Fig 2: Phosphorylated PLK1 (p-PLK) and total PLK1 (t-PLK) expression as determined by Western blot in DLD-1 and HT-29 cells treated with Epo (100 IU·mL-1), LFM-A13 (LFM 100 µM) and their combination for 48 h. Samples used for electrophoresis consisted of 20 µg of protein from six pooled cell extracts (n = 6). The band staining was quantified by densitometry. Bands of phospho-proteins are normalized to respective total proteins (A). Active and pro-caspase 3 expression as determined by Western blot in DLD-1 and HT-29 cells treated with Epo, LFM-A13 for 48 h. Samples used for electrophoresis consisted of 20 µg of protein from six pooled independent cell extracts (n = 6). Band staining was quantified by densitometry. Bands of phospho-proteins were normalized to respective total proteins (B). Effects of LFM-A13 on spindle assembly in DLD-1 cells. LFM-A13-treated DLD-1 cells showed aberrant microtubule assembly during mitosis and developed abnormal monopolar mitotic spindles with highly dense and hyperextended microtubules. Orange, ?-tubulin; red, a-Tubulin; blue, DNA/chromosomes (C). Effects of Epo, LFM-A13 and simultaneous use of both compounds (Epo + LFM-A13) on cell cycle progression of DLD-1 (D) and HT-29 cells (E). Cells were cultured in the presence of 100 IU·mL -1of Epo, 100 µM of LFM-A13 and their combination for 24 h at 37°C and then examined by DNA flow cytometry, as described in the Methods section. Epo + LFM-A13 treated DLD-1 and HT-29 cells show S phase fraction reduction and slight increase in phase G0/G1. Percentages of events at different stages in the cell cycle are shown.

Fig 3: Centrosome-targeted Plk1 inhibitors perturb early mitotic events.(A) Schematic depicting bipolar (top), abnormal bipolar (mid), and monopolar (bottom) mitotic spindle classifications. (B) Representative composite (left) images show a-tubulin (green), DNA (blue), and SNAP (magenta) or a-tubulin (right, gray) staining alone for each spindle type in U2OS cells. (C–E) Spindle profile measurements of U2OS (C) RPE (D) and HeLa (E) cells treated with 250 nM CLP-BI2536 for 4 hr. Spindle profiling indicates the % of each spindle type in drug-treated LoKI-on and LoKI-off cells. Number of cells analyzed per condition are indicated; (C) n = 3, *p=0.0214; (D) n = 3, *p=0.0269. (F) Schematic depicting that Plk1 phosphorylation of pericentriolar substrates facilitates accumulation of ?-tubulin at centrosomes and microtubule nucleation. (G, H) Representative composite (left) images show ?-tubulin (yellow) and DNA (blue) in U2OS cells expressing LoKI-off (G) and LoKI-on (H) treated with 250 nM CLP-BI2536 for 4 hr, followed by a 1 hr washout. 5X magnified grayscale images of centrosomal ?-tubulin (right). (I) Quantification of centrosomal ?-tubulin immunofluorescence in LoKI-expressing cells. Points represent individual cells (n). Data normalized to DMSO. A ROUT (Q = 1%) outlier test was performed and two values were removed prior to performing statistical tests; LoKI-off, n = 66, LoKI-on, n = 74, ****p<0.0001. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant. Source files for spindle profile analyses are available in Figure 3—source data 1.Figure 3—source data 1.Spindle profile analyses.

