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. 2019 Dec 4;5:148. doi: 10.1038/s41420-019-0229-8

Fig. 6. Regulation of intracellular signalling pathways and their functional role in mCD40L-mediated tumour cell apoptosis.

Fig. 6

a ACHN, 786-O and A-704 cells were treated with mCD40L for the indicated time periods (1.5, 3 and 6 h) and expression of phosphorylated-MAPKs MKK4 (p-MKK4), MKK7 (p-MKK7), JNK (p-JNK) and p38 (p-p38) was detected in controls (‘C’) vs. mCD40L-treated cells (‘mL’) by immunoblotting (40 µg protein/lane). Equal loading for human epithelial cell lysate was confirmed by CK18 detection (see Methods). As positive controls for p-MKK4/7, p-JNK and p-p38 protein expression induction, lysates from HCT116 cells that were treated with control (‘C’) or treated with mCD40L (‘mL’) for 6 h were included. Lysate from effector (3T3CD40L) cell monocultures served as negative control (NC) and confirmed the human-protein specificity of the antibodies. b–f ACHN, 786-O and A-704 cells were treated with mCD40L in the absence (vehicle control—denoted ‘Control’) or presence of the indicated concentration (12.5, 25 and 50 μM) of JNK inhibitor SP600125 (b), p38 inhibitor SB202190 (c), AP-1 inhibitor NDGA (d), MEK/ERK inhibitor U0126 (e) and NF-κB inhibitor (PDTC). Cell death was detected 48 h later using the CytoTox-Glo assay (see Methods). Results are presented as Cell death fold increase in background-corrected RLU readings relative to control (mCD40L treatment vs. controls) and are representative of three independent experiments. Bars show mean fold change of 5–6 technical replicates ± SEM. g ACHN, 786-O and A-704 cells were treated with mCD40L for the indicated time periods (3 and 6 h) in the presence of 25 μM JNK inhibitor SP600125 or p38 inhibitor SB202190 and expression of phosphorylated-MAPKs JNK (p-JNK) and p38 (p-p38) was detected in controls (‘C’) vs. mCD40L-treated cells (‘mL’) by immunoblotting (40 µg protein/lane). ACHN, 786-O and A-704 cells treated with mCD40L for 6 h in the absence of inhibitor (vehicle controls) were also included (denoted as positive control, ‘PC') for each experiment. Equal loading for human epithelial cell lysate was confirmed by CK18 detection (see Methods).