I: Initial autophagic vacuole; AVd: degradative autophagic vacuole; M: mitochondrion; Nu: nucleus; NM: nuclear membrane; PM: plasma membrane. Bars: 1 , 200 nm. Original blots see Figure S4.Cancers 2021, 13,14 of3.five. PKC Signaling Interferes with Autophagy Converging on ERK1/2 Pathway To clarify the molecular mechanisms underlying the involvement of PKC in the autophagic procedure, we focused our focus on MTOR, which is regarded as the key damaging regulator of autophagy also in pancreatic cancer cells [2,14]. Western blot analysis revealed that the phosphorylation of MTOR, also as that of its substrate S6K, evident just after FGF2 stimulation specifically in PANC-1 cells (Figure 6A), had been strongly dampened by PKC knockdown (Figure 6A). Surprisingly, no corresponding effects had been observed around the AKT phosphorylation (Figure 6B). Given that AKT could be the upstream substrate typically accountable for MTOR activation, our unexpected results indicated that PKC could activate MTOR through an alternative pathway. This possibility appears to become consistent together with the peculiar capability, previously described for PKC in other cellular contexts, to converge on MTOR by means of the Psalmotoxin 1 Inhibitor activation of Raf/MEK/ERK signaling [25]. Essentially, the significant contribution of ERK1/2 signaling in MTOR activation and consequent autophagy inhibition has been broadly described in pancreatic cancer cells [2]. Based on these assumptions, we investigated the influence of PKC signaling on ERK1/2 pathway. Biochemical evaluation showed that, in consequence of PKC depletion, the enhance of ERK1/2 phosphorylation in response to FGF2, visible in each pancreatic cell lines (Figure 6C), was reduced in Mia PaCa-2, which maintained a significant residual ERK phosphorylation (Figure 6C), but fully abolished in PANC-1 (Figure 6C). The se benefits indicate that the distinct expression of FGFR2c displayed by the two PDAC cell lines influence around the dependence on PKC of ERK1/2 signaling. It is also worth noting that shFGFR2c transduced MiaPaCa-2 cells displayed a larger responsiveness to FGF2 when it comes to ERK1/2 phosphorylation in comparison to non-transduced ones (see Figure 1B in comparison with Figure 6C), even when this phosphorylation remains considerably reduce than that shown by PANC-1 cells. This variability of MiaPaCa-2 cell response to FGF2 may be the consequence of distinct culture circumstances. The se benefits indicated that, only in PANC-1 cells, the activation of ERK1/2 pathway upstream depends upon PKC activation. Because ERK1/2 can also be a wellknown pathway involved in EMT of PDAC cells [4], our outcomes suggest the possibility that, in this tumor context, PKC signaling, when activated in consequence of hugely expression of FGFR2c, could simultaneously repress autophagy and orchestrate the EMT plan straight converging on ERK1/2 pathway.Cancers 2021, 13,15 ofFigure six. PKC signaling shut-off by PKC protein depletion interferes with each MTOR and ERK1/2 signaling pathways. PANC-1 and Mia PaCa-2 cells stably transduced with PKC shRNA or with an unrelated shRNA were left untreated or stimulated with FGF2 as above. (A) Western blot evaluation shows that the boost of phosphorylation of MTOR and S6K, evident right after FGF2 stimulation only in PANC-1 cells, are strongly dampened by PKC knockdown. (B) No correspondingCancers 2021, 13,16 ofeffects are observed around the AKT phosphorylation. (C) The raise of ERK1/2 phosphorylation in response to FGF2, visible in both pancreatic cell lines, is considerably Rilpivirine Formula greater.