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. 2021 Nov 27;12(12):1111. doi: 10.1038/s41419-021-04405-0

Fig. 2. MLK4 inhibition sensitizes TNBC cells to chemotherapy.

Fig. 2

A Kinase-inactive MKK7 and purified GST-MLK4 kinase domain (isolated from baculovirus-infected insect cells) were subjected to in vitro kinase assay in the presence or absence of CEP-5214 inhibitor. B HEK293T cells were transiently transfected with MLK4-WT vector or with the empty vector and incubated with increasing concentrations of CEP-5214 for 1 h. Next, whole cell lysates were collected and analyzed by immunoblotting. C–F HCC1806 (C-D) and SUM149PT (E-F) cells were incubated with CEP-5214 or DMSO for 72 h and doxorubicin for 48 h. After treatment, cells viability was assessed by crystal violet staining and quantified by absorbance measurements. Error bars indicate ±SEM from three independent experiments. Analysis of combination efficacy and synergy was performed using HSA model with Combenefit software, *p < 0.05. G–H HCC1806 and SUM149PT cells were incubated with CEP-5214 at concentrations 500 nM and 250 nM, respectively or DMSO for 72 h and doxorubicin for 48 h. Next, cells were stained with AnnexinV-FITC, and measured by flow cytometry. Error bars indicate ±SEM from two independent experiments, performed in triplicates. Significance was calculated using one-way ANOVA followed by Tukey multiple comparisons, **p < 0.01, ****p < 0.0001.