Figure 5
MFAP-4 is essential to the assembly of elastic fibers.
NHDFs were transfected with siRNAs for non-specific sequences (Control) or MFAP-4 twice during the culture for 8 days. Control siRNA-transfected cells were treated with or without 10 nM human recombinant MFAP-4 during the culture. (a) Quantitative RT-PCR analysis was performed with MFAP-4-specific TaqMan Gene Expression Assays after the reverse transcription of total RNAs from cultured NHDFs. The mRNA expression of the target gene was normalized against the expression of GAPDH mRNA and is relatively presented. The values reported represent means ± SD. ***p<0.001. (b) Western blotting analysis with antibodies specific for MFAP-4 or fibrillin-1 was conducted to confirm the impact of MFAP-4 silencing on their protein abundance using the supernatants from cultured NHDFs. (c) Immunofluorescence staining was performed with an anti-human tropoelastin antibody (green) and an anti-human fibrillin-1 antibody (red). Nuclear staining (DAPI) and merged images are also shown in the diagram. Scale bars = 50 μm. (d) Quantitative RT-PCR analysis of the MMP-12 mRNA expression in cultured NHDFs was performed as described in the legend of Fig. 5a. The values reported represent means ± SD. ***p<0.001, **p<0.01. (e) Western blotting analysis with an anti-human MMP-12 antibody was performed for the supernatants from cultured NHDFs. The obtained bands of the active form of MMP-12 were analyzed using a densitometer and the values in the graph are represented relatively. The values reported represent means ± SD. *p<0.05. (f) NHDFs were cultured for 8 days with or without 20 nM human recombinant MFAP-4 during the culture in FBS-free medium. Immunofluorescence staining was performed as described in the legend of Fig. 5c. Scale bars = 50 μm.