Fig 4: Combined Plk1 and AurA suppression at centrosomes more profoundly delays mitosis than global kinase inhibition.(A) Chemical structure of AurA kinase inhibitor CLP-MLN8237. (B) Dose-response curve of in vitro AurA kinase inhibition with CLP-MLN8237. (C) Pulse-chase experiments measuring CLP-MLN8237’s ability to block CLP-rhodamine conjugation to LoKI-on. In-gel rhodamine fluorescence (top), immunoblot of SNAP loading controls (mid), and fluorescence quantification of pulse-chase experiments (bottom). (D) Immunofluorescence of representative mitotic LoKI-on U2OS cells treated with DMSO or 100 nM CLP-MLN8237 for 4 hr. Composite images (left) show a-tubulin (green), DNA (blue), and SNAP (magenta). Immunofluorescent detection of pT288-AurA (right, gray) as an index of kinase activity is depicted. (E) Quantification of centrosomal pT288-AurA immunofluorescence after 4 hr CLP-MLN8237 treatment; 50 nM, LoKI-off, n = 18, LoKI-on, n = 53, **p=0.0014; 100 nM, LoKI-off, n = 18, LoKI-on, n = 48, **p=0.0026. (F) Schematic of dual LoKI-on moiety conjugated to CLP-BI2536 and CLP-MLN8237. Inset depicts MLN8237 in the ATP-binding pocket of AurA. (G) Representative time-lapse images of mitotic LoKI-off (top) and LoKI-on (bottom) cells transiently expressing GFP-H2B. Cells were treated with a combination (both drugs) of 250 nM CLP-BI2536 and 100 nM CLP-MLN8237 and monitored for 18 hr. (H) Quantification of time-lapse experiments depicts duration of mitosis from nuclear envelope breakdown to the onset of anaphase; DMSO, LoKI-off, n = 264, both drugs, LoKI-off n = 353, ****p<0.0001; DMSO, LoKI-on, n = 292, both drugs, LoKI-on n = 331, ****p<0.0001; both drugs, LoKI-off, n = 353, both drugs, LoKI-on, n = 331, ****p<0.0001. Points in (E) and (H) represent individual cells (n). Points in (E) are normalized to DMSO. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant. Source files for analysis of pulse-chase experiments are available in Figure 4—source data 1.Figure 4—source data 1.Analysis for pulse-chase experiments with CLP-MLN8237 in SNAP-PACT cells.

Fig 5: Validation of the LoKI platform.(A) Schematic of a centrosome-directed LoKI platform. SNAP-PACT fusion proteins conjugate CLP-linked Plk1 inhibitors at centrosomes. Inset depicts BI2536 in the ATP-binding pocket of Plk1. (B) Chemical structure of CLP-BI2536. (C) Dose-response curve of in vitro Plk1 inhibition with CLP-BI2536. (D) Structured illumination microscopy (SIM) of a LoKI-on U2OS cell labeled with CLP-fluorescein. Immunofluorescent detection of a-tubulin (green), DNA (blue), mCherry-SNAP-PACT (magenta) and CLP-fluorescein (yellow). Magnification of SNAP and CLP-fluorescein co-distribution at a centrosome (inset). (E) SIM micrographs of LoKI-off (left) and LoKI-on (right) U2OS cells. SNAP expression (top, magenta), CLP-fluorescein conjugation (mid, yellow) and composite images (bottom) are depicted. (F) Pulse-chase experiments measuring CLP-BI2536’s ability to block CLP-rhodamine conjugation to LoKI-on. In-gel rhodamine fluorescence (top), immunoblot of SNAP loading controls (mid), and fluorescence quantification of pulse-chase experiments (bottom). (G, H) Immunofluorescence of representative mitotic LoKI-off (G) and LoKI-on (H) U2OS cells treated with DMSO or 250 nM CLP-BI2536 for 4 hr. Composite images (left) show a-tubulin (green), DNA (blue), and SNAP (magenta). Immunofluorescent detection of pT210-Plk1 (mid, gray) as an index of kinase activity. 5X magnification of centrosomal pT210-Plk1 signals and surface plots measuring integrated intensity of pT210-Plk1 signal (insets). (I, J) Quantification of centrosomal pT210-Plk1 immunofluorescence for LoKI-expressing cells. Points represent individual cells (n). Data normalized to DMSO. Application of DMSO or CLP-BI2536 for 4 hr, (I) 100 nM, LoKI-off, n = 46, LoKI-on, n = 59, **p=0.0059; 250 nM, LoKI-off, n = 46, LoKI-on, n = 46, ****p<0.0001 and drug treatment followed by 1 hr washout (J) 250 nM, LoKI-off, n = 24, LoKI-on, n = 42, ****p<0.0001. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant. Source files for analysis of pulse-chase experiments are available in Figure 2—source data 1 and for quantification of pT210-Plk1 are available in Figure 2—source data 2.Figure 2—source data 1.Analysis for pulse-chase experiments with CLP-BI2536 in SNAP-PACT cells.Figure 2—source data 2.Raw analysis for pT210-Plk1 signal.