Niche-Driven Overgrowth of KPC Figure 4. IL-6 increases Oct-4 and telomerase expression in KPCs and SKPs. A, KPCs and SKPs were cultured in 1% FBS for 24 hours followed by exposure to different concentrations of IL-6, and BrdU incorporation in KPCs and SKPs was determined (mean6SEM). ** P,0.01. B, Expression of Oct-4 mRNA and protein were determined by Western blotting (WB) and RT-PCR, respectively. C, Immunofluorescence studies of Oct-4 expression in SKP and KPC after incubated with 20 ng/ml IL-6 for 24 hours. Scale bars, 20 mm. D, Expression of hTERT mRNA and protein were determined by Western blotting (WB) and RT-PCR, respectively. E, Immunofluorescence studies of hTERT expression in SKP and KPC following incubation with 20 ng/ml IL-6 for 24 hours. Scale bars, 20 mm. F, Telomerase enzyme activity of SKPs and KPCs in response to IL-6 as determined by TeloTAGGG Telomerase PCR ELISA. G and H, Treatment with neutralizing antibody for IL-6 (IL-6Ab) decreased the basal level of hTERT as determined by Western blotting (G) and TeloTAGGG Telomerase PCR ELISA (H). An isotype-matched normal mice IgG (mIgG) was used as negative control (mean6SEM). * P,0.05; ** P,0.01; *** P,0.001; ns, no significance. The results are representative of at least five independent experiments using KPCs and the matched SKPs from different patient donors (n = 5). doi:10.1371/journal.pone.0007798.g004 6, or both (Figure 5D). However, IL-6-induced up-regulation of hTERT and Oct-4 expression was only negated by pre-treatment with IL-6 neutralizing antibody, not by IL-17 neutralizing antibody (Figure 5E). Meanwhile, we examined whether treatment with IL-6 and IL-17 neutralizing antibodies had any effects on cell proliferation and cell viability of SKPs and KPCs. Our results showed that treatment with IL-6, but not IL-17, neutralizing antibodies moderately inhibited BrdU incorporation (P,0.05) (Figure S5C), but had no obvious effects on cell viability (Figure S5B). We observed no apparent morphologic change character- istic of apoptosis after treatment with IL-6 and/or IL-17 neutralizing antibodies (data not shown). In all experiments, an isotype-matched antibody (mouse IgG) was used as negative controls. Taken together, these results suggest that IL-17 induces hTERT and Oct-4 expression in SKPs and KPCs by orchestrating the downstream IL-6 paracrine loop. IL-17/IL-6 axis contributes to the fibroproliferative growth of KPCs-generated transplants In order to recapitulate the distinct ‘‘pathological’’ niche of keloid, we engineered the in vitro inflammatory niche by incorporating IL-6 non-covalently into the hydrogel carrier. KPCs and SKPs (26106) were mixed with IL-6-linked carrier and immediately injected subcutaneously on the dorsal surface of immunocompromised mice. Our results showed that KPCs- generated transplant grew faster than that from SKPs, and the PLoS ONE | www.plosone.org 9 November 2009 | Volume 4 | Issue 11 | e7798