Fig. 4

Mortaparib treatment caused oxidative stress and targeted mortalin-PARP1 interactions. JC-1 staining showed lower mitochondrial membrane poteintial as signified by increase in its monomers and decrease in aggregates in Mortaparib-treated HeLa cells (Scale bar = 20 μM); microscopic images (a) and their spectrophotometeric analysis (b) are shown. Mortaparib treatment showed decrease in total cellular ATP concentration (c) and increase in ROS in HeLa cells (Scale bar = 20 μM) (d). Western blot analysis of control and Mortaparib-treated cells showing decrease in p38, p-JNK and NF-κB; Quantitation is shown on the right (e). Co-immunoprecipitation of PARP1-mortalin (f) revealed that they exist in a complex, and Mortaparib abrogated their interaction. PARP1 interacting domain of mortalin was defined by immunoprecipitation of the two proteins from lysates of COS7 cells transfected with mortalin deletion mutants tagged with V5 (g). Deletion mutants (amino acids residues shown in the Table) that showed binding with PARP1 are marked with red squares. The ones that did not bind to PARP1 are marked by dotted black squares. Mortalin-PARP1 interactions were assigned to two domains (i) amino acid residues 1–104 and (ii) 252–390. The quantitative data represents mean ± SD obtained from, at least, three independent experiments; p-values were calculated using Student’s t-test. * < 0.05, ** < 0.01 and *** < 0.001 represent significant, very significant and very very significant, respectively