Fig. 5. Expression of laminin-332 is upregulated upon scratch wounding and correlates with HIF1 induction. (A) Histograms representing the quantification obtained by microarray analysis of the LAMA3 transcripts at different time points after cell wounding (left panel). Results were confirmed by real time Q-PCR (right panel). Values are mean ± s.e.m. of three independent experiments performed in triplicate. NW, not-wounded; W, wounded. (B) Immunofluorescence detection of HIF1 (green) and laminin-332 (BM165 antibody; red), 15 hours after wounding (W), in NHKs (upper panel) and HaCaT (lower panel), compared with nonwounded counterparts (NW). Nuclei were detected using DAPI (blue). The dotted line in the upper panel highlights the original position of the scratch wound edge, and the arrows indicate the direction of cell migration. The lower panel shows HaCaT cells in a nonwounded area of the cell culture far from the wound site. Scale bar: 20 µm. (C) Left panel, HaCaT cells untreated (NS) or treated with Cobalt (Co²⁺) for 15 hours to increase HIF1 protein. HIF1 protein that was measured by western blot analysis. GAPDH was detected as a loading control. Right panel, real-time quantitative PCR showing normalised LAMA3 mRNA expression in HaCaT cells nonstimulated (NS) or treated with Co²⁺ for 15 hours. (D) HaCaT (lower panel) and NHK cells (upper panel) were either left untreated (NS) or treated with Co²⁺. After 24 hours, cells were fixed and immunofluorescence analysis was carried out to detect HIF1 protein (green) and laminin-332 (red). Nuclei were stained using the DAPI. Scale bar: 20 µm.