Fig. 3. MLK4 promotes chemoresistance in 3D cell culture and in vivo models.

A–B MLK4 knock-down was induced by doxycycline in HCC1806_sh6 and SUM149PT_sh2 cells grown in non-adherent conditions and subsequently cells were treated with doxorubicin at indicated concentrations for 48 h. The activity of caspases 3/7 was measured using bioluminescence assay. Representative pictures of mammospheres upon doxorubicin treatment are shown. Error bars indicate ±SEM from three independent experiments, performed in triplicates (n = 9). Significance was calculated using an unpaired two-tailed t-test, *p < 0.05, **p < 0.01, ****p < 0.0001. C HCC1806_sh6 cells were injected into mammary fat pads of RAG2^−/− mice. Doxycycline was administered one day after the injection to induce MLK4 knock-down. Mice were treated with doxorubicin (4 mg/kg, i.p.) or saline at 5-day intervals, starting from day 7 of the experiment. Tumors were measured twice a week. Error bars indicate ±SEM (n = 10 for control group, n = 8 for doxorubicin-treated group, n = 8 for doxycycline-treated group, n = 10 for combination treatment group). Statistical comparison was performed using two-way ANOVA, *p < 0.05, **p < 0.01, ****p < 0.0001. D Weight of tumors resected at the end of the study. Significance was calculated using one-way ANOVA followed by Tukey multiple comparisons test, *p < 0.05, **p < 0.01, ****p < 0.0001.