Figure 2.

CXCR4 silencing induces autophagy in LM8 cells, while CXCR4 overexpression suppresses autophagy in Dunn cells. (A) The expression levels of CXCR4, and of the autophagy-related proteins beclin 1 and LC3B were determined by western blotting, and the protein bands were quantified and subjected to statistical analysis. The protein bands of GAPDH for CXCR4 silencing and overexpression here are the same as Fig. 1B, as all protein bands in the Fig. 1B and Fig. 2A are from the same blot. (B) Autophagosomes and autolysosomes were detected by transmission electron microscopy in LM8 and Dunn cells after CXCR4 silencing and overexpression respectively. Red arrows indicated autophagosomes, while blue arrows indicated autolysosomes. The number of autophagosomes and autolysosomes was calculated and subjected to statistical analysis. (C) LM8 and Dunn cells were transfected with mRFP-GFP-LC3 adenovirus before treatment. The colocalization of RFP and GFP puncta was examined by confocal microscopy. Yellow puncta represented autophagosomes, while red represented autolysosomes. The percentage of red fluorescence was calculated and analyzed with ImageJ software (National Institutes of Health, version 1.8.0). ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001. CXCR4, C-X-C motif chemokine receptor 4; LC3B, light chain 3B; RFP, red fluorescent protein; GFP, green fluorescent protein.