Because qualitatively all transfected cells confirmed LC3 GFP puncta in mock treated in addition to rapamycin, wortmannin, rapamycin/wortmannin treated cells it variance between non and puncta puncta wasn’t possible. Next, we quantified the amounts of LC3 puncta per cell. We observed an increase of LC3 GFP puncta per cell upon rapamycin treatment, and a upon the inhibition of autophagy in G361, HeLa and U2OS cells. While GFP WIPI 1 believed diffusely distributed cytoplasmic Doxorubicin structure localization, using myc WIPI 1/LC3 GFP coexpressing cells, a punctate status was kept by LC3 GFP at problems of autophagy inhibition. 3. 3. GFP WIPI 1 puncta formation assay analyzing different autophagy modulating agencies Induction of autophagy by notable inducers such as rapamycin, amino acid deprivation, thapsigargin and gleevec was apparent employing the GFP WIPI 1 puncta formation assay in HeLa cells. WIPI 1 puncta/non puncta percentages increased upon 3 h and more prominently upon 24 h solutions. Amino acid starvation generated the induction of autophagy in transiently transfected HeLa cells. Likewise, inhibition of autophagy by LY294002 peptide and the inhibitors wortmannin correlated with reduced results in GFP WIPI 1 puncta formation. 3. 4. Myc marked WIPI 1 colocalizes with LC3 GFP Previously, we demonstrated that accumulated endogenous WIPI 1 partially colocalized with accumulated LC3 GFP in individual G361 cells. Here, we coexpressed myc tagged WIPI 1 and LC3 GFP in G361, U2OS and HeLa cells and established a prominent Eumycetoma WIPI 1/LC3 colocalization at LC3 GFP noted autophagosomal membranes. We localized endogenous WIPI 1 or transiently expressed GFP WIPI 1 in autophaging individual G361 cells by immunogold staining on ultra-thin cryosections, respectively using anti WIPI 1 antiserum or antiGFP antibodies. Specifically, we discovered that WIPI 1 localized to multiple membrane structures that closely resemble autophagosomal cup-shaped solitude filters. Up to now, we were unable to identify WIPI 1 at complete autophagosomes. We conducted phospholipid protein overlay assays and demonstrate that human WIPI 1 specifically binds PI P and PI P2, nevertheless, PI P binding occurred more prominently. In order to produce a bindingdeficient WIPI 1 mutant that should keep the demands for propeller folding, we wiped the FRRG motif by changing the corresponding beta sheet 5d with a reproduction of beta sheet 1d lacking the FRRG motif. The GFP 5d1d Clindamycin dissolve solubility mutant showed paid off PI P binding and was completely puncta formationincompetent, shown by quantitative confocal microscopy. There’s an need for new markers to monitor mammalian autophagy. Recently, difficulties in using LC3 GFP as a marker for autophagy were discovered. LC3 GFP protein was reported to aggregate, therefore maybe not showing autophagosomal components. We also report here LC3 GFP to localize to punctate components independent of the cellular autophagic state.