Puncta formation assay
WebJun 29, 2024 · Autophagic flux assay, performed by measuring MAP1LC3 protein turnover, showed that autophagy was significantly decreased 6–8 h after CaOx ... We found that MAP1LC3 puncta formation was still intact at 1–2 d after GOX injection with rapamycin, whereas the number of MAP1LC3 puncta decreased at 1–2 d after GOX ... WebThis sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. ... Thus, the lysosomal …
Puncta formation assay
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WebMar 30, 2024 · For colony formation assays, cells were plated into 6-well culture plates at 1000–3000 cells/well, cultured for 14 days before being fixed with 4% paraformaldehyde ... A–D The impact of ASS1 knockdown on the formation of LC3B puncta in KYSE30 cells (A) and KYSE450 cells (B) with PSMD2 overexpression. WebCell growth and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. A549 cells were then treated with 200 µ M ... (Thermo Fisher Scientific). After A549 cells were treated with or without resveratrol or SRT1720, the number of puncta formation of GFP-LC3 was determined under fluorescent microscopy.
WebAug 8, 2024 · Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g. Halo-LC3 and Halo-GFP) are proteolytically processed … WebJun 6, 2016 · During autophagy, cytoplasmic LC3 is processed and recruited to the autophagosomal membranes; the autophagosome then fuses with the lysosome to form …
WebAug 31, 2012 · Both RNAi lines for Atg16 show a similar block of Lysotracker puncta formation in L3 (panels A and C), while the size of these dots is only reduced significantly by Atg16 HMS in L2 (compare D to B; see also panel E for statistics). * indicates a significant difference (p<0.05), ** indicates a very significant difference (p<0.01), based on two ... WebDec 1, 2024 · In fact, some of the autophagy activators identified through the use of the LC3 puncta formation assay later turned out to be autophagy inhibitors at downstream steps [22]. While cell-based autophagy screens have the advantage of discovering cell-active chemical probes to aid basic research, rigorous downstream work is therefore needed to …
WebThe formation of puncta was decreased in HSP27 knockdown (HSP27KD) cells after thapsigargin (TG) treatment. Finally, we confirmed that the reduction of STIM1 after HSP27 silencing may be due to a loss of STIM1 stability instead of transcription. HSP27 may interact with STIM1 but not Orai1, as shown by immunoprecipitation assays.
WebApr 10, 2024 · Glycyrrhizin treatment for 24 h at different concentrations (50, 100, 200 μM) did not show significant toxicity. Further, we confirmed glycyrrhizin-induced autophagy by immunofluorescence of LC3B. Drug treatment for 4 h showed a significant enhancement of LC3B puncta formation by 3.2 fold change (unpaired t-test and p value: 0.0071) (Fig. 1B boland playground equipmentWebJan 1, 2009 · Figure 16.1. Schematic demonstration of the WIPI‐1 puncta‐formation assay ( Proikas‐Cezanne et al., 2007) 3. Indirect Immunofluorescence to Visualize Endogenous … gluten free certified snacksWebJul 16, 2012 · Autophagic puncta formation was observed by fluorescense microscopy. ... This study indicated that the RAW264.7 cell line stably expressing GFP-LC3 is available … gluten free cereal colesWebAtg5 is also essential for LC3–PE formation and LC3 puncta formation (Mizushima et al., 2001). We tested the requirement of Atg5 for the recruitment of p62 to the autophagic membrane. In this assay, we used an upstream factor, WIPI-1, which is present on isolation membranes in a PI3-kinase activity dependent manner ( Proikas-Cezanne et al., 2007 ; … boland printersWebJul 24, 2007 · GFP-WIPI-1 puncta-formation assay investigating different autophagy modulating agentsInduction of autophagy by prominent inducers such as rapamycin, amino acid deprivation, gleevec [16] and thapsigargin [17] was apparent employing the GFP-WIPI … gluten free cfiaWebApr 11, 2024 · Cell counting kit-8 (CCK8) (Vazyme, A311-01) assay was performed to examine the cytotoxicity of rapamycin or 3-MA to RD cells. Western blotting. Western blotting was performed as described previously . ... In cells transfected with GFP-LC3, the level of LC3 puncta formation was increased by echovirus infection (Figures 3A, B). gluten free chalkboard signWebFeb 2, 2010 · GFP-LC3 and mRFP-GFP-LC3 Puncta Formation Assays (A) NIH 3T3 cells in culture stably expressing GFP-LC3 with (right) or without (left) 2 hr of starvation (depletion … gluten free certifiers