Fig 1: Mechanisms hypothesized in the crosstalk between pancreatic cancer cells and adipocytesAdipocytes in pro-inflammatory conditions produce high levels of IL6, that could be a potential regulator of WNT5a expression and secretion in MiaPaCa2 through IL6-STAT3 signaling pathway. The secretion of WNT5a protein is associated to an increased signal of c-Jun and AP1 in 3T3-L1 after co-culture, representing one of the pathways activated by WNT5a up-regulation. The activation of WNT5a receptor and downstream signaling is prevented by the binding with its inhibitor SFRP5. Abbreviations: IL6- interleukine 6, AP-1- activator protein-1, SFRP5-secreted frizzled related-protein 5.
Fig 2: Inhibition of WNT5a signal prevents 3T3-L1 dedifferentiation into fibroblast-like cells after co-cultured with MiaPaCa2 cellsEffect of SFRP-5 addition to the medium on the dedifferentiation of adipocytes in co-culture (C). Number and area of adipocytes (D, E) as well as number of fibrobast-like cells in co-culture with SFRP5 (F) compared to co-culture alone. Data are presented as mean+standard error (m + SE). Abbreviations: PID- post-induction day, SFRP5-secreted frizzled related-protein 5.
Fig 3: Activation of WNT5a pathway in MiaPaCa2 cells and adipocytes in co-cultureWB analysis showed that the WNT5a protein expression in total cell extract of MiaPaCa2 was augmented after 3 (PID8) and 6 days (PID11) of co-culture (CoCo), when compared to control conditions (Ctr) (A). Additionally, RT-PCR analysis showed an increase expression of WNT5aS in MiaPaCa2 cells co-cultured with 3T3-L1 adipocytes, especially at PID 8 and a lower expression of WNT5aL (B). In order to investigate the pathway that might explain WNT5a up-regulation in our experimental conditions, we registered a higher expression by WB of STAT3 in total cell extract of MiaPaCa2 cells in co-culture with 3T3-L1 when compared to control conditions (C). Moreover, PCR analysis of WnT5aS and WNT5aL in 3T3-L1 alone (Ctr) and co-cultured (CoCo) with MiaPaCa2 showed an increased expression of WNT5aL in co-culture at PID 11 and 14, while WNT5a-S was not found in adipocytes maintained in control medium (D). RT-PCR data are expressed considering as control, gene expression in 3T3-L1 at PID 8, the results are presented as mean + standard error (m + SE). Abbreviations: WB- Western blot assay PID- post-induction day, RT-PCR – real time PCR; CoCo-co-culture condition; Ctr- control condition; WNT5aS-WNT5a short; WNT5aL- WNT5a long.
Fig 4: Inhibition of the WNT5a pathway may control the adipocytes' phenotype shiftIn order to investigate the pathway that might be activated by WNT5a up-regulation in our experimental setting, expression of activated c-Jun and AP1 in 3T3-L1 adipocytes (A) and MiaPaCa2 cells (B) at PID8, PID11 and PID14 was examined by EMSA. As the c-Jun expression appeared augmented in co-culture at PID11, we treated it for 24h with a WNT5a neutralizing antibody. Our EMSA results proved that c-Jun activation was inhibited by this treatment in 3T3-L1 adipocytes (C) and MiaPaCa2 cells (D). Abbreviations: PID- post-induction day, EMSA- Electrophoretic Mobility Shift Assay, AP-1: activator protein-1.
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