Fig 2: Characterization of KCLi001-A line. A, The colonies display typical morphology of pluripotent stem cells under feeder-dependent and feeder-free conditions. B, The absence of 181 bp positive SeV band at 45 and 60 days of culture indicates that the cells are SeV free. β-actin 455 bp band serves as an internal control. C, The line is heterozygous for c.2282del4 mutation in FLG as indicated with restriction enzyme digestion and Sanger sequencing of PCR product spanning the mutation site. Mutation generates a site for DraIII that is not present in wildtype allele. The enzyme digestion cuts 811 bp product in mutated allele into 667 bp and 134 bp, whereas 811 bp remains intact in wildtype. hESC line KCL038 is used as a negative control. D, Pluripotency markers. Alkaline phosphatase activity (AP) was restricted to the iPSC colony (green) growing on AP-negative feeder cells. Both iPSC colony and feeders are positive for actin (phalloidin) staining (red). iPSC colonies are positive for POU5F1 (red), TRA-1-81 (green), NANOG (red) and TRA-1-60 (green) pluripotency markers. E, Spontaneous differentiation in vitro. The cells can differentiate into all three germ layers as demonstrated with markers specific for ectoderm (TUBB3), endoderm (AFP) and mesoderm (ACTA2). Nuclei are visualized with Hoechst 33342. F, Spontaneous differentiation in vivo. Gross anatomy and staining for human-specific MTCO2 marker iPSC indicated that the teratoma is incapsulated and did not invade surrounding tissues. Teratoma contained cells from all three germ layers as demonstrated with markers specific for ectoderm (TUBB3, GFAP), endoderm (AFP, GATA4) and mesoderm (AB-PAS, DESMIN). G, Directed differentiation into keratinocytes. The cells expressed keratinocyte-specific markers KRT14, KRT18, TP63 (ΔNp63) in time-dependent manner.

Fig 3: Rapid induction of the mitochondrial (intrinsic) apoptotic pathway and role of caspase activation in cell death. (a) Expression of Bax and Bak proteins was detected in controls (‘C’) versus mCD40L-treated (‘mL’) HCT116 and SW480-CD40 cells by immunoblotting at the indicated time points. Equal loading for human epithelial cell lysate was confirmed by CK18 detection. (b) Control (‘C’) and mCD40L-treated (‘mL’) HCT116 and SW480-CD40 cells were used to prepare cytoplasmic (‘Cyto’) and mitochondrial (‘Mito’) sub-cellular fractions for the detection of cytochrome c (Cyto c) protein by immunoblotting at 6 h post CD40 ligation. Detection of Bcl-2 and GAPDH proteins was employed to confirm sub-cellular fractionation (mitochondrial and cytoplasmic, respectively). (c) HCT116 cells were treated with mCD40L in the absence (vehicle control–denoted ‘Control’) or presence of 100 µM inhibitor of caspase-8 (z-IETD-FMK), caspase-9 (z-LEHD-FMK), caspase-10 (z-AEVD-FMK) or pan-caspase inhibitor (z-VAD-FMK). Cell death was detected 24 h later using the CytoTox-Glo assay. Results are presented as Cell death Fold increase in background-corrected RLU readings relative to control (mCD40L treatment versus controls) and are representative of 3 independent experiments. Bars show mean fold change (comparing caspase inhibitor-treated versus vehicle control cultures) for 5–6 technical replicates ± SEM. NS denotes non-significance (p > 0.05) and *** p < 0.001.

Fig 4: mCD40L-mediated signalling induces rapid up-regulation of DR5 receptor and induction of intracellular TRAIL expression. (a) Expression of TRAIL and FasL proteins was detected in controls (‘C’) versus mCD40L-treated (‘mL’) HCT116 and SW480-CD40 cells by immunoblotting at the indicated time points. Equal loading for human epithelial cell lysate was confirmed by CK18 detection. (b) DR5 receptor expression in HCT116 and SW480-CD40 cells at the indicated time points post CD40 ligation was detected by immunoblotting as in (a). (c) HCT116 and SW480-CD40 cells were treated with or without mCD40L in the presence of 10 μg/mL anti-FasL mAb (NOK1), anti-TRAIL mAb (RIK2) or isotype control mAb (Control Ig) and cell death was detected at 24 h using the CytoTox-Glo assay. Results are presented as Cell death fold increase in background-corrected RLU relative to control (mCD40L treatment versus controls) and are representative of 2 independent experiments. Bars show mean fold change (comparing mAb-treated cells versus untreated ‘Control cells’) for 4–5 technical replicates ± SEM. NS denotes non-significance (p > 0.05). (d) HCT116 cells were treated with the indicated concentration of killerTRAIL for 24 h in the presence of anti-TRAIL mAb (RIK-2) or isotype control mAb (Control Ig) and cell viability was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. Results are presented as % viability for all experimental conditions relative to untreated (control) cells. Bars show mean % Cell Viability for 5–6 technical replicates ± SEM. NS denotes non-significance (p > 0.05) and *** p < 0.001.

Fig 5: Role of caspase activation and induction of the mitochondrial (intrinsic) pathway during mCD40L-mediated tumour cell apoptosis.a ACHN, 786-O and A-704 cells were treated with mCD40L in the absence (vehicle control—denoted ‘Control’) or presence of 100 μM of inhibitor of caspase-10 (z-AEVD-FMK), caspase-8 (z-IETD-FMK), caspase-9 (z-LEHD-FMK) or pan-caspase inhibitor (z-VAD-FMK). 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 4–6 technical replicates ± SEM. b ACHN, 786-O and A-704 cells were treated with mCD40L for the indicated time periods (6, 12 and 24 h) and expression of Bak and Bax 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 Bak and Bax protein expression induction, lysates from HCT116 cells that were treated with control (‘C’) or treated with mCD40L (‘mL’) for 24 h were included. Lysate from effector (3T3CD40L) cells alone served as negative control (NC) and confirmed the human-protein specificity of the antibodies. c ACHN, 786-O and A-704 cells were treated with mCD40L for the indicated time periods (12 and 24 h) in the presence of 25 μM JNK inhibitor SP600125 or p38 inhibitor SB202190 and expression of Bak and Bax 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 24 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).