ShRNA. The efficiency of PKC gene silencing was confirmed by Western blot evaluation (Supplementary Figure S2). Real time RT-PCR showed that the induction of your three EMT-related transcription components downstream FGFR2c, induced in PANC-1 cells by FGF2 (Figure 4B), was considerably repressed by PKC depletion (Figure 4B). Furthermore, biochemical experiments highlighted that PKC knockdown also counteracted the repression of E-cadherin, at the same time as the upregulation of vimentin induced by FGF2 in these cells (Figure 4C), confirming the interference with EMT induction. Ultimately, immunofluorescence approaches showed how PKC depletion was in a position to counteract either the enhancement of vimentin expression (Figure 4D) or the morphological modifications in favor in the Methiothepin Technical Information mesenchymal feature displayed by PANC-1 cells in response to FGF2 (Figure 4D). The se outcomes indicated that PKC-mediated signaling downstream FGFR2c significantly contribute for the establishment of receptor-dependent EMT phenotype.Cancers 2021, 13,11 ofFigure 4. The depletion of PKC interferes with FGF2-triggered EMT phenotype. PANC-1 and Mia PaCa-2 cells have been left untransduced or stably transduced with PKC shRNA or with an unrelated shRNA, as negative handle. Cells were left untreated or stimulated with FGF2 in presence or absence of SU5402 as above. HaCaT cells and HFs were employed as positive controls for epithelial/mesenchymal marker expression, as reported above. (A) Western blot evaluation shows that the increaseCancers 2021, 13,12 ofof phosphorylation of PKC is observed upon FGF2 stimulation only in PANC-1 cells and this effect is abolished by SU5402. Equal loading was assessed together with the anti-actin antibody. Final results are expressed as mean value SD (n = three). The densitometric analysis was performed as reported above. ANOVA with Tukey’s numerous comparison test: p 0.05. (B) Real-time RT-PCR shows that the induction of Snail1, STAT3 and FRA1 only in PANC-1 cells in response to FGF2 is repressed upon PKC depletion. Final results are expressed as imply value SD (n = 3). ANOVA with Tukey’s multiple comparison test: p 0.05. (C) Western blot evaluation highlights that PKC knockdown also counteracted the repression of E-cadherin, also because the upregulation of vimentin induced by FGF2 in PANC-1 cells. Equal loading was assessed with the anti-actin antibody. Outcomes are expressed as mean value SD (n = 3). The densitometric analysis was performed as reported above. ANOVA with Tukey’s a Digoxigenin Formula number of comparison test: p 0.05. (D) Immunofluorescence analysis shows that PKC silencing interferes using the enhancement of vimentin expression, as well as with all the tendency of PANC-1 cells to assume the mesenchymal morphology in response to FGF2. Bar: ten . Original blots see Figure S4.three.4. PKC Signaling Negatively Impacts around the Autophagic Course of action We’ve got recently proposed a role of PKC-mediated signaling not just in FGFR2cmediated induction of EMT, but in addition in FGFR2c-dependent inhibition of your autophagic process in human keratinocytes [21]. The refore, we investigated here the feasible contribution of PKC on autophagy also inside the specific context of pancreatic cancer. Western blot analysis showed that PKC knockdown abolished the lower from the widely recognized autophagic marker LC3-II, induced by FGF2 stimulation exclusively in PANC-1 cells (Figure 5A). Also, in these cells, PKC depletion also counteracted the accumulation in the autophagy substrate SQSTM1 in response to FGF2 (Figure 5A), confirming the effici.