Supplementary Table S1. Selection of further investigated targets.
Supplementary Figure S1. A, Chemical structure of ingenol mebutate. B, Confirmation of inhibition by MEK and ERK inhibitor. Cells were treated with MEK and ERK inhibitor to confirm their inhibitory potential. WB for pERK confirmed the inhibitory effect. Confirmation of functionality of MEK and ERK inhibitor.
Supplementary Figure S2. Conformation of knock-down for siRNA experiments. Cells were transfected with indicated siRNAs for 30h followed by protein extraction or RNA extraction. Knock-down was confirmed by western blotting or qPCR. Actin was used as loading control for western blotting.
Supplementary Figure S3. Effect of IM on fibroblasts and melanoma cells and effect of TPA on SCC13 cells. A, Fibroblasts and melanoma cells were treated for 24h with indicated concentrations of IM followed by assessing cell viability by MTT assay. Graphs represent mean and standard deviation of three independent experiments from two different fibroblast or melanoma cell cultures. B, SCC13 cells were treated for 24h with indicated concentrations of TPA followed by assessing cell viability by MTT assay. Graphs represent mean and SD of three independent experiments.
Supplementary Figure S4. Gene expression analysis of IM-treated primary keratinocytes and patient-derived SCC cells. A, Heatmap showing differentially expressed genes between DMSO-treated and IM-treated primary keratinocytes and patient-derived SCC cells. Cells were treated with either DMSO or 10 µM IM or DMSO and 1 nM IM respectively for 24h following RNA extraction and performance of the microarray. Upregulated genes are displayed in red, downregulated genes are displayed in blue. B, GeneGo analysis of downregulated genes after treatment with 100 nM of IM. The top 10 pathways that are most affected by IM-dependent gene downregulation are cell cycle-associated. C, GeneGo analysis of upregulated genes after 100 nM of IM treatment. List of network objects that are most involved in processes affected by IM-dependent gene upregulation. D, Validation of selected genes from the microarray by qPCR. Primary keratinocytes, patient-derived SCC cells, SCC12 and SCC13 cells were treated for 24h with DMSO or 100 nM of IM and organ cultures from normal skin and SCC treated with either vehicle or IM 0.05% followed by RNA extraction. Gene expression was analyzed by qPCR.
Supplementary Figure S5. ERK is activated after IM treatment in a PKC-dependent manner. A, Primary keratinocytes and patient-derived SCC cells from three different donors were treated with either DMSO or 100 nM IM for 10 min or 30 min followed by protein extraction. Phosphorylation was detected with the PathScan Intracellular Signaling Kit. Antibody spots are present in duplicates. Red arrow = ERK, blue arrow = JNK, arrow mark = p38. B, ERK target gene expression was analyzed by qPCR after 24h of IM treatment +/- MEK or ERK inhibitor in primary keratinocytes and SCC cells and in SCC13 cells. All graphs represent mean +/- SD of at least three independent experiments. Data were analyzed by one-way ANOVA followed by Dunnet’s multiple-comparison test. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.
Supplementary Figure S6. Time course experiment for gene expression analysis of IL13RA2 and IL1R2. Primary keratinocytes and organ cultures of normal skin were treated with either 100 nM of IM or 0.015% or 0.05% of IM respectively for 2h, 6h or 12h followed by RNA extraction. Gene expression was analyzed by qPCR.