. In c , n = 3; P = 0.00784. In f , n = 3; P = 0.0109. Uncropped gels are shown in Supplementary Fig. . b , e , On ELISA, SPP1 levels increased in day 5 cultures of P56 Tyr-Nras Q61K ( b ) and P69 Tyr-CreER T2 ;Braf V600E ( e ) melanocytes. In b , n = 3 in WT and n = 4 in Tyr-Nras Q61K ; P = 0.00072. In e , n = 4; P = 0.00224. See Extended Data Fig. . g , Unlike WT, Tyr-Nras Q61K skin contained Trp2 + Spp1 + melanocytes adjacent to HF bulges. h , Anagen HF quantification in Tyr-Nras Q61K ; Spp1 −/− versus Tyr-Nras Q61K ; Spp1 +/− control mice. At P44, n = 12 in control and n = 14 in Tyr-Nras Q61K ; Spp1 −/− ( P = 0.0000000191); at P56, n = 12 in control and n = 15 in Tyr-Nras Q61K ; Spp1 −/− ( P = 0.0000195). i , Tyr-CreER T2 ; Braf V600E ; Spp1 fl/fl mice showed hair cycle quiescence rescue. Representative samples (left) and quantification (right) are displayed. n = 9; P = 0.000731. Arrowheads mark anagen HFs. j , On ELISA, SPP1 levels were reduced in day 5 cultures of Tyr-CreER T2 ; Braf V600E ; Spp1 fl/fl versus Tyr-CreER T2 ; Braf V600E melanocytes. n = 4; P = 0.00242. k , Spp1 −/− mice showed reduced wound-induced hair growth. Representative samples (left) and quantification (right) are displayed. n = 8 in WT and n = 7 in Spp1 −/− ; P = 0.0000575. l , Unlike BSA-soaked beads (blue), SPP1-soaked beads induced anagen in WT skin 12 days after injection. Representative samples (left) and quantification (right) are displayed. n = 5; P = 0.00562. m , n , Unlike control, doxycycline (dox)-treated P54 Tyr-rtTA ; tetO-Spp1 mice displayed premature anagen. Representative mice ( m ) and quantification ( n ) are displayed. In n , n = 9; P = 0.000000377. In b , c , e , f , j , n refers to independent experiments. In a , d , h , i , k , l , n , n refers to biologically independent samples. Data are mean ± s.d. P values were calculated using unpaired two-tailed Student’s t -test. NS, P ≥ 0.05, * P ≤ 0.05 and ** P ≤ 0.01. Scale bars, 100 μm ( g ), 200 μm (histology; i , m ) and 500 μm (wholemount; i , k , l , m ).

    Techniques Used: Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Two Tailed Test

    a-c , Osteopontin expression is increased in Tyr-Nras Q61K skin. a , Spp1 reporter activity was increased in Tyr-Nras Q61K skin. LacZ staining (blue) on Tyr-Nras Q61K ;Spp1 +/− vs . control Spp1 +/− P56 reporter mouse skin showed broad increase in LacZ + cells. Dermal and dermal papilla (DP) expression sites are marked. For each panel, representative wholemount and histology samples are shown on the left and on the right, respectively. b–c , Co-immunostaining for KRT5 (red) and SPP1 (green) in P56 WT control ( b ) and Tyr-Nras Q61K skin (c) . Tyr-Nras Q61K skin showed prominently increased SPP1 expression in the dermal compartment, including around bulge regions of HFs (inserts). d-g , Osteopontin deletion rescues hair cycle quiescence in Tyr-Nras Q61K mice. Tyr-Nras Q61K ;Spp1 −/− mice showed rescue of hair cycle quiescence. Unlike Tyr-Nras Q61K mice (see Extended Data Fig. ), Tyr-Nras Q61K ;Spp1 −/− mice showed synchronized catagen at P18 ( d ), synchronized anagen at P30 ( e ), synchronized telogen at P44 ( f ) and largely synchronized telogen P52 ( g ). For each time point, representative Spp1 -/- control and Tyr-Nras Q61K ;Spp1 -/- mutant skin samples are shown. Wholemount samples are shown on the right and histology on the left of each panel. Scale bars, b, c – 100 μm; a, d–g (wholemount) – 500 μm; a, d–g (histology) – 200 μm.
    Figure Legend Snippet: a-c , Osteopontin expression is increased in Tyr-Nras Q61K skin. a , Spp1 reporter activity was increased in Tyr-Nras Q61K skin. LacZ staining (blue) on Tyr-Nras Q61K ;Spp1 +/− vs . control Spp1 +/− P56 reporter mouse skin showed broad increase in LacZ + cells. Dermal and dermal papilla (DP) expression sites are marked. For each panel, representative wholemount and histology samples are shown on the left and on the right, respectively. b–c , Co-immunostaining for KRT5 (red) and SPP1 (green) in P56 WT control ( b ) and Tyr-Nras Q61K skin (c) . Tyr-Nras Q61K skin showed prominently increased SPP1 expression in the dermal compartment, including around bulge regions of HFs (inserts). d-g , Osteopontin deletion rescues hair cycle quiescence in Tyr-Nras Q61K mice. Tyr-Nras Q61K ;Spp1 −/− mice showed rescue of hair cycle quiescence. Unlike Tyr-Nras Q61K mice (see Extended Data Fig. ), Tyr-Nras Q61K ;Spp1 −/− mice showed synchronized catagen at P18 ( d ), synchronized anagen at P30 ( e ), synchronized telogen at P44 ( f ) and largely synchronized telogen P52 ( g ). For each time point, representative Spp1 -/- control and Tyr-Nras Q61K ;Spp1 -/- mutant skin samples are shown. Wholemount samples are shown on the right and histology on the left of each panel. Scale bars, b, c – 100 μm; a, d–g (wholemount) – 500 μm; a, d–g (histology) – 200 μm.

    Techniques Used: Expressing, Activity Assay, Staining, Immunostaining, Mutagenesis

    a , b , Epithelial HF cells in both WT control ( a ) and Tyr-Nras Q61K ( b ) mice strongly expressed CD44. Samples were also stained for the epithelial keratin marker KRT14. c , d , Co-staining for SPP1 and CD44 in Tyr-Nras Q61K ( c ) and Tyr-CreER T2 ; Braf V600E ( d ) skin revealed SPP1 high clusters of dermal cells adjacent to CD44 + bulge cells with weaker colocalizing SPP1 signal (yellow arrows). e , Cd44 −/− mice showed significantly reduced anagen activation in response to SPP1-soaked beads compared with WT mice. Representative samples (left) and quantification (right) are displayed. n = 5; P = 0.00938. f , Cd44 −/− mice showed reduced wound-induced hair growth compared with WT mice. Representative samples (left) and quantification (right) are displayed. n = 6 in WT and n = 5 in Cd44 −/− ; P = 0.0494. g , h , Tyr-Nras Q61K ; CD44 −/− mice lacking Cd44 showed rescue of hair cycle quiescence. At P44, Tyr-Nras Q61K ; Cd44 −/− HFs were in coordinated telogen ( g ). Only rare anagen HFs (arrowheads) were present at P52 ( h ). i , Quantification of anagen HFs in Tyr-Nras Q61K versus Tyr-Nras Q61K ; Cd44 −/− mice. Double mutants showed reduced ectopic anagen at P44 and P52. At P44, n = 12 and P = 0.00000000249; at P56, n = 12 and P = 0.0000166. j , k , Both constitutive epithelial-specific K14-Cre ; Cd44 fl/fl ( j ) and tamoxifen-induced K14-CreER T ; Cd44 fl/fl ( k ) mice showed significantly reduced anagen activation in response to SPP1-soaked beads compared with control mice. Representative samples (left) and quantification (right) are displayed. In j , n = 4 in control and n = 6 in mutant; P = 0.0352. In k , n = 4 in control and n = 3 in induced mutant; P = 0.0476. In e , f , i – k , n refers to biologically independent samples. P values are calculated using unpaired two-tailed Student’s t -test. * P ≤ 0.05 and ** P ≤ 0.01. Scale bars, 50 μm ( c , d ), 100 μm ( a , b ), 200 μm (histology; g , h ), 300 μm ( j , k ), 500 μm ( e , f ) and 1 mm (wholemount; g , h ).
    Figure Legend Snippet: a , b , Epithelial HF cells in both WT control ( a ) and Tyr-Nras Q61K ( b ) mice strongly expressed CD44. Samples were also stained for the epithelial keratin marker KRT14. c , d , Co-staining for SPP1 and CD44 in Tyr-Nras Q61K ( c ) and Tyr-CreER T2 ; Braf V600E ( d ) skin revealed SPP1 high clusters of dermal cells adjacent to CD44 + bulge cells with weaker colocalizing SPP1 signal (yellow arrows). e , Cd44 −/− mice showed significantly reduced anagen activation in response to SPP1-soaked beads compared with WT mice. Representative samples (left) and quantification (right) are displayed. n = 5; P = 0.00938. f , Cd44 −/− mice showed reduced wound-induced hair growth compared with WT mice. Representative samples (left) and quantification (right) are displayed. n = 6 in WT and n = 5 in Cd44 −/− ; P = 0.0494. g , h , Tyr-Nras Q61K ; CD44 −/− mice lacking Cd44 showed rescue of hair cycle quiescence. At P44, Tyr-Nras Q61K ; Cd44 −/− HFs were in coordinated telogen ( g ). Only rare anagen HFs (arrowheads) were present at P52 ( h ). i , Quantification of anagen HFs in Tyr-Nras Q61K versus Tyr-Nras Q61K ; Cd44 −/− mice. Double mutants showed reduced ectopic anagen at P44 and P52. At P44, n = 12 and P = 0.00000000249; at P56, n = 12 and P = 0.0000166. j , k , Both constitutive epithelial-specific K14-Cre ; Cd44 fl/fl ( j ) and tamoxifen-induced K14-CreER T ; Cd44 fl/fl ( k ) mice showed significantly reduced anagen activation in response to SPP1-soaked beads compared with control mice. Representative samples (left) and quantification (right) are displayed. In j , n = 4 in control and n = 6 in mutant; P = 0.0352. In k , n = 4 in control and n = 3 in induced mutant; P = 0.0476. In e , f , i – k , n refers to biologically independent samples. P values are calculated using unpaired two-tailed Student’s t -test. * P ≤ 0.05 and ** P ≤ 0.01. Scale bars, 50 μm ( c , d ), 100 μm ( a , b ), 200 μm (histology; g , h ), 300 μm ( j , k ), 500 μm ( e , f ) and 1 mm (wholemount; g , h ).

    Techniques Used: Staining, Marker, Activation Assay, Mutagenesis, Two Tailed Test

    a , Bulk RNA-seq reveals prominent differences between hairy nevi and adjacent normal facial skin in humans. A principal component analysis plot is shown. See Extended Data Fig. . b , Selected upregulated (by 2× or more; green) and downregulated (by 2× or more; red) differentially expressed genes in nevus versus normal human skin. Bold and underlined genes were validated by qRT-PCR. c , qRT–PCR of selected differentially expressed genes from bulk RNA-seq data. n = 3. d , e , SPP1 and TRP2 co-staining. In normal skin, TRP2 + melanocytes did not express SPP1 ( d ), whereas in nevus skin, clusters of TRP2 + SPP1 + cells were seen next to HF bulge regions ( e ). f , SPP1 and SOX10 co-staining. Nevus skin contained SOX10 + SPP1 + cell clusters next to HF bulge regions. g , h , SPP1 and KRT5 co-staining. Unlike in normal skin ( g ), SPP1 + cell clusters were seen next to HFs in nevus human skin ( h ). i , j , SPP1 microinjections induced precocious growth by human scalp HFs (arrowheads). Representative samples of human HFs on day 50 post-grafting ( i ) and quantification of human HFs in anagen ( j ) are shown. In j , n = 7 for control and n = 11 for SPP1; P = 0.00034. In c , n refers to independent experiments. In j , n refers to biologically independent samples. P values were calculated using unpaired two-tailed Student’s t -test. ** P ≤ 0.01. Scale bars, 100 μm ( d – h ) and 1 mm ( i ).
    Figure Legend Snippet: a , Bulk RNA-seq reveals prominent differences between hairy nevi and adjacent normal facial skin in humans. A principal component analysis plot is shown. See Extended Data Fig. . b , Selected upregulated (by 2× or more; green) and downregulated (by 2× or more; red) differentially expressed genes in nevus versus normal human skin. Bold and underlined genes were validated by qRT-PCR. c , qRT–PCR of selected differentially expressed genes from bulk RNA-seq data. n = 3. d , e , SPP1 and TRP2 co-staining. In normal skin, TRP2 + melanocytes did not express SPP1 ( d ), whereas in nevus skin, clusters of TRP2 + SPP1 + cells were seen next to HF bulge regions ( e ). f , SPP1 and SOX10 co-staining. Nevus skin contained SOX10 + SPP1 + cell clusters next to HF bulge regions. g , h , SPP1 and KRT5 co-staining. Unlike in normal skin ( g ), SPP1 + cell clusters were seen next to HFs in nevus human skin ( h ). i , j , SPP1 microinjections induced precocious growth by human scalp HFs (arrowheads). Representative samples of human HFs on day 50 post-grafting ( i ) and quantification of human HFs in anagen ( j ) are shown. In j , n = 7 for control and n = 11 for SPP1; P = 0.00034. In c , n refers to independent experiments. In j , n refers to biologically independent samples. P values were calculated using unpaired two-tailed Student’s t -test. ** P ≤ 0.01. Scale bars, 100 μm ( d – h ) and 1 mm ( i ).

