1. MiR-125b, a MicroRNA Downregulated in Psoriasis, Modulates Keratinocyte Proliferation by Targeting FGFR2 
Ning Xu, Petter Brodin, Tianling Wei, Florian Meisgen, Liv Eidsmo, Nikoletta Nagy, Lajos Kemeny, Mona Ståhle, Enikö Sonkoly, Andor Pivarcsi 
Journal of Investigative Dermatology 
Volume 131, Issue 7, Pages (July 2011)
DOI: /jid
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
The expression of microRNA-125b (miR-125b) in healthy and psoriasis skin. (a) MiR-125b expression was analyzed in healthy (n=27) and psoriasis lesional skin samples (n=25) using real-time quantitative reverse transcription-PCR (qRT-PCR). ***P< (b) In situ hybridization was performed on healthy (n=6, left panel) and psoriasis lesional skin sections (n=8, right panel) using miR-125b-specific locked nucleic acid (LNA) probe (upper panel) or scrambled probe (lower panel). The blue–purple color indicates miR-125b expression. Bar=50μm. (c) MiR-125b expression was analyzed on RNA from epidermis of healthy (n=5) and psoriasis lesional skin (n=3) using qRT-PCR. *P<0.05. (d) Expression of primary miR-125b transcripts was analyzed in healthy (n=7) and psoriasis lesional skin samples (n=9) using qRT-PCR. Results for individual patients and mean are shown. **P<0.01. (e) qRT-PCR analysis of primary miR-125b transcripts in primary keratinocytes.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
MicroRNA-125b (miR-125b) suppresses the proliferation of keratinocytes. Primary human keratinocytes were transfected with (a) miR-125b precursor RNAs (Pre-miR-125b), (b) miRNA precursor control (Pre-miR-ctrl), (c) miR-125b inhibitor oligonucleotides (Anti-miR-125b), or (d) miRNA inhibitor control (Anti-miR-ctrl). The percentage of cells that underwent cell division was assessed by anti-5-ethynyl-2′-deoxyuridine (anti-EdU) Alexa Fluor 647 staining 48hours post-transfection. The representative flow cytometry histograms gated on live propidium iodide (PI)-negative cells are shown and the percentages of EdU-positive cells are indicated. (e) The data of one representative experiment with quintuplicate are shown. The experiment was repeated five times. **P<0.01, ***P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
MicroRNA-125b (miR-125b) promotes the differentiation of keratinocytes. Primary human keratinocytes were transfected with miR-125b precursor RNA (Pre-miR-125b) or miRNA precursor control (Pre-miR-ctrl; left panel); miR-125b inhibitor oligonucleotide (Anti-miR-125b) or miRNA inhibitor control (Anti-miR-ctrl; right panel). Both RNA and protein were collected at 24, 48, 72, and 96hours after transfection. The involucrin messenger (m)RNA (a, upper panel) and cytokeratine 5 mRNA (b) were analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR) and normalized to 18S RNA. Data are expressed as relative units (RU). The data of one representative experiment with triplicates are shown. The experiment was repeated three times. *P<0.05, **P<0.01. (a, lower panel) The expression of involucrin protein was analyzed by western blotting. β-Actin was detected on the same blots as loading control.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Fibroblast growth factor receptor 2 (FGFR2) is a direct target of microRNA-125b (miR-125b). (a) Venn diagram depicting the number of potential targets of miR-125b predicted by three bioinformatics methods. (b) Schematic representation of the 3′ untranslated region (3′UTR) of FGFR2 mRNA (gray bar) with the predicted target sites for miR-125b (red lines). Mfe, the minimal free energies for miR-125b binding. (c) Nucleotide resolution of the predicted target sites: the seed sequence (green letters); the target sequence (red letters); the evolutionarily conserved regions (gray boxes); the deleted miR-125b-binding sites (black boxes). (d) HeLa cells were transfected with firefly luciferase (FL) reporter construct containing FGFR2 3′UTR together with miR-125b precursor RNA (Pre-miR-125b) or miR-125b inhibitor oligonucleotide (Anti-miR-125b). (e) The FL constructs containing the wild-type (WT) or mutant (MUT) 3′UTR or empty FL vector (Vector) were transfected to HeLa cells together with Pre-miR-125b or miRNA precursor control (Pre-miR-ctrl). Means±SD of three independent experiments are shown. *P<0.05, **P<0.01, ***P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Fibroblast growth factor receptor 2 (FGFR2) is regulated by microRNA-125b (miR-125b) in keratinocytes and overexpressed in psoriasis. Keratinocytes were transfected with miR-125b precursor RNA (Pre-miR-125b) or miR-125b inhibitor oligonucleotide (Anti-miR-125b) and both RNA and protein were collected. (a) The expression of FGFR2 mRNA was analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR). (b) FGFR2 protein was detected by western blotting and the results for the protein collected 48hours after transfection are shown. β-Actin was detected on the same blots as loading control. (c) The expression of FGFR2 in healthy (n=3) and psoriasis skin (n=4) was detected by immunohistochemistry and the rectangular areas marked in upper figures are shown at higher magnification below. The red–brown color indicates FGFR2 expression. Bar=50μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
The effects of silencing fibroblast growth factor receptor 2 (FGFR2) expression on keratinocyte proliferation and differentiation. Keratinocytes were transfected with 30nM small interfering RNA (siRNA) for FGFR2 (siFGFR2) or siRNA-negative control (siRNA-Ctrl) and both RNA and protein were collected 48 and 72hours post-transfection (hpt) and analyzed by (a) real-time quantitative reverse transcription-PCR (qRT-PCR) and (b) western blotting. (c) The percentage of 5-ethynyl-2′-deoxyuridine (EdU)-positive cells was analyzed by flow cytometry at 48 and 72hpt. (d) The mRNAs of involucrin and cytokeratine 10 were analyzed by qRT-PCR at 24–96hpt. The expression of FGFR2, involucrin, and cytokeratine 10 was normalized to 18S RNA and data are expressed as relative units (RU). The data of one representative experiment with triplicates are shown. The experiment was repeated three times. *P<0.05, **P<0.01, ***P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions