Fig 2: Plk1-Plk4 expression in pediatric B-ALL patients. The boxplot displays median relative mRNA expression of (A) Plk1, (B) Plk 2, (C) Plk3 and (D) Plk4 in B-ALL cell lines (red), patient samples (blue) and PBMCs from healthy blood donors (green). The obtained values were plotted as log2 of relative quantification (RQ) values using ggplot. Plk1 and Plk4 expression in primary patient cells was significantly higher (***p = 0.0006/0.0003 respectively) compared to PBMCs. Upper box line represents 75th, middle -50th (median), and lower - 25th percentile (*p < 0.05). Bold black dots represent outliers. GAPDH was used as an internal control.

Fig 3: Apolo1 is a bona fide PLK1 regulator(A) Kinase activity measurement of PLK1 in the presence of Apolo1, Bora, Aurora A, and their combination. Bacterially recombinant PLK1 exhibits no kinase activity and requires activation by Bora and Aurora A. An aliquot of BI2536 was included to determine minimal PLK1 kinase activity. Apolo1 alone did not activate PLK1 kinase activity.(B) Endogenous PLK1 immunoprecipitates from interphase (2 mM thymidine, 16 h) and mitosis (100 ng/mL nocodazole, 16–18 h) HeLa cells were detected with antibodies against Apolo1, PLK1, and Cyclin B1.(C) Schematic illustration of full-length and deletion mutants of Apolo1. Specifically, Apolo1-N indicates 1–176 aa, Apolo1-M indicates DUF (177–728 aa), and Apolo1-C indicates 729–853 aa.(D) Coimmunoprecipitation assay of FLAG-PLK1 and GFP-Apolo1 truncation fragments in 293T cells.(E) Schematic illustration of PLK1 full-length and domain regions. Specifically, PLK1-KD represents the PLK1 kinase domain (1–303 aa) and PLK1-PBD represents the PLK1 polo-box binding domain (304–603 aa).(F) Coimmunoprecipitation assay of FLAG-Apolo1 with GFP-PLK1 full-length and truncation fragments in 293T cells.See also Figures S2 and S3.

Fig 4: PLK1 interacts with NUAK1 at the CDK priming Ser445 site and phosphorylates NUAK1 at Ser476 and Ser480 and targets it for phosphorylation-mediated degradation(A) U2OS cells stably expressing HA–NUAK1 were treated with the indicated inhibitors for 4 h with or without 50 nM calyculin A treatment. Cell lysates were subjected to immunoblotting with the indicated antibodies. The inhibitors were used at 1 µM final except for MLN-4924 (3 µM), BI605906 (5 µM), WZ4003 (10 µM) and D4476 (30 µM). (B) Endogenous NUAK1 was immunoprecipitated (IP) from 1 mg of U2OS cell lysates treated with calyculin A (50 nM) and with or without the indicated concentrations of BI2536 and GSK461364 over the indicated periods of time prior to lysis. Immunoblotting was carried out to detect NUAK1 levels in the immunoprecipitates. Pre-immune IgG was used as a control. (C) U2OS cells stably expressing NUAK1 WT were treated with the indicated inhibitors for 1 h. HA–NUAK1 was immunoprecipitated and analysed by immunoblotting with the indicated antibodies. (D) HA–NUAK1 with or without BI2536 (1 µM) treatment was immunoprecipitated (IP) from U2OS Flp/In cells stably expressing HA–NUAK. XIC analysis of the Ser476- and Ser480-containing phosphopeptide (R.ESGYYSSPER.S+1P) was carried out. The m/z value corresponding to the Ser476- or Ser480-containing phosphopeptide was detected in both samples. In the BI253-treated cells, the m/z value corresponding to the Ser476- or Ser480-containing phosphopeptide was found to possess a ~3-fold-higher signal intensity than the control immunoprecipitate. (E) HEK-293 cells were transfected with expression plasmids for the HA-tagged NUAK1 WT or the indicated mutants. At 36 h post-transfection cells were lysed and HA-tagged proteins were immunoprecipitated from 1 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with indicated antibodies. (F) HEK-293 cells were transfected with expression plasmids for the HA-tagged NUAK1 WT or S445A mutant. At 36 h post-transfection cells were lysed and HA-tagged proteins were immunoprecipitated from 1 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with indicated antibodies. (G) HEK-293 cells transiently overexpressing NUAK1 WT or the S445A mutant were treated with 50 nM calyculin A over the indicated periods of time. The cell lysates were analysed by immunoblotting with the indicated antibodies.

Fig 5: AURKA knockdown induces volasertib sensitivity while reversely PLK1 knockdown causes phthalazinone pyrazole sensitivity in DMG cells(A) Western blot analysis showing PLK1 expression in shRNA-control and PLK1-shRNA expressing DMG cells (left) and AURKA expression in shRNA-control and AURKA-shRNA expressing DMG cells (right).(B) Dose-response curves illustrating the cytotoxic effect of volasertib in empty vector versus AURKA-shRNA expressing DMG cells (left) and PP in empty vector versus PLK1-shRNA expressing DMG cells (right). Viability-curves depicting circles represent empty vector cells while squares indicate knockdown. Error bars indicate SD.