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Staining, Two Tailed Test

    a , Limited accumulation of senescent cells adjacent to normal, intact stem cell niche can augment it and result in stem cell activation. Mechanism, effects, and examples are listed below the schematic drawing. Tissue stem cells – orange (quiescent) and green (activated); normal niche cells – blue; senescent cells – purple. b , Schematic representation of the mechanism driving hair growth hyperactivation in skin nevus. Dermal clusters of senescent melanocytes (purple) secrete SASP factors (colored geometric shapes). SPP1 (blue squares) is the leading SASP factor of senescent melanocytes. It signals via CD44 receptor (black Y shape) to epithelial stem cells in adjacent hair follicles, inducing them into precocious growth (green arrow).
    Figure Legend Snippet: a , Limited accumulation of senescent cells adjacent to normal, intact stem cell niche can augment it and result in stem cell activation. Mechanism, effects, and examples are listed below the schematic drawing. Tissue stem cells – orange (quiescent) and green (activated); normal niche cells – blue; senescent cells – purple. b , Schematic representation of the mechanism driving hair growth hyperactivation in skin nevus. Dermal clusters of senescent melanocytes (purple) secrete SASP factors (colored geometric shapes). SPP1 (blue squares) is the leading SASP factor of senescent melanocytes. It signals via CD44 receptor (black Y shape) to epithelial stem cells in adjacent hair follicles, inducing them into precocious growth (green arrow).

    Techniques Used: Activation Assay



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    a-b , Compared to control, Tyr-CreER T2 ;Braf V600E mice induced with tamoxifen at P2-4, showed prominent hair growth. Representative P56 skin samples are shown in ( a ). Anagen HFs are marked in (a) and quantified in ( b ). In b , at P44 ( n = 12 in control, n = 20 in mutant; P = 0.00218), at P56 ( n = 12 in control, n = 21 in mutant; P = 0.00000000804), at P69 ( n = 12 in control, n = 16 in mutant; P = 0.0000526), at P100 ( n = 12 in control, n = 16 in mutant; P = 0.00000662). c, d , Following tamoxifen-induction at P21-25, Tyr-CreER T2 ;Braf V600E mice developed nevi by P44 and started to display ectopic hair growth from P56 onward. Representative wholemount and histology samples at five time points between P44-100 are shown in ( c ), and anagen HF density quantification is shown in ( d ). In d , n = 9. e , On ELISA, <t>SPP1</t> levels became significantly higher in the supernatant from day 5 cultures of primary sorted Tyr-CreER T2 ;Braf V600E melanocytes at five indicated timepoints from P56 onward. Data from P69 cells is also shown in main Fig. . In e , at P44 ( n = 4; P = 0.2686), at P56 ( n = 4; P = 0.0000269), at P62 ( n = 4; P = 0.003095), at P69 ( n = 4; P = 0.00224), at P100 ( n = 4; P = 0.0035). f-i , On cytometry, SPP1 levels in permeabilized cells ( f, h ) as well as surface-bound SPP1 levels in non-permeabilized cells ( g, i ) were significantly higher in melanocytes from induced Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf) compared to wild type control mice (pWT, sWT) at indicated time points. Representative cytometry plots are shown in ( f, g ) and quantification is show in ( h, i ). Data from P69 cells is also shown in main Fig. . In h , at P44 ( n = 3 for pWT, n = 4 for pBraf; P = 0.000318), at P56 ( n = 3; P = 0.0000533), at P62 ( n = 3; P = 0.00426), at P69 ( n = 3; P = 0.001397), at P100 ( n = 3 for pWT, n = 4 for pBraf; P = 0.00000386). In i , at P44 ( n = 3; P = 0.0531), at P56 ( n = 3; P = 0.0912), at P62 ( n = 3; P = 0.2495), at P69 ( n = 3; P = 0.291), at P100 ( n = 3; P = 0.00399). j–k , Cytometry of permeabilized ( j ) and non-permeabilized melanocytes ( k ) showed significantly higher levels of SPP1 compared to isotype control both in Tyr-Nras Q61K mice (pNras, sNras) and Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf). Representative cytometry plots are shown on the left and quantification on the right of j and k . In j , for pNras ( n = 3; P = 0.00000175), for pBraf ( n = 3; P = 0.00000213). In k , for sNras ( n = 3; P = 0.00297), for sBraf ( n = 3; P = 0.000000536). l , Skin of P69 Tyr-CreER T2 ;Braf V600E mice contained Trp2 + /Spp1 + melanocytes in upper dermis adjacent to bulge regions of HFs. In b, d , n = biologically independent samples. In e, h, i, j, k , n = independent experiments. Data are mean ± s.d. P values are calculated using unpaired one-tailed (in b at P56) or two-tailed (in b at P44, P69, P100, e, h, i, j, k ) Student’s t-test. NS, P ≥ 0.05, * P ≤ 0.05, ** P ≤ 0.01. Scale bars, a (wholemount) – 1 mm; c (wholemount) – 300 μm; c (histology) – 200 μm; a (histology), l – 100 μm.
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    a-b , Compared to control, Tyr-CreER T2 ;Braf V600E mice induced with tamoxifen at P2-4, showed prominent hair growth. Representative P56 skin samples are shown in ( a ). Anagen HFs are marked in (a) and quantified in ( b ). In b , at P44 ( n = 12 in control, n = 20 in mutant; P = 0.00218), at P56 ( n = 12 in control, n = 21 in mutant; P = 0.00000000804), at P69 ( n = 12 in control, n = 16 in mutant; P = 0.0000526), at P100 ( n = 12 in control, n = 16 in mutant; P = 0.00000662). c, d , Following tamoxifen-induction at P21-25, Tyr-CreER T2 ;Braf V600E mice developed nevi by P44 and started to display ectopic hair growth from P56 onward. Representative wholemount and histology samples at five time points between P44-100 are shown in ( c ), and anagen HF density quantification is shown in ( d ). In d , n = 9. e , On ELISA, SPP1 levels became significantly higher in the supernatant from day 5 cultures of primary sorted Tyr-CreER T2 ;Braf V600E melanocytes at five indicated timepoints from P56 onward. Data from P69 cells is also shown in main Fig. . In e , at P44 ( n = 4; P = 0.2686), at P56 ( n = 4; P = 0.0000269), at P62 ( n = 4; P = 0.003095), at P69 ( n = 4; P = 0.00224), at P100 ( n = 4; P = 0.0035). f-i , On cytometry, SPP1 levels in permeabilized cells ( f, h ) as well as surface-bound SPP1 levels in non-permeabilized cells ( g, i ) were significantly higher in melanocytes from induced Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf) compared to wild type control mice (pWT, sWT) at indicated time points. Representative cytometry plots are shown in ( f, g ) and quantification is show in ( h, i ). Data from P69 cells is also shown in main Fig. . In h , at P44 ( n = 3 for pWT, n = 4 for pBraf; P = 0.000318), at P56 ( n = 3; P = 0.0000533), at P62 ( n = 3; P = 0.00426), at P69 ( n = 3; P = 0.001397), at P100 ( n = 3 for pWT, n = 4 for pBraf; P = 0.00000386). In i , at P44 ( n = 3; P = 0.0531), at P56 ( n = 3; P = 0.0912), at P62 ( n = 3; P = 0.2495), at P69 ( n = 3; P = 0.291), at P100 ( n = 3; P = 0.00399). j–k , Cytometry of permeabilized ( j ) and non-permeabilized melanocytes ( k ) showed significantly higher levels of SPP1 compared to isotype control both in Tyr-Nras Q61K mice (pNras, sNras) and Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf). Representative cytometry plots are shown on the left and quantification on the right of j and k . In j , for pNras ( n = 3; P = 0.00000175), for pBraf ( n = 3; P = 0.00000213). In k , for sNras ( n = 3; P = 0.00297), for sBraf ( n = 3; P = 0.000000536). l , Skin of P69 Tyr-CreER T2 ;Braf V600E mice contained Trp2 + /Spp1 + melanocytes in upper dermis adjacent to bulge regions of HFs. In b, d , n = biologically independent samples. In e, h, i, j, k , n = independent experiments. Data are mean ± s.d. P values are calculated using unpaired one-tailed (in b at P56) or two-tailed (in b at P44, P69, P100, e, h, i, j, k ) Student’s t-test. NS, P ≥ 0.05, * P ≤ 0.05, ** P ≤ 0.01. Scale bars, a (wholemount) – 1 mm; c (wholemount) – 300 μm; c (histology) – 200 μm; a (histology), l – 100 μm.

