DKK1 Antibody - #AF4600

Fig. 6 ApoEV treatment improves skin and hair follicle MSC functions. (A) Immunofluorescent images show that GFP-ApoEV (green) were engulfed by skin MSCs (SMSCs) (1 and 2), as indicated by co-staining with CD105 at 7 days post-injection. The right panel exhibits the higher magnification of the boxed region to show colocalization of GFP-ApoEV and CD105 positive SMSCs. (B) Western blotting shows GFP signals was detected in SMSCs after GFP-ApoEV injection. (C) Immunofluorescent staining of SMSC smears shows skin cells endocytosed apoEVs. (D) Western blotting shows Wnt/β-catenin signaling is activated and DKK1 expression is decreased in SMSCs and hair follicle MSCs (HF-MSCs) from MRL/lpr mice after apoEVs injection. (E, F, I and J) EdU and population doubling assay show that MRL/lpr SMSCs and HF-MSCs had reduced proliferation and passage rates when compared to the wild-type control group. After apoEV treatment, proliferation and passage rates were improved in MRL/lpr SMSCs and HF-MSCs, respectively. n = 3–5. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA test. Data shown as mean ± SD. (G, K) Compared to wild-type SMSCs, MRL/lpr SMSCs showed reduced capacity to form mineralized nodules when cultured under osteogenic inductive conditions, assessed by alizarin red staining, and reduced expression of osteogenic markers Runx2 and ALP, assessed by Western blotting. After apoEV treatment, reduced mineralized nodule formation and expression of Runx2 and ALP were rescued in MRL/lpr SMSCs (G). The osteogenic capacity of MRL/lpr HF-MSCs was also rescued after apoEV treatment (K). n = 3. ***P < 0.001, one-way ANOVA test. Data shown as mean ± SD. (H, L) Compared to wild-type SMSCs, MRL/lpr SMSCs showed reduced capacity to differentiate into adipocytes when cultured under adipogenic inductive conditions, as assessed by Oil red O staining, along with reduced expression of adipogenic markers PPARγ and LPL, as assessed by Western blotting. After apoEV treatment, reduced adipocyte formation and expression of PPARγ and LPL were rescued in MRL/lpr SMSCs (H). The adipogenic capacity of MRL/lpr HF-MSCs was also rescued after apoEV treatment (L). n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA test. Data shown as mean ± SD. Scale bars (A, C), 50 μm.

Fig. 5 Mechanical forces regulate the elimination of apoEVs through the skin. (A) Schematic diagram of mechanical forces applied to mice. Treadmill running exercise as a force augmentation model; tail suspension as a weightless model. (B) Immunofluorescent images show that the number of GFP-ApoEV migrating to the stratum corneum was increased in running group and reduced in tail-suspension group at 3 days post-injection. White dotted line represents the dividing layer between stratum corneum and the other layers of epidermis. The quantity graph shows the relative intensity of apoEVs in the skin. n = 5. **P < 0.01, ***P < 0.001, one-way ANOVA test. Data shown as mean ± SD. (C) Western blotting confirms the level of GFP in the skin tissue is increased in running group and reduced in tail suspension group. (D, E) PKH67-labeled apoEVs (4 × 106) were injected into C57BL/6 mice via the tail vein. After treadmill running exercise for 7 days, blood samples were collected. The flow cytometric calculation showed that the number of PKH67+/Annexin V+/CD62P− apoEVs accumulated in the blood was significantly reduced compared to the control group (D). After tail suspension for 3 days, flow cytometry analysis showed that the number of PKH67+/Annexin V+/CD62P− apoEVs in the blood was significantly increased compared to the control group (E). n = 3. *P < 0.05, Student's t-test. Data shown as mean ± SD. (F, G) DIR-labeled apoEVs (4 × 106) were injected into immunocompromised mice via the tail vein. Ex vivo fluorescent images showed that the elimination of apoEVs through the skin was increased after treadmill exercise while the migration of apoEVs through the skin was decreased after tail suspension compared to the control group, as indicated by quantitative graph. n = 3. *P < 0.05, Student's t-test. Data shown as mean ± SD. (H) ELISA analysis showed the level of DKK1 in the circulation of C57BL/6 mice was reduced after treadmill running, but enhanced after tail suspension. n = 3. *P < 0.05, **P < 0.01, Student's t-test. Data shown as mean ± SD. (I) Immunofluorescent images show the expression level of active-β-catenin (green) in skin was enhanced after treadmill running, but reduced after tail suspension. The DKK1 expression level in the skin showed opposite trends of active-β-catenin. SC, stratum corneum; BM, basement membrane; D, dermis. Scale bar (B), 50 μm; Scale bar (I), 40 μm.

Figure 3 DKK1 regulated LRP6 and airway inflammation, andrographolide elevated DKK1 and LRP6 and reduced airway inflammation. Notes: (A) Detection of TNF-α, IL-6 and IL-8 in the cell supernatant after treatment of cells with different concentrations of CSE. (B) Western blot and PCR showed changes of DKK1 and LRP6 after CSE treatment. (C) Cells were pre-transfected with DKK1 overexpression plasmid or Andrographolide, followed by treatment with CSE, Western blot was used to detect protein levels. (D) Changes in TNF-α, IL-6 and IL-8 in cell supernatants in different treatment groups. Andro: Andrographolide. *p<0.05 with, **p<0.01, ****p<0.0001, respect to the blank group, #p< 0.05, ####p<0.0001, with respect to the CD513B-1+2% CSE group, Δp < 0.05 with respect to the 2% CSE group.