Acute Lymphoblastic Leukemia - Supplementary Appendix - Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia

Oct 5, 2026

Uploaded by McQueen

Supplementary methods describe how Bafilomycin A1 is evaluated in pediatric B-cell acute lymphoblastic leukemia by assessing cellular proliferation, cell-cycle distribution, autophagic flux, and apoptosis. Experimental workflows include CFSE-based proliferation assays, propidium iodide cell-cycle staining, western blotting with co-immunoprecipitation and inhibitor cocktails, autophagy induction in HBSS with GFP-LC3 reporter readouts, and Annexin V-FITC apoptosis analysis by flow cytometry. Small RNA interference using AIF siRNAs and Lipofectamine transfection is used to modulate related pathways.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages8

Reading Time20 min

AccessFree to download

Supplementary Methods

Cell proliferation assay

Cell cycle analysis

Western blotting, co-immunoprecipitation and antibodies

Autophagic flux assay

Apoptosis assay

Small RNA Interference

Acute Lymphoblastic Leukemia SUPPLEMENTARY APPENDIX Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia Na Yuan, Lin Song, Suping Zhang, Weiwei Lin, Yan Cao, Fei Xu, Yixuan Fang, Zhen Wang, Han Zhang, Xin Li, Zhijian Wang, Jinyang Cai, Jian Wang, Yi Zhang, Xinliang Mao, Wenli Zhao, Shaoyan Hu, Suning Chen, and Jianrong Wang Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Jiangsu Key Laboratory for Stem Cell Research, Collaborative Innovation Center of Hematology, Affiliated Children’s Hospital, Soochow University School of Medicine, Suzhou, China ©2015 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2014.113324 The online version of this article has a Supplementary Appendix. Manuscript received on July 7, 2014. Manuscript accepted on December 5, 2014. [Correspondence: jrwang@suda.edu.cn](Correspondence: jrwang@suda.edu.cn) BafilomycinA1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia Na Yuan, Lin Song, Suping Zhang, Weiwei Lin, Yan Cao, Fei Xu, Yixuan Fang, Zhen Wang, Han Zhang, Xin Li, Zhijian Wang, Jinyang Cai, Jian Wang, Yi Zhang, Xinliang Mao, Wenli Zhao, Shaoyan Hu, Suning Chen, Jianrong Wang‡ Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Jiangsu Key Laboratory for Stem Cell Research, Collaborative Innovation Center of Hematology, Affiliated Children’s Hospital, Soochow University School of Medicine, Suzhou, China ‡Address correspondence to: Jianrong Wang, Hematology Center of Cyrus Tang Medical Institute, Soochow University School of Medicine, 199 Ren’ai Road, Suzhou 215123, China; Tel/Fax: 86-512-65884331; E-mail address: [jrwang@suda.edu.cn](jrwang@suda.edu.cn) Supplementary Methods Cell proliferation assay. Cells were collected and resuspended in serum-free 1640 medium ata density of 4×106 cells/mL and mixed gently immediately after adding the dye [5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma-Aldrich)] into the suspension to a final working concentration of 3 μM, then incubated the mixture in the dark for 15 minutes at 37ºC, after which 5 fold volume of cold 1640 medium containing 10% FBS was added to terminate reactions. The resultant cells were washed twice with culture medium. Finally, cell suspension (stained with CFSE) was dispensed into 24 well plate and added different drugs into corresponding wells. Cells were harvested at appropriate time, washed twice and resuspended with PBS, followed by analysis on a BD Calibur flow cytometer. Cell group not treated with CFSE served as a negative control. Cell cycle analysis. To analyze cell-cycle status, cells were pelleted by centrifugation for 5 minutes at 200 g, washed in PBS, and resuspended in 70% ice-cold ethanol. Cells were fixed overnight and then stained with propidium iodide. The pretreated cells were analyzed using a BD Calibur Flow Cytometer. Western blotting, co-immunoprecipitation and antibodies. Primary and secondary antibodies were provided by Cell Signaling Technology (Danvers, MA, USA) . Signals were detected with the ECL Plus reagent by Perkin Elmer (Boston, MA, USA) . Cell was collected andresuspended in lysis buffer containing protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail (Roche, USA) . The resuspended cell pellet was vortexed for 20 seconds and then incubated on ice for 30 min and centrifuged at 13,000 g for 20 min at 4°C. The supernatants were collected and the protein concentration was determined using the BCA protein assay kit (Thermo, USA) and heated at 95 °C for 5 min. Equal amounts of protein were subjected to SDS-PAGE. Samples were loaded on a polyacrylamide gel for electrophoresis separation and transferred to a polyvinylidenediﬂuoride membrane (Millipore, USA) . Membranes were blocked in 5% non-fat dry-milk and incubated overnight at 4°C with the primary

FAQs

How is cell proliferation measured in these experiments?

Cells are labeled with CFSE and incubated, then washed and analyzed by flow cytometry to quantify proliferation-associated dilution. A non-CFSE-treated group serves as a negative control.

What method is used to evaluate cell-cycle status?

Cells are fixed in 70% ice-cold ethanol, stained with propidium iodide, and analyzed by flow cytometry using a BD Calibur system.

How is autophagic flux assessed?

Cells are incubated in nutrient-depleted HBSS, and a stable reporter line expressing GFP-LC3 is used. Autophagic flux is quantified by counting green fluorescence dots from imaging analysis.

Oct 5, 2026

Uploaded by McQueen