    Journal: Nature

    Article Title: Signalling by senescent melanocytes hyperactivates hair growth

    doi: 10.1038/s41586-023-06172-8

    Figure Lengend Snippet: a-b , Compared to control, Tyr-CreER T2 ;Braf V600E mice induced with tamoxifen at P2-4, showed prominent hair growth. Representative P56 skin samples are shown in ( a ). Anagen HFs are marked in (a) and quantified in ( b ). In b , at P44 ( n = 12 in control, n = 20 in mutant; P = 0.00218), at P56 ( n = 12 in control, n = 21 in mutant; P = 0.00000000804), at P69 ( n = 12 in control, n = 16 in mutant; P = 0.0000526), at P100 ( n = 12 in control, n = 16 in mutant; P = 0.00000662). c, d , Following tamoxifen-induction at P21-25, Tyr-CreER T2 ;Braf V600E mice developed nevi by P44 and started to display ectopic hair growth from P56 onward. Representative wholemount and histology samples at five time points between P44-100 are shown in ( c ), and anagen HF density quantification is shown in ( d ). In d , n = 9. e , On ELISA, SPP1 levels became significantly higher in the supernatant from day 5 cultures of primary sorted Tyr-CreER T2 ;Braf V600E melanocytes at five indicated timepoints from P56 onward. Data from P69 cells is also shown in main Fig. . In e , at P44 ( n = 4; P = 0.2686), at P56 ( n = 4; P = 0.0000269), at P62 ( n = 4; P = 0.003095), at P69 ( n = 4; P = 0.00224), at P100 ( n = 4; P = 0.0035). f-i , On cytometry, SPP1 levels in permeabilized cells ( f, h ) as well as surface-bound SPP1 levels in non-permeabilized cells ( g, i ) were significantly higher in melanocytes from induced Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf) compared to wild type control mice (pWT, sWT) at indicated time points. Representative cytometry plots are shown in ( f, g ) and quantification is show in ( h, i ). Data from P69 cells is also shown in main Fig. . In h , at P44 ( n = 3 for pWT, n = 4 for pBraf; P = 0.000318), at P56 ( n = 3; P = 0.0000533), at P62 ( n = 3; P = 0.00426), at P69 ( n = 3; P = 0.001397), at P100 ( n = 3 for pWT, n = 4 for pBraf; P = 0.00000386). In i , at P44 ( n = 3; P = 0.0531), at P56 ( n = 3; P = 0.0912), at P62 ( n = 3; P = 0.2495), at P69 ( n = 3; P = 0.291), at P100 ( n = 3; P = 0.00399). j–k , Cytometry of permeabilized ( j ) and non-permeabilized melanocytes ( k ) showed significantly higher levels of SPP1 compared to isotype control both in Tyr-Nras Q61K mice (pNras, sNras) and Tyr-CreER T2 ;Braf V600E mice (pBraf, sBraf). Representative cytometry plots are shown on the left and quantification on the right of j and k . In j , for pNras ( n = 3; P = 0.00000175), for pBraf ( n = 3; P = 0.00000213). In k , for sNras ( n = 3; P = 0.00297), for sBraf ( n = 3; P = 0.000000536). l , Skin of P69 Tyr-CreER T2 ;Braf V600E mice contained Trp2 + /Spp1 + melanocytes in upper dermis adjacent to bulge regions of HFs. In b, d , n = biologically independent samples. In e, h, i, j, k , n = independent experiments. Data are mean ± s.d. P values are calculated using unpaired one-tailed (in b at P56) or two-tailed (in b at P44, P69, P100, e, h, i, j, k ) Student’s t-test. NS, P ≥ 0.05, * P ≤ 0.05, ** P ≤ 0.01. Scale bars, a (wholemount) – 1 mm; c (wholemount) – 300 μm; c (histology) – 200 μm; a (histology), l – 100 μm.

    Article Snippet: The following primary antibodies were used: rabbit anti-γH2AX (1:300; 9718, Cell Signaling), rabbit anti-TRP2 (1:200; ab74073, Abcam), rabbit anti-TRP2 (1:200; ab103463, Abcam), mouse anti-PCNA (1:1,000; ab29, Abcam), rat anti-CD34 (1:100; 14-0341-82, Thermo Fisher), rabbit anti-SOX9 (1:200; AB5535, Millipore), goat anti-SPP1 (1:100; AF808, R&D), goat anti-SPP1 (1:300; AF1433, R&D), rabbit anti-KRT14 (1:2,000; ab119695, Abcam), rabbit anti-CD44 (1:100; PA5-94934, Thermo Fisher), rabbit anti-SOX10 (1:100; ab180862, Abcam), rabbit anti-KRT5 (1:1,000; 905501, BioLegend) and goat anti-Pcad (1:200; AF761, R&D Systems).

    Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Cytometry, One-tailed Test, Two Tailed Test

