Figure 4.

Cyclophilin inhibitor treatment increases unfolded proteins and PDI aggregates formation in BRCA1-def cancer cells

(A) Photomicrographs of DMSO- or CsA-treated MDA-MB-436 (+ or def) cells labeled with TPE-MI (light/blue) and quantitative analysis of TPE signal intensity of each cell preparation. Scale = 20 μm. TPE = TPE-MI. + = BRCA1-replete. def = BRCA1-deficient.

(B) Representative flow dot plots of TPE signals in DMSO- or CsA-treated MDA-MB-436 (+ or def) breast cancer cells and quantitative analysis of TPE-high cell population of each cell preparation.

(C and D) Quantitative analysis of TPE signal intensity of each cell preparation.

(E) Confocal microscopy images of DMSO- or CsA-treated MDA-MB-436 (+ or def) breast cancer cells. Cells were co-immunostained with CypB (red) and PDI (green) antibodies. Top, 2 days post-treatment. Bottom, 3 days post-treatment. All cells were counterstained with DAPI (blue). Scale = 5 μM. Bar charts are quantitative analyses of cell populations with PDI aggregate formation.

(F) Depletion of PDI induced severe synthetic lethality in MDA-MB-436 BRCA1-def cancer cells. Top, Western blot analysis. Middle, representative images of colony forming units (CFUs) from control or CypB knock-down cancer cells after 14 days culturing. Bottom, quantitative analysis of the clonal colony formation assay. Data are displayed as mean ± SD. P values were calculated by Student’s two-tailed, unpaired t-test. See also Figure S8.