Fig. 3

Enhanced autophagic flux and G-cleave LC3B biosensor luciferase activity in MDA-MB-231 cells through proteosome-related degradation. A IVIS images show the decline of bioluminescence in MDA-MB-231 cells expressed pEGFP-LC3B^pepABLuc with 4 h of EBSS and serum starvation treatments. Quantification of total photons flux was analyzed (n = 3 replicates; student-t test; **, p < 0.01; bars represent mean ± SE). MDA-MB-231 cells expressing pEGFP-LC3B^pepABLuc were exposed to varying concentrations of B EBSS and C serum starvation for 1 to 4 h, or pre-treatment with or without 10 μM MG132 followed by indicated incubation with D EBSS and E serum starvation. The luciferase degradation activity (convert to autophagy activity) was assessed as described in materials and methods (n = 3 replicates; student-t test; *, p < 0.05; **, p < 0.01; error bars represent mean ± SE). The lysates of 4 h F EBSS-treated and G serum starvation-treated were also collected and immunoblotted with anti-luciferase antibody. The full-length luciferase appeared at around 63 KDa, while the cleavage form was around 48 KDa on SDS-PAGE. H The level of cleaved luciferase and autophagy degradation was evaluated by immunoblotting in MDA-MB-231 cells expressing pEGFP-LC3B^pepABLuc, treated with EBSS in the presence or absence of 30 μM CQ for 24 h. GAPDH was used as a loading control. I A schematic diagram of the G-cleave LC3B biosensor is presented. The fusion protein of pepA-N’luc2 and pepB-C’luc2 were linked with a 3X LC3B cleavage sequence (TFGMKLS). The bioluminescence expression of the cleavage form of luciferase mediated by ATG4B was detected upon the addition of luciferin. This bioluminescence is predominantly degraded in a proteosome-dependent manner during the activation of autophagy. The luciferase activity reflects the degradation ratio of luciferase upon complete autophagy activation