Keratinocyte differentiation promotes ER stress-dependent lysosome biogenesis

a BF analysis of control and CaCl2-incubated (at ~60？h) cells. b CLH (in μm) was measured (~60 cells, n？=？3) in each condition and then plotted (mean？±？s.e.m). c IFM analysis of LAMP-1 stained control and differentiated keratinocytes. Arrowheads point to LAMP-1 compartments and arrow indicates their distribution and increased number in differentiated cells. DL (in μm) in each condition was measured (~60 cells, n？=？3) and indicated (mean？±？s.e.m.). d The cellular distribution of LAMP-1 organelles was quantified visually (~100 cells, n？=？3) and then plotted (mean？±？s.e.m). e–h IFM analysis of keratinocytes internalized with either lysotracker or DQ-BSA, or transfected with Arl8b-GFP or GFP-Rab7, fixed and stained for LAMP-1. Arrowheads point to the LAMP-1-positive organelles. The degree of colocalization (Pearson’s coefficient, r) between markers is indicated separately (mean？±？s.e.m., n？=？3). Nuclei are stained with Hoechst 33258 and the insets are magnified view of the white boxed areas. Scale bars, 10？μm. i Glucocerebrosidase (GBA) activity was normalized with cell number and then plotted (fold change in mean？±？s.e.m., n？=？3). j Immunoblotting analysis of keratinocytes. The fold change in protein levels is indicated. * Indicates non-specific bands detected by the antibodies. k Cell surface levels of LAMP-1 and ？2 in control and differentiated cells. Fold change in mean fluorescence intensity (MFI) was calculated (n？=？3 in quadruplicates) and then plotted (mean？±？s.e.m.). *p？≤？0.05 and ***p？≤？0.00