Journal: The Journal of investigative dermatology

Article Title: Consequences of depleted SERCA2-gated calcium stores in the skin.

doi: 10.1038/sj.jid.5700091

Figure Lengend Snippet: Figure 3. Establishment of intercellular adhesion in response to elevated extracellular calcium. (a) Cells incubated for the indicated length of time in high calcium medium are shown after dispase II treatment for 15 minutes. Scale bar: 200 mm. (b) Double immunofluorescence labeling demonstrates distribution of adhesion molecules and cytoskeletal components after a 24-hour incubation in high calcium medium (green: desmoglein 3 or E-cadherin, red: keratin or actin). Scale bar: 20 mm. (c) Intercellular adhesiveness was quantified during the 24 hours after incubation in high calcium medium using the adhesion assay. Results show the mean ratio of total over single cells (high values indicate high intercellular adhesive strength) from two independent experiments performed in triplicates. Bars represent standard error (L vs NL and C after 24 hours, Po0.001 using a Bonferroni-corrected Kruskal–Wallis test). (d) The steady-state levels of adherens junction and desmosomal proteins were assessed by Western blot analysis in total cell lysates from cultures after 3 days in high calcium medium. (e) In parallel to the experiment in (d), the steady-state level of junctional mRNA was analyzed by Northern blot analysis. (f) Western blots with total lysates from cells after 5 days in high calcium were assessed for steady-state expression of differentiation markers. Experiments depicted in (d–f) were performed at least four times. Abbreviations as in Figure 1b and Dsg 3 and Dsg 1, 2, desmoglein 3 and 1/2; E-, N-, P-cad, E-, N- and P-cadherin; Dsc 1, 2, 3, desmocollin 1, 2, 3; PG, plakoglobin; PPh 1, 3, plakophilin 1, 3; DP, desmoplakin; a-cat, a-catenin; b-cat, b-catenin; tub, tubulin; NL, non-lesional cells; L, lesional.

Article Snippet: Primary antibodies were against desmoplakin I and II (for Western blot analysis: NW 161, K. Green, Northwestern University, Chicago, IL; for immunofluorescence: multi-epitope cocktail 2.15, 2.17, 2.20 Progen, Heidelberg, Germany), plakophilin 1 (11C6), plakoglobin (Pg5.1) and b-catenin (10C4; the latter three from M. Wheelock, Nebraska Medical Center, Omaha, NE), a-catenin (Zymed, San Francisco, CA), p120 (clone 98, Transduction Laboratories, Lexington, KY, Germany), E-cadherin (DECMA, R. Kemler, Max-Planck Institute, Freiburg i.Br., Germany), P-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA), N- cadherin and Dsc2 (the latter two from P. Wheelock, Nebraska Medical Center, Omaha, NE), Dsg 1/2 (DG 3.10, Progen, Heidelberg, Germany), Dsg 3 (RDI, Flanders, Netherlands), Dsc 1 (D.R.

Techniques: Incubation, Immunofluorescence, Labeling, Cell Adhesion Assay, Adhesive, Western Blot, Northern Blot, Expressing

Journal: The Journal of investigative dermatology

Article Title: Molecular Signatures and Signaling Interactions of the Hair Follicle Stem Cell Niche

doi: 10.1016/j.jid.2025.11.021

Figure Lengend Snippet: ( a ) Schematic of a first-cycle telogen (~P21-P22) follicle with key cell populations. ( b ) Labeling and isolation strategy of seven skin and HF populations from K14-H2BGFP;Crabp1- GFP;Lef1-RFP reporter back skin using FACS purification for bulk RNA sequencing. ( c ) Fluorescence image of a first-cycle telogen follicle from K14-H2BGFP;Crabp1-GFP;Lef1-RFP mice back skin (P22). Follicle shows strong K14-H2BGFP expression in epithelial BuSC, HGSC and Foll cells above and RFP expression in the DP. At high exposure GFP from the Crabp1-GFP reporter is detected in the DP (inset). Scale bars, 25 μm. ( d ) Immunofluorescence for HGSC marker PCAD and BuSC marker CD34 on WT P22 back skin. Scale bar, 25 μm ( e ) FACS plots for isolating DF, DP, Foll, HGSC, BuSC and negative cells (Neg) from the dermal fraction of transgenic reporter back skin after immunofluorescence staining for PCAD and CD34. ( f ) qRT-PCR analysis of various known marker genes for each cell population. Data are mean ± SD. N = 2 mice. ( g ) Schematic of historic hair cycle propagation wave in the mouse from anterior head region to posterior tail region, divided into four zones. ( h ) Schematic of first-cycle regeneration wave depicting hair follicles (HFs) in four distinct hair development stages. ( i ) Immunofluorescence staining for KI67 and activated Caspase-3 (CASP3*) on K14-H2BGFP mouse back skin across the four zones. White arrows highlight positively stained cells. Scale bar, 50 μm. ( j ) Quantification of relative proportion of hair cycle phases with respect to each zone. Data are mean ± SD. Data points are individual averages from N = 10 mice. Statistical significance calculated by unpaired t-test. ( k ) Relative proportion of distinct hair stage distributions across four zones based on j. ( l ) Schematic of the observed first-cycle regeneration wave from Z1-Z4 showing higher levels of anagen II near the anterior, catagen near the posterior, and a mixed distribution of anagen I and telogen across all zones.

