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. 2020 Apr 2;10:5800. doi: 10.1038/s41598-020-62430-z

Figure 5.

Figure 5

EIPA-mediated cytotoxicity involves DNA-damage, ER-stress, dysregulation of autophagy and reduced proliferation. (A–C) Top panel: Representative Western blots of MCF-7 spheroids treated with EIPA (10 µM) for 48 h. Lower panel: Quantification of band intensities normalized to control. (A) Retinoblastoma protein (pRb) phosphorylation (S807/811). 4 n; (B) PARP cleavage. 3 n; (C) Caspase 7 cleavage. (MCF-7 cells do not express caspase 3). 3 n. Error bars denote SEM. A paired (A) or ratio paired t-test (B) was used to test for statistically significant differences between conditions. (D) Representative Western blot of MCF-7 spheroids treated with cariporide (10 µM), EIPA (10 µM) or HMA (10 µM) on day 2 and 4. Lysates were prepared after 7 days of growth and blotted for γH2AX. 3 n. (E) PI-stained MCF-7 spheroids grown for 9 days and treated with EIPA (0–10 µM) on day 2, 4 and 7. Representative of 3 independent experiments. Scale bar: 200 µm. Image overlays and adjustments of the intensities were performed using ImageJ software. (F) Representative Western blots of MCF-7 cells treated with cariporide (10 µM), EIPA (10 µM) or HMA (10 µM). DMSO serves as a vehicle for HMA. β-actin: loading control for CHOP and PDI; DCTN11: loading control for p53 and γH2AX; DCTN12: loading control for fPARP, cPARP, p62 and LC3B. (G) CHOP-stained MCF-7 cells treated with cariporide (10 µM) or EIPA (10 µM) for 4 days. Representative images of 3 independent experiments. Scale bar: 20 µm. Image overlays and adjustments of the intensities were performed using ImageJ software. (H) Representative Western blots of MCF-7 cells treated with EIPA (10 µM), Akti (1 µM), U0126 (10 µM) or a combination thereof. DMSO serves as a vehicle for Akti and U0126. *The Ctrl, DMSO and EIPA lanes were additionally run on a separate gel to allow longer exposure to visualize cPARP. β-actin: loading control for fPARP* and cPARP*. DCTN11: loading control for pERK, ERK, fPARP and cPARP; DCTN12: loading control for pAkt, Akt and CHOP. fPARP: full length PARP; cPARP: cleaved PARP. Full length blots are presented in Supplementary Fig. S6.