Fig 1.
Silencing of PIN1 and PTOV1 decrease the proliferation, colony formation, and migration of MDA-MB-231 cells.
(A) Cell viability was estimated by using CTG assay and luminescence signal was recorded in microtiter plate ELISA reader. (B) Representative images of the colony of cells transfected with siRNAs in 6 well plates after 2 weeks of incubation. (C) Densitometry representation of colony number using Image J software. (D) Representative images of the wound area of each treated group after 0, 6, and 12 hours. (E) Densitometry representation of Wound area using Image J software. (F) Representative image of PIN1 and PTOV1protein knockdown after transfecting cells with respective siRNAs. β- actin was used as a loading control. Data are represented as mean ± SD of three independent experiments. *** Significant difference from scramble groups (p < 0.001).
Fig 2.
Silencing of PTOV1 and PIN1 lead to the G2/M arrest in MDA-MB-231 cells.
(A) Histogram representing the cell cycle distribution of MDA-MB-231 cells transfected with PTOV1 siRNA for 48 hours (left panel) and statistical representation of percentage distribution of the population in each phase (right panel). (B) Histogram representing the cell cycle distribution of MDA-MB-231 cells transfected with PIN1siRNA for 48 hours (left panel) and statistical representation of percentage distribution of the population in each phase (right panel). Data are represented as mean ± SD of three independent experiments. *, *** Significant difference from scramble groups (p < 0.05 & 0.001).
Fig 3.
Silencing of PIN1 and PTOV1 alter cell morphology, apoptosis, and intracellular ROS production.
(A) Representative images of cells transfected with the siRNAs after 72 hours and stained with Hoechst 33342. (B) Microscopic evaluation of intracellular ROS production in each group after 72 hours using CM-H2DCFDA. Images were captured using Nikon fluorescent microscope, right panel shows the graphical representation of the ROS production in different set of experiments. *** represents the significant difference from scramble (p< 0.001).
Fig 4.
Silencing of PIN1 and PTOV1 drive cells towards apoptosis and PTOV1 knockdown further induces autophagy.Western blot analysis of (A) Bcl-2, (B) Bcl-xL, and (C) BAX after 72 hours of transfection. Expression analysis of autophagy markers (D) LC3, and (E) Beclin-1after transfecting cells with scramble and PTOV1 siRNA for 72 hours (F) Beclin-1 after transfecting cells with respective scramble and PIN1 siRNAs. The density was calculated by image J software. β- actin was used as a loading control. The experiments were performed in triplicates.
Fig 5.
Silencing of PIN1 and PTOV1 share similar expression profile for the majority of genes at the transcriptional level.
(A) The upper panel represents the expression of mRNA after transfecting cells with PIN1 siRNA for 72 hours. (B) The lower panel represents the expression of mRNA after knockdown with PTOV1 siRNA for 72 hours. Relative expression was calculated using delta-delta CT method. The expression in scramble group was considered 1.00. Experiments were performed in triplicates. Data are represented as mean ± SD.*, **, *** represents significant difference from the scramble with p< 0.05, 0.01 and 0.001 respectively.
Table 1.
The genes used for the mRNA expression analysis with their cellular functions are listed in the table below.
Fig 6.
Silencing of PIN1 and PTOV1 cooperatively target multiple proteins and share similar expression status at the translational level.
A. (i) PIN1 and PTOV1 interaction studied by co-immunoprecipitation in MDA-MB-231 cells. (ii& iii) Western blot analysis of PIN1 and PTOV1 after transfecting cells with siRNAs for 72 hours, (iv) with Juglone (5 μM) for 48 hours, and (v) Overexpression with PIN1-XPRESS vector. Expression analysis of B. (i) β- catenin, (ii) Cyclin D1, (iii) c-Myc, & further analysis of expression of C. (i) RACK1, (ii) Cyclin B1, (iii) MAPK8, and (iv) p-ERK after 72 hours of siRNA transfection. The densitometry was calculated by using image J software. β-actin was used as a loading control. The experiments were performed in triplicates.
Fig 7.
Overexpression of PIN1 induces the expression of PTOV1, which suggest that PIN1 might be the upstream protein which regulates the expression of PTOV1. Both the gene cooperatively share MAPK pathway including the expression of ERK, C-Jun and the master regulator C-Myc as well as induces the expression of apoptosis and autophagy related genes. PTOV1 interacts with RACK1 which is involved for protein synthesis in cancer similarly knockdown of PIN1 also inhibits RACK1 and thus PIN1 might contribute to oncogenic signalling via PTOV1-RACK1 interaction.