1. Stratifin-Induced Matrix Metalloproteinase-1 in Fibroblast Is Mediated by c-fos and p38 Mitogen-Activated Protein Kinase Activation 
Eugene Lam, Runhangiz T. Kilani, Yunyuan Li, Edward E. Tredget, Aziz Ghahary 
Journal of Investigative Dermatology 
Volume 125, Issue 2, Pages (August 2005)
DOI: /j X x
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Stratifin stimulates the expression of c-fos, c-jun, and matrix metalloproteinase-1 (MMP-1) messenger ribonucleic acid (mRNA). Confluent human dermal fibroblasts in Dulbecco's modified Eagle's medium and 2% fetal bovine serum were incubated with stratifin (2.5 μg per mL) for 0, 0.5, 1, 2, 4, 6, 12, 24 h. Total RNA (10 μg per lane) was electrophoresed on 1% agarose gels, transferred to nitrocellulose, and hybridized with MMP-1 cDNA. Levels of MMP-1, c-jun, and c-fos mRNA were determined by northern blot analysis. (A) The same blots were re-hybridized with cDNA specific for 18S ribosomal RNA and were used as an RNA loading control. Quantitative densitometry of the ratio of MMP-1 mRNA expression/18S ribosomal RNA with various treatments is shown as a function of time (h) (B).
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Stratifin treatment increases the matrix metalloproteinase-1 (MMP-1) transcriptional rate, but not ribonucleic acid (RNA) stability, in dermal fibroblasts. (A) A representative autoradiogram of two separate experiments obtained from hybridization of the newly synthesized stratifin-treated and untreated radio-labeled RNA with either 2 or 8 μg of MMP-1 cDNA. (B) A representative of three separate autoradiograms of MMP-1 mRNA expression in cells initially treated with nothing (control) or 2.5 μg stratifin for 24 h and subsequently treated with actinomycin D at indicated time points. A representative of the corresponding ethidium bromide stained 18 and 28 S ribosomal RNA used as a loading control is also shown. (C) Autoradiograms and ethidium bromide-stained 18S ribosomal RNA from three blots prepared from different experiments were quantified, and the ratio of MMP-1/18 S ribosomal RNA was calculated as a percent of that of 0 time point and plotted. Solid and open bars are representative of stratifin-treated and non-treated samples, respectively.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Stimulation of matrix metalloproteinase (MMP)-1 expression by stratifin is mediated by p38 mitogen-activated protein kinase (MAPK). Fibroblasts in Dulbecco's modified Eagle's medium (DMEM) and 2% fetal bovine serum (FBS) were incubated with 10 μM of specific inhibitors for each MAPK kinase (PD98059 (PD), SB (SB), and SP (SP)) for 1 h before stimulation stratifin (2.5 μg per mL) for 24 h (A). (B) Fibroblasts were treated in DMEM plus 2% FBS with 1, 5, 10, 50 μM of SB for a period of 24 h. For both sets of experiment, total ribonucleic acid (RNA) was extracted and northern blot analysis was performed to determine the expression of MMP-1. The same blots were re-hybridized with cDNA specific for 18S ribosomal RNA and were used as an RNA loading control.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Stratifin induces p38 mitogen-activated protein kinase (MAPK) phosphorylation in dermal fibroblasts. Fibroblasts were treated with stratifin (2.5 μg per mL) for various time intervals as indicated. The levels of activated p38 (p-p38) were determined by western blot analysis using phospho-specific antibodies for p38 MAPK. The levels of total p38 were also determined in the same samples by western blot analysis using specific antibodies. C6 cell extracts treated with anisomycin were used as positive control for phospho-p38 (A). (B) Fibroblasts were treated with either stratifin (2.5 μg per mL), SB (SB) (10 μM), or a combination of both. Fibroblasts were pretreated with SB (10 μM) for 1 h before treatment with stratifin. The membrane was immunoblotted using antibodies against total and phosphorylated forms of p38 MAPK and subsequently immunoblotted with β-actin antibody as loading control. Quantitative densitometry of the ratio of intracellular p-p38 MAPK protein/total p38 MAPK protein was performed and is shown in (C). All p-values less than represent very significant differences.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Stimulation of intracellular matrix metalloproteinase (MMP)-1 protein levels by stratifin is mediated by p38 mitogen-activated protein kinase. Fibroblasts were treated with stratifin (2.5 μg per mL) for different periods of time as indicated (A). The intracellular MMP-1 protein levels were determined by western blot analysis using specific antibodies. The levels of β-actin were also determined in the same samples by western blot analysis for loading controls. (B) Fibroblasts were treated for 24 h with nothing (N), stratifin (σ), SB (SB), or a combination of both (σ+SB). Fibroblasts were pretreated with SB for 1 h before treatment with σ. β-actin is shown as loading controls. Quantitative densitometry of the ratio of intracellular MMP-1 protein/β-actin was performed and is shown in (C). The values of p<0.05 and p<0.005 represent significant and very significant differences, respectively.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Microarray analysis of messenger ribonucleic acid (mRNA) extracted from stratifin-treated fibroblasts. Fibroblasts were treated with stratifin (2.5 μg per mL) for 90 min. Total mRNA was extracted, reverse-transcribed, and the corresponding cDNA biotin-labeled according to the manufacturer's directions. (A) Shows the results of untreated and stratifin-treated samples of MAP kinase signaling pathway gene array. Cyclophilin A or peptidylprolyl isomerase A (PPIA) expression was used as positive control and the DNA of PUC18 plasmid was used as negative control. The highlighted box illustrates the expression of Elk4/Sap1a and autotaxin (ENPP2). A key to gene coordinates is also shown in (B) in addition to a table indicating the fold increases of Elk4/Sap1a and autotaxin from stratifin treatment.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

8. Figure 7
Confirmation of array results by northern analysis of Elk4/Sap1a. Fibroblasts were treated with stratifin (2.5 μg per mL) for different time intervals. The total ribonucleic acid (RNA) was extracted and northern blot analysis was performed to determine the expression of Elk4/Sap1a. The same blot was re-hybridized with cDNA specific for 18S ribosomal RNA and was used as an RNA loading control.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions