tubulin polymerization assay kit  (Cytoskeleton Inc)

Journal: Cancer Research Communications

Article Title: Targeting Cancer-Associated PCNA with AOH1996 Induces Mitotic Catastrophe and Enhances Cisplatin Therapy in Cervical Cancer

doi: 10.1158/2767-9764.CRC-25-0648

Figure Lengend Snippet: AOH1996 disrupts centrosome organization and spindle integrity. A, Representative immunofluorescence microscopy images of HFK, HeLa, and CaSki cells treated with DMSO or 1 μmol/L AOH1996 for 24 hours, stained for β-tubulin (red), γ-tubulin (green), and DNA (DAPI, blue). In DMSO-treated cells, mitotic spindles and centrosomes appear organized and bipolar. In contrast, AOH1996-treated cells exhibit multipolar, shortened, and disorganized spindles with supernumerary centrosomes. B, Quantification of spindle length shows a significant reduction in AOH1996-treated cells across all lines, confirming spindle collapse or failure. C, Quantification of centrosome number per cell reveals a significant increase following AOH1996 treatment, most pronounced in cervical cancer (CaCx) cells. D, Chemical structures of AOH1996 and its structurally related analog AOH1996-8Nq, which contains a nitrogen substitution in the aromatic ring and lacks cytotoxic or mitosis-disrupting activity. E, Protein thermal shift assay of recombinantly expressed 6xHis PCNA with AOH1996-8Nq. A PCNA-stabilizing shift of 0.5°C at each concentration indicates binding interaction. F, Brightfield and immunofluorescence microscopy images of HeLa cells treated with 200 nmol/L AOH1996 or AOH1996-8Nq. AOH1996 causes cell rounding and centrosome/spindle disruption, whereas AOH1996-8Nq does not. G, Immunofluorescence microscopy of β-tubulin (red), γ-tubulin (green), and DAPI (blue) in HeLa cells. AOH1996 significantly decreases colocalization between γ-tubulin and β-tubulin, as shown by quantification (right). AOH1996-8Nq has no effect. Bottom, pixel-based colocalization maps show overlapping γ/β-tubulin signal (white). H, Western blot analysis of centrosome components (pericentrin and γ-tubulin), spindle protein (β-tubulin), and mitotic marker (p-H3) from HeLa cells treated with DMSO, AOH1996, or AOH1996-8Nq. AOH1996 decreases centrosomal protein levels and increases p-H3 abundance. AOH1996-8Nq does not alter these levels. Nucleolin serves as a loading control. Bar graph represents corresponding quantification. Bar graphs represent the mean ± SEM from three independent experiments. Statistical comparisons were performed using two-way ANOVA. ****, P < 0.0001; ***, P < 0.001; **, P < 0.0001.

Article Snippet: Tubulin polymerization was measured using an optical density–based tubulin polymerization kit (Cytoskeleton, Inc., BK004P).

Techniques: Immunofluorescence, Microscopy, Staining, Activity Assay, Thermal Shift Assay, Concentration Assay, Binding Assay, Disruption, Western Blot, Marker, Control

Journal: Cancer Research Communications

Article Title: Targeting Cancer-Associated PCNA with AOH1996 Induces Mitotic Catastrophe and Enhances Cisplatin Therapy in Cervical Cancer

doi: 10.1158/2767-9764.CRC-25-0648

Figure Lengend Snippet: AOH1996 binds to tubulin and disrupts PCNA–γ-tubulin interactions. A, Tubulin polymerization assay performed in vitro using purified tubulin. AOH1996 partially inhibits polymerization relative to DMSO and paclitaxel (positive control) but more effectively than the inert analog AOH1996-8Nq and the microtubule-depolymerizing agent colcemid (negative control). B, SPR analysis of AOH1996 and AOH1996-8Nq binding to purified tubulin. Sensorgrams (top) and fitted binding curves (bottom) demonstrate that AOH1996 binds tubulin with higher affinity than AOH1996-8Nq. C, Apparent dissociation constants (Kd) derived from SPR data. AOH1996 binds tubulin with a mean Kd of ∼30 μmol/L, whereas AOH1996-8Nq shows modest, yet significantly weaker, binding with a mean Kd of ∼70 μmol/L. Graphs represent the mean and range of nine independent experiments. D, Co-immunoprecipitation (co-IP) of endogenous PCNA from HeLa lysates treated with or without AOH1996 or AOH1996-8Nq, followed by immunoblotting for α-, β-, or γ-tubulin. AOH1996 treatment reduces the interaction between PCNA and γ-tubulin by ∼50%, whereas AOH1996-8Nq has no effect. Normal rabbit IgG was used as a negative IP control. Right, quantification of γ-tubulin co-IP signal normalized to DMSO. Bar graphs represent the mean and range from at least two biological replicates. E, Dose–response curve from MTT assay shows reduced impact of AOH1996-8Nq (IC 50 value not reached) on HeLa viability compared with AOH1996-treated (IC 50 = 125 nmol/L) cells. Statistical comparisons were made using unpaired two-tailed t tests. **, P < 0.01.

Article Snippet: Tubulin polymerization was measured using an optical density–based tubulin polymerization kit (Cytoskeleton, Inc., BK004P).

Techniques: Polymerization Assay, In Vitro, Purification, Positive Control, Negative Control, Binding Assay, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Control, MTT Assay, Two Tailed Test