Fig. 5

Mislocalization of integrin α6 in Beclin 1-silenced primary keratinocytes. a Primary keratinocytes were transfected with the indicated siRNAs, and after 3 days, cells were differentiated by the addition of 2 mM CaCl₂. Then, cells were harvested and lysed on the indicated days. Immunoblot analysis was performed using the indicated antibodies. Representative images of three independent experiments are shown. b, c Primary keratinocytes were transfected with the indicated siRNAs, and after 3 days, cells were differentiated by the addition of CaCl₂. Cells were then assayed for internalized anti-integrin α6 antibodies after 6 h from CaCl₂ addition. Nuclei were counterstained with DAPI (blue). Representative images are shown in b. Scale bars = 20 µm. Note that the puncta of integrin α6 were observed in Beclin 1-silenced cells, but were scarce in control cells. c The number of cells with integrin α6 puncta was counted (n = 3, mean ± SD). Asterisk indicates a significant difference at p < 0.05. (Student’s t test) d Similar experiments with b were performed using anti-Rab11 (green) and anti-integrin α6 (magenta) antibodies. Cells were counterstained with DAPI (blue). Representative images are shown. Scale bars = 5 µm. A magnified image of an integrin α6 punctum in Beclin 1-silenced keratinocytes are shown in the right panels. Scale bars = 5 µm. e Similar experiments with d were performed using an anti-EEA1 antibody instead of an anti-Rab11 antibody. Uncropped immunoblot images are provided in Supplementary Figure 5a. Data sources for c are provided in Supplementary Data 3