Figure 5

The alteration of autophagy gene expression when high glucose inhibited the generation of cranial neural crest cells.

HH9 chick embryonic neural tube tissues were incubated in vitro for 48 hours. (A-C) The bright-field images of the CNCCs that emigrated from the primary cultures of neural tubes in the presence of simple saline. Control, (A), L-Glucose (B) and D-Glucose (C), respectively. (A’–C’) The high magnification images from the sites indicated by the white dotted squares in (A–C), respectively. (D) The bar chart showing the comparison of the migratory cell areas from the cultured neural tubes between the control and high glucose-treated embryos. (E–G) Immunofluorescent staining against AP-2α was performed on the primary cultures as shown in (A–C), respectively. The merged images of AP-2α staining + DAPI were taken at the same position relative to the neural tubes (A–C) in the control (E), L-Glucose (F) and D-Glucose (G) treated primary cultures. (H) The bar chart showing the comparison of AP-2α positive cranial neural crest cell numbers among control and high glucose-treated embryos. (I–K) Immunofluorescent staining against LC3B was performed on the primary cultures of neural tube as shown in (A–C), respectively. The fluorescent images of LC3B were taken at the same position related to the neural tubes in the control (I), L-Glucose (J) and D-Glucose (K) treated primary cultures. (I’–K’) The merged images of I-K + DAPI, respectively. (L) Western blots showing the expression of LC3B in the control and high glucose-treated embryos. (M) Western blots showing the protein expression of Beclin1, Atg5-Atg12, FL-Atg5, Truncated Atg5 and LC3B in HEK293 cells in the presence of simple saline (control) and high glucose. (N–O) Transmission electron microscopy of the control and D-Glucose treated HH10 chick cranial neural crest cells, the autophagosomes are indicated by the black arrowheads. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate significant differences between the experimental and control embryos. Abbreviations: Con, control; L-Glc, L-Glucose; D-Glc, D-Glucose; PI, Propidium iodide; CNCC, cranial neural crest cell; n: nucleus; mi: mitochondrion. Scale bars = 500 μm in (A–C); 100 μm in (A’–C’), 50 μm in E-G and 50 μm in (I–K), (I’–K’), 200 nm in (N–O).