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. 2022 Nov 18;33(14):ar135. doi: 10.1091/mbc.E21-08-0420

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© 2022 Gasparian et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 1: — Depletion of the COPI complex inhibits mitochondrial activity and activates ROS production in tumor cell lines. (A) Measurements of mitochondrial metabolic activity. PC3 cells were transfected with control (siCONT) or anti COPZ1 (siCOPZ1) siRNAs and plated in Seahorse XF24 microplates. ATP production (upper panel), and maximum respiration capacity (lower panel) were detected by the OCR, see Supplemental Figure S1 for details. The bar graph shows the mean of four independent transfections ± SE. (B) Measurement of induced mitochondrial ROS production in PC3 cells. PC3 cells were transfected with control (siCONT) or anti COPA (siCOPA) siRNAs. 72 h posttransfection ROS were detected with DCFDA, and mitochondria were stained with MitoNIR in live cells. Fluorescent images of separate and merged images are presented. (C) Summary of flow cytometry analysis of ROS production in COPI-depleted PC3, DU145, and U2OS cells and the effects of MitoQ and JNK inhibitor SP600125, labeled as JNKi (examples of flow cytometry profiles are presented in Supplemental Figure S2). Plots represent the DCFDA mean fluorescent intensity in siCONT, siCOPA, and siCOPZ1-transfected cells ± MitoQ or SP600125 treatments. Results shown are three independent experiments per data point ± SD. Scale bar = 25 μm.