Pharmacological modulators of this pathway have been extensively utilized in a wide range of basic research and pre-clinical studies. Bafilomycin A1 and chloroquine are commonly used compounds that inhibit autophagy by targeting the lysosomes but through distinct mechanisms. Plaquenil and sulfasalazine Aralen dosage In an in vitro experiment of autophagic flux assay by western blot using chloroquine, is it correct to use the LC3-II/LC3-I ratio as a manner to represent the effects of a treatment or should I. Example of IC data by Autophagy Watch Starvation induces increasing the amount of LC3-II. lane1 vs. lane 2 Additionally, the amount of LC3-II increases in the presence of Chloroquine or Bafilomycin A1.lane 2 vs. lane3, 4 These results suggest the accumulation of LC3-II under starvation condition indicates the enhancement of the autophagic flux. These results were further confirmed using chloroquine and GFP-RFP-LC3 in autophagic flux assays in HepG2 cells Fig. 5C and 5D. Tauro-UDCA further increased LC3-II in the presence of chloroquine, which was independent of FXR. Furthermore, tauro-UDCA leads to the formation of red dots in the LC3-GFP-RFP assay, indicating enhanced autophagic flux. To address this, we cultured primary rat cortical neurons from E18 embryos and used the Seahorse XF96 analyzer and a targeted metabolomics approach to measure the effects of bafilomycin A1 and chloroquine on bioenergetics and metabolism. Since it is now clear that mitochondrial quality control, particularly in neurons, is dependent on autophagy, it is important to determine whether these compounds modify cellular bioenergetics. Chloroquine autophagic flux Suppression of autophagy by chloroquine sensitizes 5., For Autophagy Flux Assay - Nature Biotech Er pr her breast cancer chloroquine Autophagic flux is a measure of efficiency or throughput of the pathway. Here, we describe a method for determining autophagic flux in vitro and in vivo using the autophagosomal/lysosomal fusion inhibitors chloroquine or bafilomycin A1 and then probing for the autophagosomal marker LC3-II via Western Blot. Measuring Cardiac Autophagic Flux In Vitro and In Vivo. FXR-dependent Rubicon induction impairs autophagy in.. Tenovin-6 impairs autophagy by inhibiting autophagic flux.. Indeed, Chloroquine is a potent blocker of autophagy and has been demonstrated in a lab setting to dramatically enhance tumor response to radiotherapy, chemotherapy and even anti-hormonal therapy. This is why Chloroquine has become part of many clinical trials, in combination with chemotherapy and other form of cancer treatments. Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Mauthe M12, Orhon I1, Rocchi C13, Zhou X14, Luhr M5, Hijlkema KJ1, Coppes RP13, Engedal N5, Mari M12, Reggiori F12. The term “autophagic flux” is used to denote the dynamic process of autophagosome synthesis, delivery of autophagic substrates to the lysosome, and degradation of autophagic substrates inside the lysosome and is a more reliable indicator of autophagic activity than measurements of autophagosome numbers.