Fig 2: Models for KIF4A function chromosome alignment and anaphase spindle formation. (A) The model summarizes Aurora-regulated kinetochore-dependent and -independent pathways contributing to chromosome congression. At kinetochores, Aurora B destabilizes kinetochore–microtubule attachments for nonbioriented chromosomes. Aurora A phosphorylates the condensin I–bound pool of KIF4A at polar chromosomes, promoting KIF4A motor activity and chromosome congression. (B) In anaphase, PRC1 and KIF4A control microtubule growth and the organization of the anaphase spindle, ensuring symmetric furrowing. The KIF4A mutant FF1154AA shows this interaction is essential for regulated microtubule growth and organized cytokinesis.

Fig 3: Aurora A forms a complex with H-Ras and Raf-1, acting through H-Ras to enhance ERK activationA. Aurora A directly interacts with Raf-1. TR-FRET was performed using HEK 293T lysates in which GST Raf-1 and Venus-Flag Aurora A along with vector controls were co-expressed. TR-FRET signals were recorded using an EnVision multilabel plate reader. Data shown are average signals with SD from duplicate samples. B. Aurora A associates with Raf-1. GST pull-down (as described in Figure 1B) between GST Raf-1 and Venus-Flag Aurora A with corresponding western blot analysis of inputs to assess changes in pERK compared to total ERK 48-hours post-transfection in HEK 293T cells. C. Aurora A/H-Ras/Raf-1 interactions stabilize the protein signaling complex. GST pull-down comparing the ability of wild-type (H-Ras WT) or dominant negative (H-Ras S17N) H-Ras to isolate either co-expressed Aurora A, Raf-1, or both proteins. Western blot analysis demonstrates binding of Aurora A or Raf-1 to H-Ras and the induced effect on pERK. Since both epitope-tagged proteins resolve around the same size, anti-Aurora A and anti-Raf-1 antibodies were used instead of anti-Flag. GST-tagged H-Ras WT and H-Ras S17N were detected using anti-GST antibody. Changes in pERK were detected using anti-pERK antibody. D. Proposed model for the role of Aurora A in the Aurora A/H-Ras/Raf-1 oncogenic singaling complex.

Fig 4: Bexarotene decreases tumor incidence in a tumor xenograft model of RCC. (A) Schematic depicting the workflow outlining the treatment regimen using bexarotene. (B) Rate of tumor take plotted as a percentage of lesions greater than 200 mm3 in size (tumor positive) at each of the indicated time points [week (W)1–W8] for vehicle (circles) and bexarotene (squares)-treated groups. (C) Representative H&E stained images (10× objective) of xenograft tumors from vehicle and bexarotene-treated animals. Scale bars: 500 µm. (D) Representative Ki67-stained images (10× objective) of xenograft tumors from vehicle- and bexarotene-treated animals. Scale bars: 500 µm. (E) Final tumor volume (mm3) measured following euthanasia of the mice treated with vehicle (circles) or bexarotene (squares). (F) Final tumor weight (g) measured for each tumor excised from mice treated with either vehicle (circles) or bexarotene (squares) at the time of euthanasia as shown. (G) Lysates from tumors of vehicle- and bexarotene-treated xenografts immunoblotted for the indicated antibodies. (H) Densitometric analysis showing a ratio of phospho-AURKA to total AURKA in vehicle- (circles) and bexarotene (squares)-treated xenograft tumors. * denotes statistical significance between the groups as indicated.

Fig 5: Phosphorylation of PKM2 at T45 promotes glycolysis and serine synthesis pathway.A, endogenous PKM2 was knocked down in H1299 cells and shRNA resistant Flag-PKM2 WT, Flag-PKM2 T45A, or Flag-PKM2 K433E mutant were expressed. Seahorse assays were performed to evaluate the glycolytic flux. ECAR over time (left panel) and ECAR in different stages of the measurement (right panel) were shown (n = 3). B, H1299 cells were treated with or without Aurora A kinase inhibitor, MLN8237 (100 nM). Seahorse assays were performed to evaluate the glycolytic flux. ECAR over time (left panel) and ECAR in different stages of the measurement (right panel) were shown (n = 3). C, the glycolytic tracing assay was performed with 13C-labeled glucose in cells used in (A) (left panel). The relative abundance of the 13C-labeled glycolytic metabolites: G6P, PEP, serine, and IMP were shown (right panel). D, the glycolytic tracing assay was performed with 13C-labeled glucose in cells with or without Aurora A overexpression. The relative abundance of serine was shown. E, Flag-Aurora A was coexpressed with HA-PKM2 in H1299 cells. Extracts of H1299 cells with or without the treatment of serine/glycine starvation for 24 h were incubated with Flag beads. The elutes were immunoblotted with the indicated antibodies (left panel). Quantified data were shown (right panel) (n = 3). F, proximity ligation assay (PLA) was performed in H1299 cells with Aurora A and PKM2 antibodies upon the treatment of serine/glycine starvation for 24 h. DAPI and PCNA were cell cycle indicators (left panel). The scale bar represents 10 µm. The PLA signals per cell were quantified (right panel) (0 h, n = 29; 24 h, n = 27). DAPI, 4',6-diamidino-2-phenylindole; ECAR, extracellular acidification; G6P, glucose 6-phosphate; IMP, inosine monophosphate; PEP, phosphoenolpyruvate.