Figure 6: Lcn2 is the key secreted mediator of Tcf3-induced cell migration and wound healing.

(a) Images of skin wound sites taken immediately after wounding and 10 days post wounding. After full-thickness wounds of 1 cm² were created on the dorsal skins of wild-type mice (n=5), CM from Tcf3-overexpressing cells or control cells was applied topically onto the wound sites daily for a week. Surface areas of the wounds were measured at the initial time point and 10 days post wounding. (b) Graph quantifying the surface areas of the wounds as a percentage of the original wounds 10 days post wounding. Data are mean±s.d. *P<0.05 (Student’s t-test). (c) Real-time PCR analysis of Lcn2 expression in keratinocytes that were transduced to express tet-inducible Lef/Tcf members. Keratinocytes were transduced with GFP-tagged, tet-inducible lentivectors carrying myc-tagged Lef/Tcf members. Transduced cells were isolated by FACS (fluorescence-activated cell sorting) and were treated with Dox or vehicle control for 24 h prior to being harvested for messenger RNA analysis. Mean expression values from biological triplicates were calculated. Data are mean±s.d. (d) Migration assays were performed on keratinocytes that were incubated with control CM or Tcf3-CM together with anti-Lcn2 antibody or IgG isotype control. Graph quantifying the relative area migrated by keratinocytes treated with Tcf3-CM incubated with IgG or anti-Lcn2 normalized over area migrated by cells treated with control CM. For each sample, over 30 non-overlapping fields were measured at each time point; and each experiment was repeated twice. Data are mean±s.d. **P<0.001 (Student’s t-test). (e) Images of the wound sites were taken 10 days post wounding. 1 cm² full-thickness wounds were created on dorsal skins of nude mice and were treated daily with topical application of conditioned media from control (empty vector) CM (cont-CM) or Tcf3-CM untreated or pre-incubated with antibodies against Lcn2 or isotype control. (f) Graph quantifying the surface areas of the wounds as a percentage of the original wounds 10 days post wounding, cont-CM (n=7) or Tcf3-CM untreated (n=7) or preincubated with antibodies against Lcn2 (n=8) or isotype control (n=6). Surface areas of the wounds were measured at the initial time point and 10 days post wounding. Data are mean±s.d. *P<0.05 (Student’s t-test).