Fig. 1: Super-resolution imaging of lysosome clustering upon stimulation.

a Schematic representation of the formation of lysosome clusters in CCCP-induced mitophagy. b Formation of lysosome clusters in HeLa cells with or without CCCP-treated for 12 h. Lysosomes were stained by 200 nM commercial LysoTracker Red (LTR) at 37 °C for 30 min and then observed using SIM. White rectangles 1 and 2 show an enlarged area; white dotted lines indicate cell membranes; N, nucleus. c Lysosome puncta count in HeLa cells with and without CCCP treatment. d Distributions of lysosome areas in untreated HeLa cells. e Ratio of clustered lysosome clusters to the total number of lysosomes in untreated and CCCP-treated HeLa cells. f SIM revealing the dynamic process of the formation of lysosome clusters in CCCP-treated HeLa cells. The white arrow shows a representative dynamic formation of a lysosome cluster. The lower right number indicates the ratio of lysosome clusters to lysosomes. g SIM images and (h) qualification of the formation of lysosome clusters in RM-, EBSS-, and O/A-treated HeLa cells. i Specific lysosome probe, Coupa-lyso, was used for revealing lysosome cluster in living cells. Cells were stained with Coupa-lyso without (left) or with (right) CCCP treatment. White rectangles indicate the enlarged region. j CCCP-treated cells co-stained with Coupa-lyso and DAPG, showing lysosome cluster contact with autophagosome. White rectangles indicate representative clustered lysosomes around autophagosome. Data in c, e and h are presented as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.