Figure 2 CXXC5 attenuates ALP activity and proliferation in HFDPCs. (a) HFDPCs (n = 3) were transfected with pcDNA3.1 or pcDNA3.1-CXXC5-Myc. The cells were further cultured for 3 days before harvesting and whole cell lysates (WCLs) were subjected to western blot analyses to detect the protein levels of β-catenin, ALP, PCNA, Myc, and α-tubulin. (b) Immunocytochemical staining for β-catenin (red) with DAPI staining (blue), ALP staining (dark blue) in HFDPCs transfected with pEGFP-c3 or GFP-CXXC5 (green) (left panels) and quantitative analyses (n = 3) of immunocytochemical staining for β-catenin in GFP-positive cells (right graph). Red arrowheads indicate the expression of β-catenin and green arrowheads indicate the expression of GFP. (c) HFDPCs were transfected with pcDNA3.1, pcDNA3.1-CXXC5-Myc, or pcDNA3.1-CXXC5ΔDBM-Myc. The cells were further incubated for 3 days and WCLs were subjected to western blot analyses to examine the expression of β-catenin, ALP, PCNA, Myc, and α-tubulin. (d–g) Wnt reporter assay, ALPL reporter assay, ALP activity assay, and MTT assay (n = 3) in HFDPCs transfected with pcDNA3.1 or pcDNA3.1-CXXC5-Myc. (h) HFDPCs were transfected with pcDNA3.1, pcDNA3.1-CXXC5-Myc, or pcDNA3.1-CXXC5ΔDBM-Myc. The transfected cells were treated with or without 50 ng/ml recombinant Wnt3a for 72 hours. WCLs or cell lysates immunoprecipitated with anti-Myc were analyzed by western blotting to detect Dvl-1, Myc, β-catenin, or α-tubulin. (i, j) HFDPCs were treated with or without 50 ng/ml recombinant Wnt3a for 3 days after transfection with control siRNA or CXXC5 siRNA (CXXC5 si). (i) Western blot analyses of WCLs were performed with antibodies against β-catenin, ALP, PCNA, Myc, and α-tubulin. (j) Immunocytochemical staining for β-catenin (green) or CXXC5 (red), ALP staining (dark blue) (left panels), and mean intensity quantitation (n = 3) (right graphs) were also performed. All results presented are representative of at least three independent experiments. Scale bars = 100 μm for panels (b) and (j). Data are expressed as means ± SD. *P < 0.05, **P < 0.005, ***P < 0.0005 for panels (b), (d), (e), (f), (g), and (j). ALP, alkaline phosphatase; Dvl, Dishevelled; GFP, green fluorescent protein; HFDPC, human hair follicle dermal papilla cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCNA, proliferating cell nuclear antigen; SD, standard deviation; siRNA, small interfering RNA.