    a , d , On cytometry, SPP1 was increased in P56 Tyr-Nras Q61K ( a ) and P69 Tyr-CreER T2 ; Braf V600E ( d ) melanocytes. In a , for the permeabilized condition, n = 3 in WT and n = 5 in Tyr-Nras Q61K ( P = 0.000000115); for the surface-bound condition, n = 3 in WT and n = 5 in Tyr-Nras Q61K ( P = 0.0257). In d , for the permeabilized condition, n = 3 ( P = 0.001397); for the surface-bound condition, n = 3 ( P = 0.2888). See Extended Data Fig. . c , f , On western blot, SPP1 levels were increased in P56 Tyr-Nras Q61K ( c ) and P69 Tyr-CreER T2 ; Braf V600E ( f ) melanocytes. In c , n = 3; P = 0.00784. In f , n = 3; P = 0.0109. Uncropped gels are shown in Supplementary Fig. . b , e , On ELISA, SPP1 levels increased in day 5 cultures of P56 Tyr-Nras Q61K ( b ) and P69 Tyr-CreER T2 ;Braf V600E ( e ) melanocytes. In b , n = 3 in WT and n = 4 in Tyr-Nras Q61K ; P = 0.00072. In e , n = 4; P = 0.00224. See Extended Data Fig. . g , Unlike WT, Tyr-Nras Q61K skin contained Trp2 + Spp1 + melanocytes adjacent to HF bulges. h , Anagen HF quantification in Tyr-Nras Q61K ; Spp1 −/− versus Tyr-Nras Q61K ; Spp1 +/− control mice. At P44, n = 12 in control and n = 14 in Tyr-Nras Q61K ; Spp1 −/− ( P = 0.0000000191); at P56, n = 12 in control and n = 15 in Tyr-Nras Q61K ; Spp1 −/− ( P = 0.0000195). i , Tyr-CreER T2 ; Braf V600E ; Spp1 fl/fl mice showed hair cycle quiescence rescue. Representative samples (left) and quantification (right) are displayed. n = 9; P = 0.000731. Arrowheads mark anagen HFs. j , On ELISA, SPP1 levels were reduced in day 5 cultures of Tyr-CreER T2 ; Braf V600E ; Spp1 fl/fl versus Tyr-CreER T2 ; Braf V600E melanocytes. n = 4; P = 0.00242. k , Spp1 −/− mice showed reduced wound-induced hair growth. Representative samples (left) and quantification (right) are displayed. n = 8 in WT and n = 7 in Spp1 −/− ; P = 0.0000575. l , Unlike BSA-soaked beads (blue), SPP1-soaked beads induced anagen in WT skin 12 days after injection. Representative samples (left) and quantification (right) are displayed. n = 5; P = 0.00562. m , n , Unlike control, doxycycline (dox)-treated P54 Tyr-rtTA ; tetO-Spp1 mice displayed premature anagen. Representative mice ( m ) and quantification ( n ) are displayed. In n , n = 9; P = 0.000000377. In b , c , e , f , j , n refers to independent experiments. In a , d , h , i , k , l , n , n refers to biologically independent samples. Data are mean ± s.d. P values were calculated using unpaired two-tailed Student’s t -test. NS, P ≥ 0.05, * P ≤ 0.05 and ** P ≤ 0.01. Scale bars, 100 μm ( g ), 200 μm (histology; i , m ) and 500 μm (wholemount; i , k , l , m ).

    Journal: Nature

    Article Title: Signalling by senescent melanocytes hyperactivates hair growth

    doi: 10.1038/s41586-023-06172-8

    Figure Lengend Snippet: a , d , On cytometry, SPP1 was increased in P56 Tyr-Nras Q61K ( a ) and P69 Tyr-CreER T2 ; Braf V600E ( d ) melanocytes. In a , for the permeabilized condition, n = 3 in WT and n = 5 in Tyr-Nras Q61K ( P = 0.000000115); for the surface-bound condition, n = 3 in WT and n = 5 in Tyr-Nras Q61K ( P = 0.0257). In d , for the permeabilized condition, n = 3 ( P = 0.001397); for the surface-bound condition, n = 3 ( P = 0.2888). See Extended Data Fig. . c , f , On western blot, SPP1 levels were increased in P56 Tyr-Nras Q61K ( c ) and P69 Tyr-CreER T2 ; Braf V600E ( f ) melanocytes. In c , n = 3; P = 0.00784. In f , n = 3; P = 0.0109. Uncropped gels are shown in Supplementary Fig. . b , e , On ELISA, SPP1 levels increased in day 5 cultures of P56 Tyr-Nras Q61K ( b ) and P69 Tyr-CreER T2 ;Braf V600E ( e ) melanocytes. In b , n = 3 in WT and n = 4 in Tyr-Nras Q61K ; P = 0.00072. In e , n = 4; P = 0.00224. See Extended Data Fig. . g , Unlike WT, Tyr-Nras Q61K skin contained Trp2 + Spp1 + melanocytes adjacent to HF bulges. h , Anagen HF quantification in Tyr-Nras Q61K ; Spp1 −/− versus Tyr-Nras Q61K ; Spp1 +/− control mice. At P44, n = 12 in control and n = 14 in Tyr-Nras Q61K ; Spp1 −/− ( P = 0.0000000191); at P56, n = 12 in control and n = 15 in Tyr-Nras Q61K ; Spp1 −/− ( P = 0.0000195). i , Tyr-CreER T2 ; Braf V600E ; Spp1 fl/fl mice showed hair cycle quiescence rescue. Representative samples (left) and quantification (right) are displayed. n = 9; P = 0.000731. Arrowheads mark anagen HFs. j , On ELISA, SPP1 levels were reduced in day 5 cultures of Tyr-CreER T2 ; Braf V600E ; Spp1 fl/fl versus Tyr-CreER T2 ; Braf V600E melanocytes. n = 4; P = 0.00242. k , Spp1 −/− mice showed reduced wound-induced hair growth. Representative samples (left) and quantification (right) are displayed. n = 8 in WT and n = 7 in Spp1 −/− ; P = 0.0000575. l , Unlike BSA-soaked beads (blue), SPP1-soaked beads induced anagen in WT skin 12 days after injection. Representative samples (left) and quantification (right) are displayed. n = 5; P = 0.00562. m , n , Unlike control, doxycycline (dox)-treated P54 Tyr-rtTA ; tetO-Spp1 mice displayed premature anagen. Representative mice ( m ) and quantification ( n ) are displayed. In n , n = 9; P = 0.000000377. In b , c , e , f , j , n refers to independent experiments. In a , d , h , i , k , l , n , n refers to biologically independent samples. Data are mean ± s.d. P values were calculated using unpaired two-tailed Student’s t -test. NS, P ≥ 0.05, * P ≤ 0.05 and ** P ≤ 0.01. Scale bars, 100 μm ( g ), 200 μm (histology; i , m ) and 500 μm (wholemount; i , k , l , m ).

    Article Snippet: The following primary antibodies were used: rabbit anti-γH2AX (1:300; 9718, Cell Signaling), rabbit anti-TRP2 (1:200; ab74073, Abcam), rabbit anti-TRP2 (1:200; ab103463, Abcam), mouse anti-PCNA (1:1,000; ab29, Abcam), rat anti-CD34 (1:100; 14-0341-82, Thermo Fisher), rabbit anti-SOX9 (1:200; AB5535, Millipore), goat anti-SPP1 (1:100; AF808, R&D), goat anti-SPP1 (1:300; AF1433, R&D), rabbit anti-KRT14 (1:2,000; ab119695, Abcam), rabbit anti-CD44 (1:100; PA5-94934, Thermo Fisher), rabbit anti-SOX10 (1:100; ab180862, Abcam), rabbit anti-KRT5 (1:1,000; 905501, BioLegend) and goat anti-Pcad (1:200; AF761, R&D Systems).

    Techniques: Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Two Tailed Test

    a-c , Osteopontin expression is increased in Tyr-Nras Q61K skin. a , Spp1 reporter activity was increased in Tyr-Nras Q61K skin. LacZ staining (blue) on Tyr-Nras Q61K ;Spp1 +/− vs . control Spp1 +/− P56 reporter mouse skin showed broad increase in LacZ + cells. Dermal and dermal papilla (DP) expression sites are marked. For each panel, representative wholemount and histology samples are shown on the left and on the right, respectively. b–c , Co-immunostaining for KRT5 (red) and SPP1 (green) in P56 WT control ( b ) and Tyr-Nras Q61K skin (c) . Tyr-Nras Q61K skin showed prominently increased SPP1 expression in the dermal compartment, including around bulge regions of HFs (inserts). d-g , Osteopontin deletion rescues hair cycle quiescence in Tyr-Nras Q61K mice. Tyr-Nras Q61K ;Spp1 −/− mice showed rescue of hair cycle quiescence. Unlike Tyr-Nras Q61K mice (see Extended Data Fig. ), Tyr-Nras Q61K ;Spp1 −/− mice showed synchronized catagen at P18 ( d ), synchronized anagen at P30 ( e ), synchronized telogen at P44 ( f ) and largely synchronized telogen P52 ( g ). For each time point, representative Spp1 -/- control and Tyr-Nras Q61K ;Spp1 -/- mutant skin samples are shown. Wholemount samples are shown on the right and histology on the left of each panel. Scale bars, b, c – 100 μm; a, d–g (wholemount) – 500 μm; a, d–g (histology) – 200 μm.

    Journal: Nature

    Article Title: Signalling by senescent melanocytes hyperactivates hair growth

    doi: 10.1038/s41586-023-06172-8

    Figure Lengend Snippet: a-c , Osteopontin expression is increased in Tyr-Nras Q61K skin. a , Spp1 reporter activity was increased in Tyr-Nras Q61K skin. LacZ staining (blue) on Tyr-Nras Q61K ;Spp1 +/− vs . control Spp1 +/− P56 reporter mouse skin showed broad increase in LacZ + cells. Dermal and dermal papilla (DP) expression sites are marked. For each panel, representative wholemount and histology samples are shown on the left and on the right, respectively. b–c , Co-immunostaining for KRT5 (red) and SPP1 (green) in P56 WT control ( b ) and Tyr-Nras Q61K skin (c) . Tyr-Nras Q61K skin showed prominently increased SPP1 expression in the dermal compartment, including around bulge regions of HFs (inserts). d-g , Osteopontin deletion rescues hair cycle quiescence in Tyr-Nras Q61K mice. Tyr-Nras Q61K ;Spp1 −/− mice showed rescue of hair cycle quiescence. Unlike Tyr-Nras Q61K mice (see Extended Data Fig. ), Tyr-Nras Q61K ;Spp1 −/− mice showed synchronized catagen at P18 ( d ), synchronized anagen at P30 ( e ), synchronized telogen at P44 ( f ) and largely synchronized telogen P52 ( g ). For each time point, representative Spp1 -/- control and Tyr-Nras Q61K ;Spp1 -/- mutant skin samples are shown. Wholemount samples are shown on the right and histology on the left of each panel. Scale bars, b, c – 100 μm; a, d–g (wholemount) – 500 μm; a, d–g (histology) – 200 μm.

    Article Snippet: The following primary antibodies were used: rabbit anti-γH2AX (1:300; 9718, Cell Signaling), rabbit anti-TRP2 (1:200; ab74073, Abcam), rabbit anti-TRP2 (1:200; ab103463, Abcam), mouse anti-PCNA (1:1,000; ab29, Abcam), rat anti-CD34 (1:100; 14-0341-82, Thermo Fisher), rabbit anti-SOX9 (1:200; AB5535, Millipore), goat anti-SPP1 (1:100; AF808, R&D), goat anti-SPP1 (1:300; AF1433, R&D), rabbit anti-KRT14 (1:2,000; ab119695, Abcam), rabbit anti-CD44 (1:100; PA5-94934, Thermo Fisher), rabbit anti-SOX10 (1:100; ab180862, Abcam), rabbit anti-KRT5 (1:1,000; 905501, BioLegend) and goat anti-Pcad (1:200; AF761, R&D Systems).

    Techniques: Expressing, Activity Assay, Staining, Immunostaining, Mutagenesis

    a , b , Epithelial HF cells in both WT control ( a ) and Tyr-Nras Q61K ( b ) mice strongly expressed CD44. Samples were also stained for the epithelial keratin marker KRT14. c , d , Co-staining for SPP1 and CD44 in Tyr-Nras Q61K ( c ) and Tyr-CreER T2 ; Braf V600E ( d ) skin revealed SPP1 high clusters of dermal cells adjacent to CD44 + bulge cells with weaker colocalizing SPP1 signal (yellow arrows). e , Cd44 −/− mice showed significantly reduced anagen activation in response to SPP1-soaked beads compared with WT mice. Representative samples (left) and quantification (right) are displayed. n = 5; P = 0.00938. f , Cd44 −/− mice showed reduced wound-induced hair growth compared with WT mice. Representative samples (left) and quantification (right) are displayed. n = 6 in WT and n = 5 in Cd44 −/− ; P = 0.0494. g , h , Tyr-Nras Q61K ; CD44 −/− mice lacking Cd44 showed rescue of hair cycle quiescence. At P44, Tyr-Nras Q61K ; Cd44 −/− HFs were in coordinated telogen ( g ). Only rare anagen HFs (arrowheads) were present at P52 ( h ). i , Quantification of anagen HFs in Tyr-Nras Q61K versus Tyr-Nras Q61K ; Cd44 −/− mice. Double mutants showed reduced ectopic anagen at P44 and P52. At P44, n = 12 and P = 0.00000000249; at P56, n = 12 and P = 0.0000166. j , k , Both constitutive epithelial-specific K14-Cre ; Cd44 fl/fl ( j ) and tamoxifen-induced K14-CreER T ; Cd44 fl/fl ( k ) mice showed significantly reduced anagen activation in response to SPP1-soaked beads compared with control mice. Representative samples (left) and quantification (right) are displayed. In j , n = 4 in control and n = 6 in mutant; P = 0.0352. In k , n = 4 in control and n = 3 in induced mutant; P = 0.0476. In e , f , i – k , n refers to biologically independent samples. P values are calculated using unpaired two-tailed Student’s t -test. * P ≤ 0.05 and ** P ≤ 0.01. Scale bars, 50 μm ( c , d ), 100 μm ( a , b ), 200 μm (histology; g , h ), 300 μm ( j , k ), 500 μm ( e , f ) and 1 mm (wholemount; g , h ).

