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 Fmoc-Ile-OH-15N manufacturer 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 process, we focused our attention on MTOR, that is considered the main unfavorable regulator of autophagy also in pancreatic cancer cells [2,14]. Western blot analysis revealed that the phosphorylation of MTOR, as well as that of its substrate S6K, evident after FGF2 stimulation especially in PANC-1 cells (Figure 6A), were strongly dampened by PKC knockdown (Figure 6A). Surprisingly, no corresponding effects have been observed around the AKT phosphorylation (Figure 6B). Because AKT is the upstream substrate typically responsible for MTOR activation, our unexpected results indicated that PKC may well activate MTOR by means of an alternative pathway. This possibility appears to become constant with all the peculiar capacity, previously described for PKC in other cellular contexts, to converge on MTOR by way of the activation of Raf/MEK/ERK signaling [25]. Truly, the vital contribution of ERK1/2 signaling in MTOR activation and consequent autophagy inhibition has been extensively described in pancreatic cancer cells [2]. Determined by these assumptions, we investigated the impact of PKC signaling on ERK1/2 pathway. Biochemical analysis showed that, in consequence of PKC depletion, the improve of ERK1/2 phosphorylation in response to FGF2, visible in both pancreatic cell lines (Figure 6C), was reduced in Mia PaCa-2, which maintained a substantial residual ERK phosphorylation (Figure 6C), but totally abolished in PANC-1 (Figure 6C). The se benefits Bisindolylmaleimide XI In Vivo indicate that the diverse expression of FGFR2c displayed by the two PDAC cell lines effect around the dependence on PKC of ERK1/2 signaling. It’s also worth noting that shFGFR2c transduced MiaPaCa-2 cells displayed a larger responsiveness to FGF2 in terms of ERK1/2 phosphorylation in comparison to non-transduced ones (see Figure 1B in comparison with Figure 6C), even though this phosphorylation remains drastically decrease than that shown by PANC-1 cells. This variability of MiaPaCa-2 cell response to FGF2 may well be the consequence of different culture circumstances. The se benefits indicated that, only in PANC-1 cells, the activation of ERK1/2 pathway upstream is determined by PKC activation. Considering that ERK1/2 is also a wellknown pathway involved in EMT of PDAC cells [4], our results recommend the possibility that, within this tumor context, PKC signaling, when activated in consequence of extremely expression of FGFR2c, could simultaneously repress autophagy and orchestrate the EMT plan directly converging on ERK1/2 pathway.Cancers 2021, 13,15 ofFigure six. 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 raise 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 on the AKT phosphorylation. (C) The boost of ERK1/2 phosphorylation in response to FGF2, visible in both pancreatic cell lines, is substantially greater.
