Analysis of gene expression reveals major rearrangement after Ca²⁺/FCS induction.

<p>Serum and calcium-dependent genes in 60%-confluent hNEK cultures. A. Histogram represents normalized mRNA levels determined by qPCR in cells cultured in basal medium (no FCS, 0.1 mM Ca²⁺) reports with black columns, or after a 3-day calcium switch (no FCS, 1.8 mM Ca²⁺) reports with dark grey column, or after a 3-day serum switch (2% FCS, 0.1 mM Ca²⁺) reports with light grey columns or after a 3-day double serum/calcium switch (2% FCS, 1.8 mM Ca²⁺) reports with white columns. Genes studied were: 1) markers of basal epidermal compartment: Keratin 5 (K5) and P2Y purinoceptor 2 (P2Y2), 2) marker of proliferative epidermal compartment: Keratin 16 (K16), 3) markers of spinal compartment: Keratin 1 (K1) and 10 (K10), 4) markers of granular compartment: Involucrin (INV), Filaggrin (FLG), Transglutaminase (TGM1), P2X purinoceptor 7 (P2X7). mRNA level has been calculated as described in <i>Materiel</i> and <i>methods</i> for the four experimental conditions of cell culturing before its normalization on the value for the -Ca²⁺ /-FCS condition. Data are presented as Mean ± SD (N=6). Significance was reached when p< 0.05 (‡). B. Same as (A) for mRNA levels of proliferating cell nuclear antigen (PCNA) and the inhibitors of cell cycle p21 (CDKN1A) and p27 (CDKN1B), left panel, and ER-stress related genes: PERK (EiF2AK3), ATF4 and ER-stress effector CHOP (DDIT3), right panel (N=6). C. Kinetics of keratinocyte differentiation reveals different serum and calcium sensitivities of gene expression. Values represent mRNA levels of the genes of interest normalized with GAPDH mRNA levels. Confluent hNEK were grown in 2% FCS + 0.1 mM Ca²⁺ for 1 day prior to an addition of 1.8 mM Ca²⁺ for 5 days (N=4). All points were significantly different from Day 0 with p<0.001.