Figure 1.
Lumican and lumican core protein promotes mice skin wound healing.
(A) Coomassie brilliant blue staining of lumican protein. Lane 1 is glycosylated lumican recombinant protein with Strep-II tag expressed in EBNA-293 cells. Lane 2 is deglycosylated lumican protein after digested by N-glycosidase F. Lane 3 is lumican core protein with His tag expressed in prokaryotic system. Lane 4 is N-glycosidase F. The molecular weight (MW) of N-glycosidase F is 35 KD, the MW of Strep-II tag is 3.7 KD, and His tag is 1 KD. So the MW of deglycosylated lumican protein is about 42 KD, and the MW of lumican core protein is 39 KD. Mr: molecular mass ladder. (B) Representative images of glycosylated lumican protein treated mice skin wound. Scale bar = 1 mm. (C) Quantification of glycosylated lumican protein treated mice wound area. Data are presented as means±SD, n = 5, *p<0.05, **p<0.01. (D) Representative images of lumican core protein treated mice skin wound. Scale bar = 1 mm. (E) Quantification of lumican core protein treated mice wound area. Data are presented as means±SD, n = 5, *p<0.05, **p<0.01.
Figure 2.
Recombinant lumican protein did not significantly influence keratinocyte proliferation.
(A) Images of PCNA stained mice wound sections at post-injury day 3 and day 7. Scale bar = 25 µm. (B) Quantification of PCNA positive cells in epidermis. Values represent means±SD, n = 5. (C) Results of in vitro CCK-8 HaCaT cells proliferation assays. Values are denoted as means±SEM, n = 3.
Figure 3.
Recombinant lumican protein did not affect keratinocyte migration statistically.
(A) Epidermal tongue in HE stained mice wound sections at post-injury day 3. The black lines outline the epidermal tongue. Scale bar = 50 µm. (B) Quantification of epidermal tongue length. Values are means±SD, n = 5. (C) Quantification of in vitro HaCaT cells scratch wound healing assays after treating with lumican at 0, 0.1, 1 and 10 nM. Values are means±SEM, n = 3.
Figure 4.
Lumican activated fibroblasts in vivo and in vitro.
(A) Low magnification images of α-SMA stained mice wound tissue. Scale bar = 100 µm. (B) High magnification images of α-SMA stained mice wound tissue. Scale bar = 25 µm. (C) Quantification of α-SMA positive cells ratio. Values are denoted as means±SD, n = 5, **p<0.01. (D) Immunofluorescence staining of α-SMA. α-SMA is shown by red fluorescence and cell nucleus is stained by DAPI. Scale bar = 25 µm. (E) Ratio of α-SMA positive HDF cells. Values means±SEM, n = 3. *p<0.05.
Figure 5.
Fibroblast proliferation was not affected by lumican.
(A) Images of PCNA stained mice wound granulation tissue at post-injury day 3 and day 7. Scale bar = 25 µm. (B) Quantification of PCNA positive cells in granulation tissue. Values represent means±SD, n = 5. (C) Results of in vitro CCK-8 HDF cells proliferation assays. Values are denoted as means±SEM, n = 3.
Figure 6.
Lumican enhanced contraction of fibroblasts-populated collagen gel lattices.
(A) Representative images of quiescent fibroblasts populated collagen gel lattices at different time points. The gels are outlined by white spots. (B) Quantification of quiescent fibroblasts populated collagen gel areas. Values are means±SEM, n = 3, *p<0.05, **p<0.01, ***p<0.001. (C) Representative images of activated fibroblasts populated collagen gel lattices at different time points. The gels are outlined by white spots. Lumican used in the assay is 10 nM. (D) Quantification of activated fibroblasts populated collagen gel areas. Values are means±SEM, n = 3, *p<0.05, **p<0.01. (E) Comparison of contraction efficiency of lumican (10 nM) on quiescent or activated HDF.
Figure 7.
Silencing of integrinα1 did not block the pro-contractility of lumican.
(A) Silencing efficiency of integrin α1 was evaluated by western blotting. (B) Representative images of contracted collagen gel lattices at different time points. Lumican used in the experiments was 10 nM. The gels are outlined by white spots. (C) Quantification of gel areas. Values are means±SEM, n = 3, *p<0.05, **p<0.01.
Figure 8.
Silencing of integrinα2 abolished the pro-contractility of lumican.
(A) Silencing efficiency of integrin α2 was evaluated by western blotting. (B) Representative images of contracted collagen gel lattices at different time points. Lumican used in the experiments was 10 nM. The gels are outlined by white spots. (C) Quantification of gel areas. Values are means±SEM, n = 3, *p<0.05, **p<0.01.