Fig. 2: Golgi stacks and lysosomes are closely apposed in differentiated keratinocytes.

A IFM analysis of proliferative and differentiated keratinocytes co-immunostained for the Golgi markers GM130 and p230. B co-immunostaining of the representative lysosome marker LAMP1 with the Golgi markers GM130 (representing cis/medial Golgi), p230 (trans-Golgi), TGN46 (trans-Golgi network) or expressed ARF1-GFP (ubiquitous in Golgi). Cells were visualized by Airyscan super-resolution microscope. Represented insets are the 2.5 times magnified, numbered (1-3) regions of the cells shown in Figs. 2A & S2A. Arrowheads represent the close apposition/contact between the Golgi and lysosomes. Scale bar=2.5 μm. Pearson’s correlation coefficient (r) and Manders’ overlap coefficients (M1 and M2) were calculated in differentiated cells (n) from at least 3 different experiments. ****(=P ≤ 0.0001) on the graphs indicating the significant difference between M1 and M2 values. C The total protein secretion from the proliferative and differentiated keratinocytes was measured by the SUnSET assay. Protein secretion at 0 h and 2 h was measured with or without the presence of cycloheximide (taken as experimental control) and Golgicide A (functional inhibitor of the Golgi). P= proliferative and D = differentiated conditions. UT= untreated, CHX= cycloheximide, GCA= Golgicide A. Puromycin signal in the lysate and in cell medium respectively indicate total synthesis and secretion. D Ultrathin cryosections of proliferative and differentiated keratinocytes were analyzed under TEM. Arrowheads represent the Golgi overlap to lysosomes. Golgi (G); Golgi Vesicles (GVs); lysosomes (L); mitochondria (M) and keratin fibers (K). Scale bar=200 nm. E The graph is representing % of lysosomes (L) that are closely apposed (within 100 nm) to the Golgi (G). F The distance (nm) between lysosomes (L) from the closest Golgi membrane in proliferative and differentiated cells. Dotted grey line on the graph was drawn on 50 nm on the X axis as a reference. The value of significance was calculated by Mann-Whitney non-parametric test. **** = P ≤ 0.0001.