Fig. 3

Mechanism of action of PPH. (a) PTX (3 nM) and PPH (1 µM) induce rounding of MCF7 breast cancer cells 24h post-treatment. White arrows indicate the rounded cells. Each biological replicate is represented with a different symbol (N = 3) and each symbol is a technical replicate (n = 3). Statistical significance between groups was determined with Kruskall Wallis test and Dunn’s multiple comparisons test. (b) 24-h treatment with PTX (100 nM) or PPH (5 µM) induces a G2/M cell cycle arrest in MCF7 breast cancer cells. Bar plot represents mean ± SD of three biologically independent experiments. Statistical significance between groups was determined with one-way ANOVA and Tukey’s multiple comparisons test. (c,d) Volcano plot depicting the differentially abundant proteins in SKOV3 spheroids after 5-day treatment with PPH (1 µM) (c) or PTX (4 nM) (d) compared to vehicle control. The vertical dotted red lines indicate a log₂ fold change > 1. The horizontal dotted red line indicates a p-value = 0.05 (log₁₀ adjusted p-value 1.30). Proteins are labelled with their corresponding gene names. Data are representative of the mean of six biological replicates (N = 6). (e) Graphs representing the upregulation of biological processes in vehicle treated spheroids versus PPH treated spheroids (top) or PPH treated spheroids versus vehicle treated spheroids (bottom) based on mass-spectometry assisted proteomics analysis and analysis with g:Profiler. (f,g) Heatmap of differentially regulated genes involved in cell cycling (GO: 0007049) (f) or microtubule-based processes (GO:0007017) (g) after PTX or PPH treatment versus vehicle control spheroids. (h) 24-h treatment with PTX (100 nM) or PPH (2.5 µm) of SKOV3 cells induces Lysine 40 acetylation (K40 Ac) of α-tubulin. GAPDH was used as loading control. Representative images from three biologically independent replicates are shown. Uncropped blots are shown in Supplementary Figure S5. Bar plot represents mean ± SD of three biologically independent experiments. Statistical significance between groups was determined with one-way ANOVA and Tukey’s multiple comparisons test. (i) Immunohistochemical K40 Ac of α-tubulin staining of ex vivo 4T1 tumor fragments after 5-day treatment with 100 nM PTX or 50 µM PPH. Levels of statistical significance are indicated as * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P < 0.0001. DMSO was used as vehicle control.