Fig. 3

RPA inhibition in NPC cells enhances autophagic flux. a γH2A staining. Nuclear γH2A intensity was measured. The graph corresponds to the quantification of 50 cells for each group with three independent experiments. Blue, DAPI. Scale bar = 50 μm. b Immunoblot analyses of cell lysates were performed to determine the levels of p-AMPK, total AMPK, p-Beclin-1, total Beclin-1, γH2A, p-RPA2, total RPA2 and β-actin and are representative of three independent experiments. c GSEA identified the autophagy‒lysosome genes significantly elevated in the low RPA1/3 expression group in the GSE12452 gene expression data. d Quantification of LC3B staining after treatment with vehicle or 15 μM HANMO for 24 h. Blue, DAPI. Scale bar = 50 μm. e NPC cell lines were treated with DMSO or HAMNO at the indicated dose for 24 h to assess flux. Immunoblot analyses of cell lysates were performed to determine the levels of LC3B, p62 and β-actin. f Electron microscopy showed the ultrastructure of autolysosomes in 5-8F and S26 cells following 24 h of HAMNO treatment. Scale bar = 1 or 2 μm as indicated. In (a, b) and (d–f), all experiments were performed with three biological replicates, and for immunoblots, a representative image is shown. The statistical analysis for (a) and (d) was performed using the Kolmogorov‒Smirnov test, and the data are presented as medians with 95% CI