mRNA was extracted from HaCaT, HEKn cells (passage 1 & 3) or HEK-CaY (passage 3 & 10) and expression of differentiation markers K10 (A) and Inv (B) or basal marker K14 (C) assessed by qRT-PCR normalised to multiple reference genes B2M and YWHAZ.

<p>Data is expressed as relative normalised expression, Mean +/- SEM, n = 3. *** = p<0.001, ns = not significant, p>0.05, ANOVA. Isolated protein was assessed by western blot to confirm expression with β-tubulin used as a loading control (D). Images are representative of three experimental repeats.</p

Neonatal human keratinocytes were cultured in CellnTec basal media (containing 0.07mM calcium) or basal media supplemented with 10μM Y-27632.

<p>Cells were assessed for cell viability at each passage, performed at 90% confluence. Trypan blue exclusion assay differentiated between viable and non-viable cells based upon membrane permeability and growth rate calculated as population doubling per day (A). Growth rate is represented graphically as population doubling over days in culture (B). Phase contrast images at 20x magnification demonstrate morphology of primary passage 3 and 10 HEKn cultured in CnT-07 alone and passage 3 and 10 HEKn-CaY supplemented with Y-27632 (C). n = 3 isolations from separate donors. Data is expressed as Mean +/- SEM. Images are representative of three experimental repeats.</p

Diagram of human skin showing differentiated keratinocytes of the epidermis and expression of stratification markers Keratin 10 (K10), Keratin 14 (K14) and Involucrin (Inv).

<p>Diagram of human skin showing differentiated keratinocytes of the epidermis and expression of stratification markers Keratin 10 (K10), Keratin 14 (K14) and Involucrin (Inv).</p

Passage 10 HEK-CaY were seeded onto LabTek II Chamber slides for the 2D differentiation model.

<p>Cells were cultured for 72hrs in either 0.07 calcium and assessed for K14 (D), K10 (E) or Inv (F) expression by immunocytochemistry or 1.2mM calcium and K14 (G), K10 (H) or Inv (I) expression assessed. DAPI nuclear counterstain was also performed on cells grown in 0.07mM (A-C) and 1.2mM (J-L) calcium to confirm the presence of live cells. All images are taken at 20x magnification and are representative of three experimental repeats.</p