Fig. 1. Kaempferide (Kaem) identified as a hit compound based on lysosomal activity promotes autophagy flux in HeLa cells.

a Chemical structure of the autophagy inducer Kaem. b, c DMSO control or Kaem-treated HeLa cells were stained with acridine orange (AO), and confocal microscopy was performed (b). Graph shows mean ± SD (n = 30) of acidic vesicles per cell (c). Kaem, 20 µM. Scale bar, 50 µm. d–f Hela cells treated with DMSO control or Kaem for 24 h. Cell extract was subjected to western blot analysis using antibodies against LC3B and p62. Representative images (d) and intensity of p62 (e) and LC3B (f) immunoblot bands normalized to ACTB. Kaem, 20 µM. Graph shows mean ± SD from three independent experiments. g–i Kaem treatment in the presence/absence of chloroquine (CQ). Cell extract was subjected to western blot analysis using antibodies against LC3B and p62. Representative images (g) and intensity of p62 (h) and LC3B (i) immunoblot bands normalized to ACTB. Kaem, 20 µM; CQ, 10 µM. Graph shows mean ± SD from three independent experiments. j, k HeLa cells transfected with mRFP-GFP-LC3 were treated with DMSO control, rapamycin (Rapa), bafilomycin A1 (Baf A1), and Kaem for 48 h, respectively, and confocal microscopy was performed. Representative images (j) and number of yellow (autophagosome) and red puncta (autolysosome) (k). Kaem, 20 µM; Rapa, 10 µM; BafA1, 10 nM. Scale bar, 10 µm. Graph shows mean ± SD (n = 5). Statistical significance was assessed by Student’s t-test. ***P < 0.001; **P < 0.01; *P < 0.05.