Fig. 4: Honokiol causes a decrease of tumor growth in soft agar by promoting PTP opening and apoptosis.

A Representative images (i) of colony formation in soft agar by CTR and IF1 KO HeLa cells treated for 15 days with 0-10 µM HK. Histograms show the mean of the total colony area/mm² (ii), the number of colonies/mm² (iii) and the average of colony area in mm² (iv). Data are mean ± SEM of at least three independent experiments, *p ≤ 0.05, **p = 0.0029, ***p = 0.001, Student’s t test. B, C Calcium retention capacity is assessed in permeabilized CTR and IF1 KO HeLa cells in a buffer promoting state 3 respiration, containing the membrane-impermeable Ca²⁺ sensor, Ca²⁺ Green-5N. Histogram represents nmols of Ca²⁺ (per μg of protein) that are necessary to induce PTP opening. B Cells are treated with 0-100 μM HK. Data are mean (expressed as % of untreated controls) ± SEM of five independent experiments. *p = 0.0103, **p = 0.0036, ***p = 0.0002, ****p ≤ 0.0001, One-way Anova. C Cells are treated with/without 100 μM HK in the presence or absence of 1.6 μM CSA. Data are mean (expressed as % of untreated controls) ± SEM of three independent experiments. *p ≤ 0.05, **p = 0.0045, One-way Anova. D Adherent CTR and IF1 KO HeLa cells are treated with 0–100 μM HK for 1 or 2 h, or with 2 μM staurosporine for 24 h (STS), detached and incubated with the annexin V-fluorescent probe to quantify the apoptotic cells by cytofluorimetric measurements. Histogram shows the mean quantification ± SEM of apoptotic annexin V-positive cells (expressed as % of the total cell number). Data are from at least four independent experiments. *p ≤ 0.05, **p = 0.0051, ****p ≤ 0.0001, One-way Anova. E Adherent CTR and IF1 KO HeLa cells are treated with 0–100 μM HK for 30 min, or with 100 μM arachidonic acid for 1 h (ARA), detached for cytofluorimetric measurements. Mitochondrial superoxide anion analysis is performed by incubating cells with MitoSOX red. In the histogram, MitoSOX mean fluorescence (a.u.) is shown. Data are mean ± SEM of three independent experiments. *p = 0.0345, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, One-way Anova.