Figure 3: SupraT induces nucleophagic degradation of unrepaired damaged DNA.

A, A representative single confocal section is shown from three independent experiments. LNCaP cells stained for γ-H2AX (red) and LC3B (green) after treatment with 1 and 5 nM R1881. Arrow-heads indicate cells harboring γ-H2AX foci with lower LC3B puncta. Each experiment had at least 5 random field images. B, Scatter plot showing a correlation between the number of LC3B puncta and the number of γ-H2AX foci counted (n=19 measurements). Counting of puncta was performed using the image analysis software Fiji. C, Confocal microscopy images for PCa cell lines treated with 10 nM R1881 (T) and hydroxychloroquine (HCQ). Each experiment had at least 5 random field images. The lower panel is an inverted and magnified image of a single cell in the view-field for better visualization. Arrows indicate the localization of cytoplasmic DNA. D, Confocal images showing mitochondrial staining in LNCaP cells treated with vehicle (C), 10 nM R1881 (T), 10 μM HCQ, or combination of T+HCQ for 72h. Lower panel shows a magnified view for enhanced visualization. Each experiment had at least 5 random field images. E, Photomicrographs showing colocalization of LC3B (green) and DAPI (Gray) in LNCaP cells treated with control (C), 10 nM R1881 (T), HCQ (10 μM) or T+HCQ treated LNCaP cells (72h). Rightmost panel shows colocalized pixels. F, Graphical representation of fluorescence intensities of LC3B and DAPI on individual autophagosomes (n=16 measurements). G, Photomicrographs showing LC3B (green) and γ-H2AX (red) in LNCaP cells after 72h of treatment with T+HCQ. Lower panels show the enlarged insets with a region of interest. Arrow-heads in the lower-left image indicate the presence of DNA (DAPI) in autophagosomes (LC3B), and lower-right image show γ-H2AX positivity in DNA present in those autophagosomes. Each experiment had at least 5 random field images. Scale bars: 5μm (A), 10μm (C), 10μm (D), 10μm (E), and 10μm (G).