Article Snippet: CD34 (rat, eBiosciences 14034185) and PCAD (goat, R&D Systems BAF761) stainings were performed on CD1 mice to visualize BuSCs and HGSCs respectively at first-cycle telogen.

Techniques: Isolation, Labeling, Purification, RNA Sequencing, Fluorescence, Expressing, Immunofluorescence, Marker, Transgenic Assay, Staining, Quantitative RT-PCR

Journal: Nature

Article Title: Stem cell function and stress response are controlled by protein synthesis

doi: 10.1038/nature18282

Figure Lengend Snippet: a , Hair cycle stages and genetic lineage marking using K19- and Lgr5 tdTomato (tdTom) mice. Cell surface markers to isolated bulge stem cells are CD34 and Itgα6. Telogen: stem cells (CD34 + /Itgα6 + ) are quiescent and resting in the bulge (BG). Early anagen: stem cells divide and give rise to committed progenitors in the hair germ (HG), which then grow downwards into the bulb (BU) surrounding the dermal papilla (DP). Late anagen: cells differentiate upwards to form the hair. Catagen: intermediate phase, when the hair bulb degenerates into a new resting bulge. IFE: interfollicular epidermis; SG: sebaceous glands. Mouse transgenes label K19 (red)- and Lgr5 (orange)-positive stem cells and their progeny. b , OP-puro detection in mouse epidermis at all hair cycle stages. Dotted lines: hair follicle and epidermal basal layer. Arrows: OP-puro high cells in the hair follicle. Arrowheads: OP-puro low cells in the interfollicular epidermis. Nuclei (DAPI). c , tdTomato and OP-puro detection in back skin of K19tdTom and Lgr5tdTom mice in telogen and late anagen. Arrows: Tomato + cells. Arrowheads: Tomato + /OP-puro high cells. Dotted line: lower bulge. Merged panels in . e , Hair follicle lineages and differentiation markers used in . ORS: outer root sheet; Cp: Companion layer; IRS: inner root sheet; He: Henle’s layer; Hu: Huxley layer; Ci: cuticle of inner root sheet; Ch: cuticle; Co: cortex; Me: Medulla. f , P-cadherin and OP-puro detection in a late anagen. Scale bars: 50 μm.

Article Snippet: Primary antibodies were used at the following dilutions: rabbit polyclonal to RFP (for tdTomato) (1:1000; Rockland inc., 600-401-379), mouse monoclonal to Dlx3 (1:200, Abnova, H00001747-A01), rabbit polyclonal to K6 (1:200, Abcam, ab24646), mouse monoclonal to Gata3 (1:50, Santa Cruz Biotech, sc-268), guinea pig polyclonal to K31 and K72 (1:200, Progen, GP-hHa1 and GP-K6irs2), rabbit polyclonal anti c-maf (1:100, Bethyl, A300-613A), mouse monoclonal to Lef1 (1:50, Santa Cruz Biotech, sc-81470), goat polyclonal to P-cadherin (1:100, R&D systems, FAB761A), rabbit monoclonal antibody to Ki67 (1:200; SP6, Vector Labs, VP-RM04), mouse monoclonal anti-mouse Keratin 15 (1:1000 ), β-Catenin (1:200, Santa Cruz Biotech, sc-7199), rat monoclonal anti-Itgβ1 (1:200, clone HMβ1-1, BioLegend, 102203), rabbit polyclonal anti mouse keratin 10 (1:500; Covance, PRB-159P), rabbit polyclonal anti-NSun2 (1:500; Aviva Systems Biology, ARP48811_P050), rabbit polyclonal anti-human NSun2 (MetA, 1:500 ), rat monoclonal anti-Itgα6 (1:500; GoH3, eBioscience, 14-0495), rat monoclonal anti-CD44 (1:200, IM7, BioLegend, 103004), anti-mouse Podoplanin (1:500, clone 8.1.1, eBioscience, 14-5381), rat monoclonal anti-BrdU (1:100; Abcam, ab6326), rabbit polyclonal anti-Laminin alpha 5 (1:100, Abcam, ab75344), mouse monoclonal anti-cytokeratin 8 (1:100, TROMA-I, DSHB, US), rabbit polyclonal to Slug (1:200, Cell Signaling, 9585P), chicken polyclonal anti-GFP (1:200, Abcam, ab13970), rabbit polyclonal anti-p53 (1:100, CM5, Novocastra, NCL-p53-CM5p), and rabbit anti human Involucrin (SY5 clone, 1:200, Abcam, ab80530).

Techniques: Isolation