Pure Mathematics

I. Supplementary materials and methods

RNA extraction and qRT-PCR

Skin biopsies were homogenized using TissueLyser LT (Qiagen, Hilden, Germany) prior to

RNA extraction. Total RNA was extracted from human tissue and cells using the miRNeasy

Mini kit (Qiagen) or TRIzol reagent (ThermoFisher Scientific, Carlsbad, CA). Reverse

transcription and PCR were performed as previously described (Meisgen et al., 2014).

Expression was either determined by TaqMan expression assays (ThermoFisher Scientific)

(IL8, CXCL5 and CCL20) or by SybrGreen expression assays (ThermoFisher Scientific)

(WAKMAR2, IL1B, MMP3, RRM2B, MALAT1, H2BK, KCNQ and SMAD3) and

normalized based on the values of the housekeeping gene 18S or GAPDH. The information for

all the primers/probes used in this study is listed in Table S7.

Cell fractionation

Cytoplasm and nucleus of keratinocytes were separated by using Nuclear Extract Kit (Active

Motif) following the manufacturer’s instructions. RNA was extracted from these fractions using

Trizol (ThermoFisher Scientific). QRT-PCR was performed to analyze the expression of

WAKMAR2, MALAT1 and GAPDH. KNCQ was used for normalization.

WAKMAR2 polyadenylation study

Total RNA was extracted from keratinocytes by using Trizol. Poly(A)+ and poly(A)- RNA

fractions were separated by Dynabeads™ mRNA Purification Kit (ThermoFisher Scientific)

following the manufacturer’s instructions. QRT-PCR were performed to analyze the expression

of WAKMAR2, GAPDH and H2BK in the poly(A)+ and poly(A)- RNA fractions.

Laser capture microdissection

Fresh frozen tissue samples were cut to 10 µm tissue sections and stained with hematoxylin.

Laser capture microdissection was performed with Leica LMD7000 (Leica, Bernried,

Germany). RNA from microdissected tissue was purified using the miRNAeasy Kit (Qiagen).

Cell culture and treatments

Human primary epidermal keratinocytes (Cascade Biologics, Portland, OR) were cultured in

EpiLife medium supplemented with 10% Human Keratinocyte Growth Supplement (HKGS)

and 1% penicillin/ streptomycin at 37°C in 5% CO2 (ThermoFisher Scientific).

To study the biological function of WAKMAR2, third passage keratinocytes at 60- 70%

confluence were transfected with 20 nM LNA-long RNA-GapmeR-antisense-oligonucleotide

(GapmeR) targeting WAKMAR2 or negative control oligos (Exiqon, Denmark) for 24 hours

with Lipofectamine™ 2000 (ThermoFisher Scientific). Then cells were treated with TNF-a (50

ng/ml, R&D systems Inc) for another 3 or 6 or 24 hours to induce inflammatory response.

Keratinocytes were transfected with siRNAs for 24 hours to deplete PERP, MMP3, RRM2B,

IL1B (ON-Target plus Human siRNA- smart pool; Dharmacon, Lafayette, CO) or SMAD3

(Silencer Select, Ambion, ThermoFisher Scientific),

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To determine which RNA polymerase transcribes WAKMAR2, keratinocytes were treated with

a-amanitin (5 µg/ml) or actinomycin-D (5 µg/ml) (Sigma-Aldrich, St Louis, MO) for 2-8 hours.

To study the mechanism regulating WAKMAR2 expression, keratinocytes were treated with

IL-1a (20 ng/ml), IL-1b (20 ng/ml), IL-6 (50 ng/ml), IL-8 (50 ng/ml), IL-17a (30 ng/ml), IL17f (30 ng/ml), IL-22 (30 ng/ml), TNF-a (50 ng/ml), TGF-b1 (20 ng/ml), TGF-b2 (10 ng/ml),

TGF-b3 (20 ng/ml), BMP-2 (100 ng/ml), EGF (20 ng/ml), KGF (20 ng/ml), IGF-1 (20 ng/ml),

FGF-1 (30 ng/ml), FGF-2 (30 ng/ml), VEGF-A (20 ng/ml), GM-CSF (50 ng/ml) or PBS as

control for 24 hours and WAKMAR2 expression was analyzed by qRT-PCR. All these

cytokines and growth factors were purchased from ImmunoTools (Friesoythe, Germany). TGFβ receptor inhibitor, SB431542 (15 µM, Tocris, Ellisville, MO) was applied 15 minutes before

adding TGF-β2.

ELISA

Conditioned medium from WAKMAR2 GapmeR or GapmeR negative control-treated

keratinocytes was collected. Protein levels of IL-8 and CXCL5 were measured by ELISA

according to the manufacturer’s instructions (BioLegend, San Diego, CA).

Analysis of cell motility

For scratch assay, keratinocytes transfected with 20 nM WAKMAR2 GapmeR were grown to

full confluence and then treated with Mitomycin C (5 µg/ml) (Roche, Basel Switzerland) to

prevent proliferation. A scratch was made with a 10µL pipette tip and the cells were incubated

with Epilife medium without HKGS and photographed at the indicated time points. The wound

areas were measured using Image J (National Institutes of Health, Bethesda, MD). Healing rate

at each time point = (initial wound area − area of the wound at this time point) x 100% / initial

wound area.

For IncuCyte™ 96-well Real-Time Cell Migration Assay, keratinocytes were transfected with

20 nM siRNA for IL1B, RRM2M, MMP3, PERP or siRNA Control and grown to full

confluence. 24 hours post-transfection, wounds were created using Essen® 96-pin

WoundMaker™ (Essen BioScience, Ann Arbor, MI). The assay plates were incubated in the

IncuCyte® (Essen BioScience) and equilibrated for at least 15 minutes before the first scan.

The IncuCyte® ZOOM software was set to scan the plates every 2 hours.

Transwell migration assay was performed using the BD BioCoat Matrigel Chamber (BD

Falcon, Erembodegem, Belgium). Keratinocytes were transfected with 20 nM GapmeR for 24

hours. 3×104 cells in HKGS-free medium were placed into the upper chamber of the insert.

Medium containing