    Journal: Nature

    Article Title: Signalling by senescent melanocytes hyperactivates hair growth

    doi: 10.1038/s41586-023-06172-8

    Figure Lengend Snippet: a , b , Epithelial HF cells in both WT control ( a ) and Tyr-Nras Q61K ( b ) mice strongly expressed CD44. Samples were also stained for the epithelial keratin marker KRT14. c , d , Co-staining for SPP1 and CD44 in Tyr-Nras Q61K ( c ) and Tyr-CreER T2 ; Braf V600E ( d ) skin revealed SPP1 high clusters of dermal cells adjacent to CD44 + bulge cells with weaker colocalizing SPP1 signal (yellow arrows). e , Cd44 −/− mice showed significantly reduced anagen activation in response to SPP1-soaked beads compared with WT mice. Representative samples (left) and quantification (right) are displayed. n = 5; P = 0.00938. f , Cd44 −/− mice showed reduced wound-induced hair growth compared with WT mice. Representative samples (left) and quantification (right) are displayed. n = 6 in WT and n = 5 in Cd44 −/− ; P = 0.0494. g , h , Tyr-Nras Q61K ; CD44 −/− mice lacking Cd44 showed rescue of hair cycle quiescence. At P44, Tyr-Nras Q61K ; Cd44 −/− HFs were in coordinated telogen ( g ). Only rare anagen HFs (arrowheads) were present at P52 ( h ). i , Quantification of anagen HFs in Tyr-Nras Q61K versus Tyr-Nras Q61K ; Cd44 −/− mice. Double mutants showed reduced ectopic anagen at P44 and P52. At P44, n = 12 and P = 0.00000000249; at P56, n = 12 and P = 0.0000166. j , k , Both constitutive epithelial-specific K14-Cre ; Cd44 fl/fl ( j ) and tamoxifen-induced K14-CreER T ; Cd44 fl/fl ( k ) mice showed significantly reduced anagen activation in response to SPP1-soaked beads compared with control mice. Representative samples (left) and quantification (right) are displayed. In j , n = 4 in control and n = 6 in mutant; P = 0.0352. In k , n = 4 in control and n = 3 in induced mutant; P = 0.0476. In e , f , i – k , n refers to biologically independent samples. P values are calculated using unpaired two-tailed Student’s t -test. * P ≤ 0.05 and ** P ≤ 0.01. Scale bars, 50 μm ( c , d ), 100 μm ( a , b ), 200 μm (histology; g , h ), 300 μm ( j , k ), 500 μm ( e , f ) and 1 mm (wholemount; g , h ).

    Article Snippet: The following primary antibodies were used: rabbit anti-γH2AX (1:300; 9718, Cell Signaling), rabbit anti-TRP2 (1:200; ab74073, Abcam), rabbit anti-TRP2 (1:200; ab103463, Abcam), mouse anti-PCNA (1:1,000; ab29, Abcam), rat anti-CD34 (1:100; 14-0341-82, Thermo Fisher), rabbit anti-SOX9 (1:200; AB5535, Millipore), goat anti-SPP1 (1:100; AF808, R&D), goat anti-SPP1 (1:300; AF1433, R&D), rabbit anti-KRT14 (1:2,000; ab119695, Abcam), rabbit anti-CD44 (1:100; PA5-94934, Thermo Fisher), rabbit anti-SOX10 (1:100; ab180862, Abcam), rabbit anti-KRT5 (1:1,000; 905501, BioLegend) and goat anti-Pcad (1:200; AF761, R&D Systems).

    Techniques: Staining, Marker, Activation Assay, Mutagenesis, Two Tailed Test

    a , Bulk RNA-seq reveals prominent differences between hairy nevi and adjacent normal facial skin in humans. A principal component analysis plot is shown. See Extended Data Fig. . b , Selected upregulated (by 2× or more; green) and downregulated (by 2× or more; red) differentially expressed genes in nevus versus normal human skin. Bold and underlined genes were validated by qRT-PCR. c , qRT–PCR of selected differentially expressed genes from bulk RNA-seq data. n = 3. d , e , SPP1 and TRP2 co-staining. In normal skin, TRP2 + melanocytes did not express SPP1 ( d ), whereas in nevus skin, clusters of TRP2 + SPP1 + cells were seen next to HF bulge regions ( e ). f , SPP1 and SOX10 co-staining. Nevus skin contained SOX10 + SPP1 + cell clusters next to HF bulge regions. g , h , SPP1 and KRT5 co-staining. Unlike in normal skin ( g ), SPP1 + cell clusters were seen next to HFs in nevus human skin ( h ). i , j , SPP1 microinjections induced precocious growth by human scalp HFs (arrowheads). Representative samples of human HFs on day 50 post-grafting ( i ) and quantification of human HFs in anagen ( j ) are shown. In j , n = 7 for control and n = 11 for SPP1; P = 0.00034. In c , n refers to independent experiments. In j , n refers to biologically independent samples. P values were calculated using unpaired two-tailed Student’s t -test. ** P ≤ 0.01. Scale bars, 100 μm ( d – h ) and 1 mm ( i ).

    Journal: Nature

    Article Title: Signalling by senescent melanocytes hyperactivates hair growth

    doi: 10.1038/s41586-023-06172-8

    Figure Lengend Snippet: a , Bulk RNA-seq reveals prominent differences between hairy nevi and adjacent normal facial skin in humans. A principal component analysis plot is shown. See Extended Data Fig. . b , Selected upregulated (by 2× or more; green) and downregulated (by 2× or more; red) differentially expressed genes in nevus versus normal human skin. Bold and underlined genes were validated by qRT-PCR. c , qRT–PCR of selected differentially expressed genes from bulk RNA-seq data. n = 3. d , e , SPP1 and TRP2 co-staining. In normal skin, TRP2 + melanocytes did not express SPP1 ( d ), whereas in nevus skin, clusters of TRP2 + SPP1 + cells were seen next to HF bulge regions ( e ). f , SPP1 and SOX10 co-staining. Nevus skin contained SOX10 + SPP1 + cell clusters next to HF bulge regions. g , h , SPP1 and KRT5 co-staining. Unlike in normal skin ( g ), SPP1 + cell clusters were seen next to HFs in nevus human skin ( h ). i , j , SPP1 microinjections induced precocious growth by human scalp HFs (arrowheads). Representative samples of human HFs on day 50 post-grafting ( i ) and quantification of human HFs in anagen ( j ) are shown. In j , n = 7 for control and n = 11 for SPP1; P = 0.00034. In c , n refers to independent experiments. In j , n refers to biologically independent samples. P values were calculated using unpaired two-tailed Student’s t -test. ** P ≤ 0.01. Scale bars, 100 μm ( d – h ) and 1 mm ( i ).

    Article Snippet: The following primary antibodies were used: rabbit anti-γH2AX (1:300; 9718, Cell Signaling), rabbit anti-TRP2 (1:200; ab74073, Abcam), rabbit anti-TRP2 (1:200; ab103463, Abcam), mouse anti-PCNA (1:1,000; ab29, Abcam), rat anti-CD34 (1:100; 14-0341-82, Thermo Fisher), rabbit anti-SOX9 (1:200; AB5535, Millipore), goat anti-SPP1 (1:100; AF808, R&D), goat anti-SPP1 (1:300; AF1433, R&D), rabbit anti-KRT14 (1:2,000; ab119695, Abcam), rabbit anti-CD44 (1:100; PA5-94934, Thermo Fisher), rabbit anti-SOX10 (1:100; ab180862, Abcam), rabbit anti-KRT5 (1:1,000; 905501, BioLegend) and goat anti-Pcad (1:200; AF761, R&D Systems).

    Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Staining, Two Tailed Test

    a , Limited accumulation of senescent cells adjacent to normal, intact stem cell niche can augment it and result in stem cell activation. Mechanism, effects, and examples are listed below the schematic drawing. Tissue stem cells – orange (quiescent) and green (activated); normal niche cells – blue; senescent cells – purple. b , Schematic representation of the mechanism driving hair growth hyperactivation in skin nevus. Dermal clusters of senescent melanocytes (purple) secrete SASP factors (colored geometric shapes). SPP1 (blue squares) is the leading SASP factor of senescent melanocytes. It signals via CD44 receptor (black Y shape) to epithelial stem cells in adjacent hair follicles, inducing them into precocious growth (green arrow).

    Journal: Nature

    Article Title: Signalling by senescent melanocytes hyperactivates hair growth

    doi: 10.1038/s41586-023-06172-8

    Figure Lengend Snippet: a , Limited accumulation of senescent cells adjacent to normal, intact stem cell niche can augment it and result in stem cell activation. Mechanism, effects, and examples are listed below the schematic drawing. Tissue stem cells – orange (quiescent) and green (activated); normal niche cells – blue; senescent cells – purple. b , Schematic representation of the mechanism driving hair growth hyperactivation in skin nevus. Dermal clusters of senescent melanocytes (purple) secrete SASP factors (colored geometric shapes). SPP1 (blue squares) is the leading SASP factor of senescent melanocytes. It signals via CD44 receptor (black Y shape) to epithelial stem cells in adjacent hair follicles, inducing them into precocious growth (green arrow).

    Article Snippet: The following primary antibodies were used: rabbit anti-γH2AX (1:300; 9718, Cell Signaling), rabbit anti-TRP2 (1:200; ab74073, Abcam), rabbit anti-TRP2 (1:200; ab103463, Abcam), mouse anti-PCNA (1:1,000; ab29, Abcam), rat anti-CD34 (1:100; 14-0341-82, Thermo Fisher), rabbit anti-SOX9 (1:200; AB5535, Millipore), goat anti-SPP1 (1:100; AF808, R&D), goat anti-SPP1 (1:300; AF1433, R&D), rabbit anti-KRT14 (1:2,000; ab119695, Abcam), rabbit anti-CD44 (1:100; PA5-94934, Thermo Fisher), rabbit anti-SOX10 (1:100; ab180862, Abcam), rabbit anti-KRT5 (1:1,000; 905501, BioLegend) and goat anti-Pcad (1:200; AF761, R&D Systems).

    Techniques: Activation Assay

    ( A–D, F–G ) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor gene pairs. Red dashed lines in figures A, B and D mark the erythroid clusters. The blow-up shown in ( G ) is to better visualize PDGFA- and PDGFB-expressing cells. ( E ) Left panel: Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), SPP1 (red), CD271 (pink), and NCAM1 (cyan) and scanned with an OlympusVS120 slide scanner. Left upper image: all markers are shown; left lower image: just NCAM1 channel is shown. Middle panel: confocal laser scanning analysis of BM biopsies co-stained with CD271 (red), NCAM1 (green), SPP1 (white), and DAPI (blue) presented as 3D orthographic cross-section view. Right panel: Single staining channel data for CD271 (red), NCAM1 (green), SPP1 (white), and corresponding blow-ups for indicated areas. NCAM1, SPP1 and CD271(NGFR). White scale bars represent 50 μm and yellow scale bars represent 25 μm. White dashed lines indicate the trabecular bone surface lining regions.

    Journal: eLife

    Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

    doi: 10.7554/eLife.81656

    Figure Lengend Snippet: ( A–D, F–G ) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor gene pairs. Red dashed lines in figures A, B and D mark the erythroid clusters. The blow-up shown in ( G ) is to better visualize PDGFA- and PDGFB-expressing cells. ( E ) Left panel: Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), SPP1 (red), CD271 (pink), and NCAM1 (cyan) and scanned with an OlympusVS120 slide scanner. Left upper image: all markers are shown; left lower image: just NCAM1 channel is shown. Middle panel: confocal laser scanning analysis of BM biopsies co-stained with CD271 (red), NCAM1 (green), SPP1 (white), and DAPI (blue) presented as 3D orthographic cross-section view. Right panel: Single staining channel data for CD271 (red), NCAM1 (green), SPP1 (white), and corresponding blow-ups for indicated areas. NCAM1, SPP1 and CD271(NGFR). White scale bars represent 50 μm and yellow scale bars represent 25 μm. White dashed lines indicate the trabecular bone surface lining regions.

    Article Snippet: For SPP1 staining, after deparaffinization, rehydration, and antigen retrieval, slides were permeabilized with 0.1% Triton X100 (Sigma Ultra, T9284) solution at 37 °C for 15 min, blocked using donkey serum (Jackson Immuno Research, 017-000-121), and stained overnight at 4 °C with goat anti-human SPP1 antibody (R&D Systems, AF1433-SP), followed by secondary staining with donkey anti-goat AF594 (Jackson ImmunoResearch, 706-585-147) and staining with CD56 (NCAM1)-AF647 plus DAPI.

    Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Staining

    ( A–E, G–H, J ) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor gene pairs. ( F ) Flow cytometric analysis of SPP1 expression of primary CD45 low/- CD235a - CD71 - CD271 + CD56 + cells (red histogram), CD45 low/- CD235a - CD71 - CD271 + CD56 - CD81 ++ cells (blue histogram) and corresponding isotype control (orange histogram). ( I ) CFU-F assay of sorted CD45 low/- CD235a - CD71 - CD271 + cells (100 cells/well) in the presence (upper panel) or absence (lower panel) of sorted bone marrow CD45 + cells (3x10 5 cells/well), respectively.

    Journal: eLife

    Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

    doi: 10.7554/eLife.81656

    Figure Lengend Snippet: ( A–E, G–H, J ) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor gene pairs. ( F ) Flow cytometric analysis of SPP1 expression of primary CD45 low/- CD235a - CD71 - CD271 + CD56 + cells (red histogram), CD45 low/- CD235a - CD71 - CD271 + CD56 - CD81 ++ cells (blue histogram) and corresponding isotype control (orange histogram). ( I ) CFU-F assay of sorted CD45 low/- CD235a - CD71 - CD271 + cells (100 cells/well) in the presence (upper panel) or absence (lower panel) of sorted bone marrow CD45 + cells (3x10 5 cells/well), respectively.

    Article Snippet: For SPP1 staining, after deparaffinization, rehydration, and antigen retrieval, slides were permeabilized with 0.1% Triton X100 (Sigma Ultra, T9284) solution at 37 °C for 15 min, blocked using donkey serum (Jackson Immuno Research, 017-000-121), and stained overnight at 4 °C with goat anti-human SPP1 antibody (R&D Systems, AF1433-SP), followed by secondary staining with donkey anti-goat AF594 (Jackson ImmunoResearch, 706-585-147) and staining with CD56 (NCAM1)-AF647 plus DAPI.

    Techniques: Expressing, Control