Figure 6

Analysis of TMEM45A gene expression level in normal human keratinocytes in vitro and in vivo. (A and B) Keratinocytes were cultured in autocrine conditions (C-2d, 2 days before culture confluence; C, confluence; C + 2d, 2 days after confluence; C + 4d, 4 days after confluence). (A) Total RNA has been extracted and retro-transcribed into cDNA. A real time PCR has been performed with specific primers for TMEM45A and for TBP and RPL0 for normalization. Results are expressed as induction level by comparison with the reference condition “C-2d”. Results are expressed as means ± 1 SD for n = 3, keratinocytes obtained from three different normal donors. ** = significantly different from C-2d (p < 0.01). (B) TMEM45A protein level was assessed by western blot analysis. RPL13a was used as the loading control. (C) Keratinocytes were cultured at low density in the presence of low (0.06 mM) or high (1.5 mM) calcium concentration for 48 hours. Total RNA has been extracted and retro-transcribed into cDNA. A real time PCR has been performed with specific primers for TMEM45A and for TBP and RPL0 for normalization. Results are expressed as induction level by comparison with undifferentiated cells (0.06 mM calcium). Results are expressed as means ± 1 SD for n = 3, keratinocytes obtained from three different normal donors. *** = significantly different from low calcium concentration ((p < 0.001, paired Student’s t test). (D) Immunohistological staining for TMEM45 in sections of human normal skin section with (b and c) or without primary antibody (a).