Fig. 5: Loss of COPZ1 induces autophagy in GBM cells in vitro.
From: Loss of COPZ1 induces NCOA4 mediated autophagy and ferroptosis in glioblastoma cell lines
a Representative fluorescence images of GFP/mCherry-LC3B puncta in si-COPZ1#1 transfected U87MG after 48 h. Scale bars, 25 μm. b Statistical analysis of the images shown in (a). Quantification represents results taken from 3 images from experiments performed in triplicate. c Representative western blots showing protein levels of LC3B, SQSTM1 (p62), ATG7 and ACTB (loading control) in si-COPZ1#1 transfected U87MG, U251, and P3#GBM cells. d Western blots showing protein levels of FTH1, NCOA4, and ACTB (loading control) in si-COPZ1#1 transfected U87MG, U251, and P3#GBM cells. e Representative fluorescence images showing intracellular localization of COPZ1 (green) and NCOA4 (red). Alexa-NCOA4 was diffusely localized in the cytoplasm and largely colocalized with FITC-COPZ1 in normal U87MG and U251 cells. Scale bar, 10 μm; scale bars of magnified images, 1 μm. f Western blots to detect levels of NCOA4 and LC3B in U87MG-sh-COPZ1#1 cells after pretreatment with 3-MA (10 mM) and CQ (3 μM) for 1 h. Data are representative of 3 independent experiments. g Relative iron levels in U87MG-sh-COPZ1#1 cells after pretreatment with 3-MA (10 mM) and CQ (3 μM) for 1 h. h Cell death ratio after pretreatment with 3-MA (10 mM) and CQ (3 μM) for 1 h in U87MG-sh-COPZ1#1 cells. i MDA levels after pretreatment of U87MG-sh-COPZ1#1 cells with 3-MA (10 mM) and CQ (3 μM) for 1 h. Student’s t test for two-group comparison: **p < 0.01; one-way ANOVA for multi-group comparisons: NS = non-significant, **p < 0.01, ***p < 0.001.
