Fig. 4: Pro-apoptotic and anti-migratory activity of Roy-Bz is mediated by selective activation of PKCδ in HCT116 cancer cells

a Cell viability dose–response curves were determined by trypan blue assay for control (Mock) and PKCδ-knockdown (PKCδ RNAi) HCT116 cells treated with 0.1–6 μM Roy-Bz for 24 h; cell viability of vehicle was set as 100%. b Annexin V-positive cells were determined in control and PKCδ-knockdown HCT116 cells after 24 h treatment with 1 μM Roy-Bz or vehicle. c Western blot analysis of PARP cleavage, p53 and Bax expression levels for 16 h treatment with 1 μM Roy-Bz or vehicle in control and PKCδ-knockdown HCT116 cells; GAPDH (loading control). d,e Wound healing assay for control and PKCδ-knockdown HCT116 confluent cells treated with 0.25 µM Roy-Bz or vehicle, at different time-points; in d, scale bar = 50 µm and magnification = ×100; in e, quantification of wound closure in five randomly selected microscopic fields. f Chemotaxis migration assay for control and PKCδ-knockdown HCT116 cells for 24 h treatment with 0.25 µM Roy-Bz; migratory cells were quantified by fluorescence intensity, which was set as 100% for untreated cells. g Western blot analysis of full-length PKCδ (PKCδ-FL) and cleaved PKCδ fragment (PKCδ-CF) in HCT116 cells treated with 1 μM Roy-Bz for up to 24 h. h,i Levels of histone H3 phosphorylation on Ser-10 (pSer-10) in control and PKCδ-knockdown HCT116 cells treated with 1 μM Roy-Bz for up to 8 h; histone H3 (loading control); in i, quantification of pSer-10 levels normalized to histone H3, values of asynchronized cells were set as 1. In c, g, and h, immunoblots represent one of three independent experiments; in a,b,e,f, and i data are mean ± SEM of three independent experiments; values significantly different are indicated (*p < 0.05, **p < 0.01, ***p < 0.001), unpaired Student’s t-test