ABSTRACT
Objective To monitor the autophagy in osteosarcoma cells by constructing three rLC3B fusion expression vectors,respectively.Methods Rat LC3B gene sequence was amplified by PCR and cloned into pEGFP-C 1 and pmCherry-C1 to construct the fusion expression vector of pEGFP-rLC3B and pmCherry-rLC3B.Subsequently,the EGFP-rLC3B sequence was obtained by PCR with the pEGFP-rLC3B as a template,and cloned into pmCherry-C 1,so the pmCherry-EGFP-rLC3B fusion expression vector was constructed.Three plasmids were transfected into U-2OS cells,and the starvation or Rapamycin was adopted to induce autophagy or the chloroquine or Baf-A1 was used to inhibit autophagy,to verify the above plasmids' function in autophagy detection by laser scanning confocal microscopy.Western blot was used to detect the endogenous LC3B and exogenous EGFPrLC3B,pmCherry-rLC3B and mCherry-EGFP-rLC3B,and to verify the correct expression of exogenous rLC3B and their function of autophagy detection.Finally,cleaved free EGFP was detected by western blot to evaluate the level of autophagic degradation.Results Three fusion expression vectors were constructed successfully through sequencing and restriction enzyme digestion validation.The starvation or Rapamycin was adopted to induce autophagy or the chloroquine or Baf-A 1 was used to inhibit autophagy in transfected U-2OS cells.Clear autophagosomes and autolysosomes were observed by laser scanning confocal microscopy.Endogenous LC3B and exogenous EGFP-rLC3B,pmCherry-rLC3B and mCherry-EGFP-rLC3B were detected through western blot.Finally,western blot verified that the expression of cleaved free EGFP was significantly up-regulated with the increase of starvation time.12 h group increased 1.05 times than the control group and 24 h group increased 1.56 times,showing that the levels of autophagic degradation increased.Conclusion EGFP-rLC3B can be used to detect autophagosome and evaluate the level of autophagic degradation.mCherry-rLC3B can be used to detect autophagosome and autolysosome,but can't distinguish autophagosome from autolysosome.The pmCherry-EGFP-rLC3B has an advantage in the detection of autophagic flux which can distinguish autophagosome from autolysosome.