Tioning (m) and hematoxylin and eosin (H E) staining at the UCSF Helen Diller Household Complete Cancer Center Mouse Pathology Core. For immunofluorescence staining, slides have been subject to heatinduced antigen retrieval (Antigen Unmasking remedy, Citrate Primarily based, Vector Labs) for minutes at C. Q autofluorescence was not evident in formalinfixed paraffin embedded sections. Slides have been cooled, blocked in goat serum, followed by overnight principal antibody incubation at C in a humidified chamber. Slides had been washed in PBS andOncogene. Author manuscript; obtainable in PMC July .Salas et al.Pageincubated with Alexa conjugated secondary antibodies (Invitrogen) for h at C. Right after washing with PBS, slides had been counterstained with Hoechst (gmL) for min, washed and mounted with Prolong Gold (Invitrogen). TUNEL assays were performed using ApopTagkits (EMD Millipore) based on manufacturer’s protocol. Phase and immunofluorescence image acquisition and TCS-OX2-29 biological activity Evaluation Phase images of H E sections were acquired on Aperio’s ScanScope XT affixed having a X and X objective making use of Aperio ImageScope software program (v.). IF images have been acquired on an Axiovert microscope (Carl Zeiss) equipped with a Spot RT camera (Diagnostic Instruments) in purchase NIK333 addition to a mercury lamp. Pictures were acquired making use of MetaMorph computer software (v.) and arranged utilizing Adobe Illustrator CS (v).CNMRAuthor Manuscript Author Manuscript Author Manuscript Author Manuscriptacquisition and analysis Cells had been grown in DMEM medium containing mmolL of C glucose to confluency, upon which medium was collected for NMR evaluation and cells were harvested for protein quantification making use of the BCA assay. Briefly, medium was lyophilized and reconstituted in mL of deuterium oxide. To acquire C spectra, the reconstituted sample was acquired on a MHz INOVA spectrometer (Varian; Palo Alto, CA) applying a flip angles repetition time, and broadband proton decoupling. Evaluation of your NMR spectra was performed applying Bruker Topspin software program version . (Bruker Biospin GmbH, Germany). The concentrations of metabolites were determined with respect to an external reference,,tetradeutero(trimethylsilyl)propionic acid (TSP), having a known concentration.Analysis of Oxidative Pentose Phosphate Pathway A single million cells were plated onto cm dishes in triplicate, grown overnight and treated with car manage (water) or with CQ or Q in the indicated doses for h at C. Cells had been washed twice with PBS and the tissue culture media was replaced with media containing .Ci of Cglucose (Perkin Elmer) or Cglucose containing car (water) or the indicated antimalarial; cells have been incubated for an more h before evaluation. To capture gaseous CO, Whatman filter paper was taped to the inside of culture dish lid as well as the plate was then sealed with Parafilm; the experiment was terminated by adding L of perchloric acid for the cells; the plate was resealed and left inside the incubator 
overnight. Filter papers have been then cautiously removed and placed into vials containing ReadySafe Liquid Scintillation Fluid (Beckman Coulter), and CO activity was measured on a Beckman LS Liquid Scintillation Analyzer. The incubation of cells PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/19297450 with Cglucose, utilized to appropriate for CO production in the citric acid cycle, yielded minimal CO activity. Glucosephosphate dehydrogenase (GPD) activity assay GPD activity was determined employing the GPD activity assay kit from Biovision (Milpitas, CA). cells have been grown overnight in triplicate on cm dishes and either treated with CQ, Q at the indicated.Tioning (m) and hematoxylin and eosin (H E) staining in the UCSF Helen Diller Household Comprehensive Cancer Center Mouse Pathology Core. For immunofluorescence staining, slides have been subject to heatinduced antigen retrieval (Antigen Unmasking resolution, Citrate Based, Vector Labs) for minutes at C. Q autofluorescence was not evident in formalinfixed paraffin embedded sections. Slides had been cooled, blocked in goat serum, followed by overnight primary antibody incubation at C inside a humidified chamber. Slides have been washed in PBS andOncogene. Author manuscript; offered in PMC July .Salas et al.Pageincubated with Alexa conjugated secondary antibodies (Invitrogen) for h at C. After washing with PBS, slides have been counterstained with Hoechst (gmL) for min, washed and mounted with Prolong Gold (Invitrogen). TUNEL assays were performed working with ApopTagkits (EMD Millipore) according to manufacturer’s protocol. Phase and immunofluorescence image acquisition and analysis Phase images of H E sections were acquired on Aperio’s ScanScope XT affixed having a X and X objective applying Aperio ImageScope software (v.). IF images were acquired on an Axiovert microscope (Carl Zeiss) equipped using a Spot RT camera (Diagnostic Instruments) plus a mercury lamp. Images were acquired applying MetaMorph application (v.) and arranged utilizing Adobe Illustrator CS (v).CNMRAuthor Manuscript Author Manuscript Author Manuscript Author Manuscriptacquisition and analysis Cells were grown in DMEM medium containing mmolL of C glucose to confluency, upon which medium was collected for NMR evaluation and cells have been harvested for protein quantification working with the BCA assay. Briefly, medium was lyophilized and reconstituted in mL of deuterium oxide. To obtain C spectra, the reconstituted sample was acquired on a MHz INOVA spectrometer (Varian; Palo Alto, CA) applying a flip angles repetition time, and broadband proton decoupling. Evaluation with the NMR spectra was performed applying Bruker Topspin software version . (Bruker Biospin GmbH, Germany). The concentrations of metabolites have been determined with respect to an external reference,,tetradeutero(trimethylsilyl)propionic acid (TSP), using a identified concentration.Evaluation of Oxidative Pentose Phosphate Pathway One particular million cells have been plated onto cm dishes in triplicate, grown overnight and treated with vehicle handle (water) or with CQ or Q at the indicated doses for h at C. Cells were washed twice with PBS plus the tissue culture media was replaced with media containing .Ci 
of Cglucose (Perkin Elmer) or Cglucose containing automobile (water) or the indicated antimalarial; cells were incubated for an more h before analysis. To capture gaseous CO, Whatman filter paper was taped to the inside of culture dish lid plus the plate was then sealed with Parafilm; the experiment was terminated by adding L of perchloric acid for the cells; the plate was resealed and left within the incubator overnight. Filter papers had been then carefully removed and placed into vials containing ReadySafe Liquid Scintillation Fluid (Beckman Coulter), and CO activity was measured on a Beckman LS Liquid Scintillation Analyzer. The incubation of cells PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/19297450 with Cglucose, utilized to right for CO production from the citric acid cycle, yielded minimal CO activity. Glucosephosphate dehydrogenase (GPD) activity assay GPD activity was determined making use of the GPD activity assay kit from Biovision (Milpitas, CA). cells were grown overnight in triplicate on cm dishes and either treated with CQ, Q in the indicated.
