Fig. 1

Characterization of MCF-7 and MDA-MB-231 spheroids. Sections of MCF-7 and MDA-MB-231 spheroids that were grown for 9 days (a, b _ii , c, d, e and f) followed by PFA fixation, embedding, and histological and immunohistochemical analysis (IHC). a: Hematoxylin and eosin (H&E) staining. b: MCF-7 spheroids (3D) and 2D cultures were grown for 4 days in parallel followed by lysis and Western blotting with PARP antibodies. Top panel shows representative Western blots, while panel b _i shows quantifications of band intensities normalized to that of corresponding 2D culture. Error bars denote SEM. 8 n. A two-tailed, paired Student’s t-test was used to test for statistically significant difference in means between the two groups. * indicates p < 0.05. Panel b _ii shows a section of a MCF-7 spheroid stained with antibodies recognizing cleaved PARP (c-PARP) only. Dashed lines indicate spheroid boundaries and arrowheads indicate c-PARP positive nuclei. Scalebar: 20 μm. c: ZO-1 and E-cadherin staining of MCF-7 spheroids. Arrowheads indicate ZO-1 puncta. Scalebars: 20 μm. d: Ezrin/Radixin/Moesin (ERM) staining. Scalebar: 20 μm. e and f: Pimonidazole (Pim) and CA IX staining was used to detect hypoxic regions. Scalebars: 100 μm and 20 μm, respectively. All images are representative of 4–5 n, except for data in d-right and e, which represent 3n. L, P and C: indicate lumen, periphery and core, respectively, of spheroids