Fig 1: Localization of phospho-PLK4 to centrosomes, kinetochores, and midbody varies with the phase of the cell cycle when an antibody to the N-terminal PEST domain is used. Phospho-serine305-PLK4 is identified first in centrosomes of interphase and prophase cells followed by kinetochores (metaphase and anaphase) and, subsequently, the cleavage furrow (telophase) and midbody (cytokinesis) of synchronized cells by immunofluorescence (IF) microscopy using an anti–phospho-serine305-PLK4 rabbit polyclonal antibody (our Ab#3 to a PLK4-serine305 peptide; red IF). Anti–α-tubulin antibody (green IF) identifies centrosomes, spindle apparatus, and midbody. DAPI stain (blue) identifies DNA. Results are illustrated for OVCAR3 ovarian cancer cells photographed at 400× original magnification. (Lower) Schematic diagram illustrates approximate location (arrows) of our rabbit polyclonal antibody (Ab#3, anti-phospho-PLK4) for phospho-PLK4. Similar results were obtained with a second, independently produced anti–phospho-serine305-PLK4 antibody (14299) from the laboratory of G.P.N.
Fig 2: PLK4 induces radioresistance in GBM. (A) RT-qPCR and (B) western blot analysis for PLK4 expression in U87 cells treated with or without radiation (12 Gy; *P<0.05, **P<0.01 vs. 0 h; one-way ANOVA followed by Dunnett's post hoc test. β-actin served as the control. (C) RT-qPCR results showed that PLK4 mRNA expression was markedly increased following radiation (12 Gy), and could be partially eliminated by shPLK4. **P<0.01, ***P<0.001; one-way ANOVA followed by Dunnett's post hoc test. (D) In vitro cell proliferation assay for U87 cells transduced with either shNT or shPLK4 lentiviruses, followed (or not) by radiotherapy (12 Gy). **P<0.01 vs. shNT; one-way ANOVA followed by Dunnett's post hoc test. (E) Flow cytometry (Annexin V and propidium iodide) determined the apoptosis of U87 cells transduced with shNT or shPLK4, followed (or not) by radiotherapy (12 Gy). (F) RT-qPCR analysis of U87 cells transduced with PLK4 overexpression or control lentivirus. **P<0.01; t-test. (G) Flow cytometry determined the apoptosis of U87 cells transduced with PLK4 overexpression or control lentivirus, followed (or not) by radiotherapy (12 Gy). GBM, glioblastoma; ANOVA, analysis of variance; RT-qPCR, reverse transcription quantitative polymerase chain reaction; ns, not significant; PLK4, polo-like kinase 4; sh, small hairpin RNA; IR, radiation.
Fig 3: PLK4 is localized to centrosomes throughout S-phase, G2, and M-phase during the cell cycle when an antibody against C-terminal PB domains is used for immunofluorescence (IF). IF microscopy using anti-PLK4 rabbit polyclonal antibody (C-PLK4, ab137398; Abcam; to aa 614–871; red IF) and an anti–α-tubulin antibody (green IF) in synchronized cell lines permitted identification of PLK4 in centrosomes throughout the cell cycle (interphase, prophase, metaphase, anaphase, telophase, and cytokinesis). Results are illustrated for OVCAR3 ovarian cancer cells photographed at 400× original magnification. (Lower) Approximate location of the commercially available (C-PLK4) antibody used for IF above. Please note differences in PLK4 distribution compared with Fig. 2 based on phosphorylation status and epitopes recognized.
Fig 4: PLK4 overexpression indicates high risk of lymph node metastasis and distant metastasis or surrounding recurrence, and poor prognosis in breast cancer. Lymph node status (A) and metastasis or recurrence status (B) was analyzed based on the PLK4 expression which was divided into low or high by the median values. PFS (C) and OS (D) curves were generated based on the PLK4 protein expression statuses in 154breast cancer samples.
Fig 5: PLK4 promotes GBM proliferation and tumorigenesis. (A) Phase-contrast and fluorescence images showing that pGFP-shPLK4 lentivirus was successfully transduced into U87 cells. (B) RT-qPCR and (C) western blot analysis of U87 cells transduced with shPLK4 or shNT (***P<0.001 vs. shNT; t-test). β-actin served as the control. (D) In vitro cell proliferation assays showed that shPLK4 reduced U87 cell proliferation in cells (**P<0.01 vs. shNT; one-way ANOVA). (E) Representative images of hematoxylin and eosin-stained mouse brain sections implanted with U87 cells transduced with shPLK4 or shNT. (F) Kaplan-Meier analysis of nude mice intracranially implanted with transduced U87 cells (n=5; P=0.0244; log-rank test). GBM, glioblastoma; ANOVA, analysis of variance; RT-qPCR, reverse transcription quantitative polymerase chain reaction; PLK4, polo-like kinase 4; sh, small hairpin RNA; NT, non-targeting control.
Supplier Page from Abcam for Anti-PLK4 antibody