Fig. 1

Schwann cell autophagy is activated in PNI. A Normal neurons in pancreatic parenchyma and perineural invasion in pancreatic cancer tissue. HE: HE staining showing the general morphology. SILVER: silver staining showing neurons. GFAP: GFAP IHC staining labeling Schwann cells. Black arrows indicate neurons. B. Representative LC3 immunohistochemistry in tissue adjacent to carcinoma (control) and PanCa (PNI) tissue, scale bar 200 μm. Black arrows indicate LC3 positive neurons at the same position compared with Fig. 1A. C. LC3 immunofluorescence (green) of Schwann cells treated with the control or PanCa-conditioned medium. White arrow: positive LC3 puncta indicating autophagic vacuoles. D. Statistics of LC3 puncta number in Schwann cells treated with the control or PanCa-conditioned medium (cell images are in Fig. 1C) (* p < 0.05). E. P62 immunofluorescence (green) of Schwann cells treated with the control or PanCa-conditioned medium. White arrow: positive P62 puncta indicating autophagic vacuoles. F. Statistics of P62 puncta number in Schwann cells treated with the control or PanCa-conditioned medium (cell images are in Fig. 1E) (* p < 0.05). G. Dorsal root ganglion (DRG) monoculture or coculture with PanCa cells or PanCa CM. DAPI labeled with blue, NF-H labeled with green, LC3 labeled with red. PANC-1 and BxPC-3 cell lines labeled with GFP are shown in green. Light microscopy indicates neurofilament and Schwann cell morphology. Red arrow: PANC-1 or BxPC-3 cells. Yellow arrow: autophagy-inactivated Schwann cells. Green arrow: autophagy-activated Schwann cells. H. Representative TEM image of sciatic nerves with or without PDAC cancer cell line injection. Green arrow: myelin sheath formed by Schwann cells. Yellow arrow: autophagosomes in Schwann cells. I. Representative TEM image of the RSC96 cell line with or without PanCa cell-conditioned medium. White arrow: cell nucleus. Black arrow: autophagosomes in Schwann cells. J. Statistics of autophagic vacuole number in RSC96 cells treated with the control or PanCa-conditioned medium (cell TEM images are in Fig. 1I) (* p < 0.05). K. Statistics of red and yellow puncta number in RSC96 cells treated with control or PanCa-conditioned medium (cell images are in Fig. 1L) (* p < 0.05). L. Autophagic flux detection of RSC96 cells treated with the control or PanCa-conditioned medium. LC3 labeled in GFP (green) and RFP (red) and merged as yellow. GFP is unstable in low pH while RFP is stable in low pH. In autophagosome when not fused with lysosomes,the GFP and RFP are both positive to be merged as yellow. When fused with lysosomes,the autophagosomes turn into autophagolysosomes and GFP will be quenched. So the autophagolysosomes are red. Red arrow: autophagolysosomes. Yellow arrow: autophagosome. PanCa-conditioned medium could promote autophagy flux in RSC96 cell line. M. Western blotting of RSC96 cells treated with the control or PanCa-conditioned medium. Several autophagy related proteins were detected. Actin was used as a loading control. PanCa-conditioned medium could inhibit p-mTOR while promote p-ULK1 and ATG5 expression in RSC96 cell line. Moreover, PanCa-conditioned medium could promote the conversion of LC3I to LC3II and the degradation of P62