Fig. 2
PanCa-related NGF activates SC autophagy. A. NGF secretion measured by ELISAs in the serum-free medium of five pancreatic cancer cell lines (* p < 0.05). B. NGF secretion measured by ELISAs in the serum-free medium of the control and shNGF PANC-1 cancer cell lines (* p < 0.05). C. NGF secretion measured by ELISAs in the serum-free medium of the control and shNGF BxPC-3 cancer cell lines (* p < 0.05). D. LC3 immunofluorescence (green) of RSC96 cells treated with the control or shNGF-PanCa CM or PanCa-conditioned medium. White arrows: positive LC3 puncta indicating autophagic vacuoles. E. Statistics of LC3 puncta number in RSC96 cells treated with the control or shNGF-PANC-1 CM or normal PANC-1 conditioned medium (* p < 0.05). F. Statistics of LC3 puncta number in RSC96 cells treated with the control or shNGF-BxPC-3 CM or normal BxPC-3 conditioned medium (* p < 0.05). G. P62 immunofluorescence (green) of RSC96 cells treated with the control or shNGF-PanCa CM or PanCa-conditioned medium. White arrows:positive P62 punctas indicating autophagic vacuoles. H. Statistics of P62 puncta number in RSC96 cells treated with the control or shNGF-PANC-1 CM or normal PANC-1-conditioned medium (* p < 0.05). I. Statistics of P62 puncta number in RSC96 cells treated with the control or shNGF-BxPC-3 CM or normal BxPC-3-conditioned medium (* p < 0.05). J. Autophagic flux detection of RSC96 cells treated with the control or shNGF-PANC-1 CM or PANC-1-conditioned medium. LC3 labeled with GFP (green) and RFP (red) and merged as yellow. Red arrow: autophagolysosomes. Yellow arrow: autophagosome. PANC-1-conditioned medium could promote autophagy flux in RSC96 cell line while inhibiting NGF secrection in PANC-1 could partially reverse the effect. K. Autophagic flux detection of RSC96 cells treated with the control or shNGF-BxPC-3 CM or BxPC-3-conditioned medium. LC3 labeled with GFP (green) and RFP (red) and merged as yellow. Red arrow: autophagolysosomes. Yellow arrow: autophagosome. PANC-1-conditioned medium could promote autophagy flux in RSC96 cell line while inhibiting NGF secrection in BxPC-3 could partially reverse the effect. L. Statistics of red and yellow puncta number in RSC96 cells treated with the control or shNGF-PANC-1 CM or PANC-1-conditioned medium (* p < 0.05). M. Statistics of red and yellow puncta number in RSC96 cells treated with the control or shNGF-BxPC-3 CM or BxPC-3-conditioned medium (* p < 0.05). N. Western blotting of NGF in the control or NGF knockdown PANC-1 cell lines. Actin was used as a loading control. Several autophagy related proteins were detected. PANC-1-conditioned medium could inhibit p-mTOR while promote p-ULK1 and ATG5 expression in RSC96 cell line. Moreover, PANC-1-conditioned medium could promote the conversion of LC3I to LC3II and the degradation of P62. Inhibiting NGF secretion of PANC-1 by knocking down NGF could partially reverse the effect. O. Western blotting of NGF in the control or NGF knockdown BxPC-3 cell lines. Actin was used as a loading control. Several autophagy related proteins were detected. BxPC-3-conditioned medium could inhibit p-mTOR while promote p-ULK1 and ATG5 expression in RSC96 cell line. Moreover, BxPC-3-conditioned medium could promote the conversion of LC3I to LC3II and the degradation of P62. Inhibiting NGF secretion of BxPC-3 by knocking down NGF could partially reverse the effect