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 Carbendazim In Vitro Figure S4.Cancers 2021, 13,14 of3.5. PKC Signaling Interferes with Autophagy Converging on ERK1/2 Pathway To clarify the molecular mechanisms underlying the involvement of PKC inside the autophagic course of action, we focused our consideration on MTOR, which is viewed as the primary unfavorable regulator of autophagy also in pancreatic cancer cells [2,14]. Western blot evaluation revealed that the phosphorylation of MTOR, at the same time as that of its substrate S6K, evident following FGF2 stimulation specifically in PANC-1 cells (Figure 6A), have been strongly dampened by PKC knockdown (Figure 6A). Surprisingly, no corresponding effects were observed on the AKT phosphorylation (Figure 6B). Considering the fact that AKT is definitely the upstream substrate commonly accountable for MTOR activation, our unexpected results indicated that PKC may well activate MTOR by means of an alternative pathway. This possibility seems to be constant together with the peculiar ability, previously described for PKC in other cellular contexts, to converge on MTOR by way of the activation of Raf/MEK/ERK signaling [25]. Basically, the significant contribution of ERK1/2 signaling in MTOR activation and consequent autophagy inhibition has been widely described in pancreatic cancer cells [2]. Based on these assumptions, we investigated the influence of PKC signaling on ERK1/2 pathway. Biochemical analysis showed that, in consequence of PKC depletion, the boost of ERK1/2 phosphorylation in response to FGF2, visible in each pancreatic cell lines (Figure 6C), was decreased in Mia PaCa-2, which maintained a considerable residual ERK phosphorylation (Figure 6C), but completely abolished in PANC-1 (Figure 6C). The se results 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 in terms of ERK1/2 phosphorylation in comparison with non-transduced ones (see Figure 1B in comparison with Figure 6C), even if this phosphorylation remains considerably reduced than that shown by PANC-1 cells. This variability of MiaPaCa-2 cell response to FGF2 could possibly be the consequence of unique culture conditions. The se results indicated that, only in PANC-1 cells, the activation of ERK1/2 pathway upstream depends on PKC activation. Because ERK1/2 is also a wellknown pathway involved in EMT of PDAC cells [4], our benefits recommend the possibility that, within this tumor context, PKC signaling, when activated in consequence of very expression of FGFR2c, could simultaneously repress autophagy and orchestrate the EMT program straight converging on ERK1/2 pathway.Cancers 2021, 13,15 ofFigure 6. PKC signaling shut-off by PKC protein depletion interferes with both 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 analysis shows that the boost of phosphorylation of MTOR and S6K, evident 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 improve of ERK1/2 phosphorylation in response to FGF2, visible in both pancreatic cell lines, is significantly greater.