Fig. 1.MEF cells stably expressing GFP-LC3 (green) are counterstained for p62/SQSTM1 (red) and acquired through super-resolution structured illumination. p62/SQSTM1 signal is present abundantly throughout the cytoplasm, it remains largely non-associated with autophagosomes (A), which robustly ch...
Recently, Zhou et al. evaluated autophagic flux in hippocampal neurons in vitro (Zhou et al., 2017). OGD resulted in decreased lysosomal number and acidity, and decreased LAMP2. Using a GFP-RFP-tf-LC3 assay, the investigators quantified autolysosomes and autophagosomes. OGD decreased the ratio...
CCK8, mRFP-GFP-LC3 assay, western blot, colony formation, wound healing, and transwell migration assays were conducted to evaluate the effects of the ACK1 inhibitor on lung cancer cells. A subcutaneous NSCLC xenograft model was used for in vivo study. Results:RNA-seq revealed the regulatory ...
BafA1 has been used to assess autophagy flux, because it is reported to completely block the lysosomal degradation of LC3-II. cAMP has been reported to have effects on lysosomal pH by promoting the assembly of v-ATPase48. Surprisingly, however, we found that cilostazol and cAMP were able to...
Flow cytometry, LC3-II expression levels, and LC3-GFP puncta formation revealed a similar lysosomotropic effect for both compounds. We used a molecular docking approach, that predicts a stronger inhibitory activity against V-ATPase-C1 and C2 domains for bafilomycin-A1 in comparison to ML9. ...
17). Furthermore, additional increase in lipidated LC3 upon bafilomycin treatment is indicative of autophagic flux. Traditionally, detection of LGG-1 or SQST-1/P62 on an immunoblot relied on using anti-GFP antibodies on lysates from GFP::LGG-1 or SQST-1/P62::GFP worms due to lack of ...
It also increased the percentage of cells containing GFP-MAP1LC3B puncta and upregulated the expression of beclin-1 and ATG5. Cotreatment with the late stage autophagy inhibitor bafilomycin A1 further increased the levels of LC3-II and p62/SQSTM1 in both cell lines. Interestingly, cotreatment ...
GFP-LC3 单荧光自噬指示体系：利用LC3在自噬形成过程中发生聚集的原理，开发出GFP-LC3指示技术：无自噬时，GFP-LC3融合蛋白弥散在胞浆中；自噬形成时，GFP-LC3融合蛋白转位至自噬体膜，在荧光显微镜下形成多个明亮的绿色荧光斑点，一个斑点相当于一个自噬体，可以通过计数来评价自噬活性的高低。但是绿色斑点增多并不...