Figure 6
Overexpression of PEG10 in SKNMC SAM D8 cells leads to detectable levels of apoptosis potentially via activation of non-canonical TGFβ pathways. (A) Schematic representation of the cell proliferation assay. (B) SKNMC SAM D8 cells overexpressing TGFBR2 and PEG10 were harvested on day 4 post transduction. The whole cell lysate was analyzed using a panel of antibodies as indicated with β-tubulin as control. TGFBR2 and PEG10 antibodies were used to confirm overexpression of the proteins. PAPR, cleaved caspase 3, 7, 8 and 9 were used as apoptosis markers (left panel). Phosphorylation levels of ERK1/2, SAPK/JNK, P38, AKT, SMAD2 were monitored to measure the activity of the different non-canonical pathways downstream of TGFβ (right panel). The panel is representative for 3 biological replicates. Image was created by using Image Lab version 6.1, https://www.bio-rad.com/en-ch/product/image-lab-software. (C) Densitometry of the Western Blot measuring PEG10, TGFBR2 protein levels, cleavage status of PARP, Caspase 3, 7, 8 and 9, phosphorylation status of ERK1/2, SAPK/JNK, P38, AKT and SMAD2 in TGFBR2 and PEG10 overexpressing SKNMC SAM cells 4 days post transduction. β-tubulin was used to normalize relative protein expression of PEG10, TGFBR2, Caspase 3 and 7. Cleaved PARP, Caspase 8 and 9 were normalized with total length PARP, Caspase 8 and 9. Phosphorylated ERK1/2, SAPK/JNK, P38, AKT and SMAD2 were normalized with total ERK1/2, SAPK/JNK, P38, AKT and SMAD2. Image was created by using GraphPad version 8, https://www.graphpad.com.
