Fig. 3: N-glycosylation of 4F2hc plays a critical role in the ferroptosis sensitivity of PDAC cells. | Cell Death & Differentiation

Fig. 3: N-glycosylation of 4F2hc plays a critical role in the ferroptosis sensitivity of PDAC cells.

From: Targeting N-glycosylation of 4F2hc mediated by glycosyltransferase B3GNT3 sensitizes ferroptosis of pancreatic ductal adenocarcinoma

Fig. 3

A Schematic plot of the 4F2hc and xCT complex and its function. B High expression of 4F2hc shows a positive correlation with resistance to ferroptosis inducers (RSL3 (Pearson’s correlation coefficient (PCC) = 0.329), ML162 (PCC = 0.348), and ML210 (PCC = 0.33)) in cancer cells. Data mined from the Cancer Therapeutics Response Portal (CTRP) (http://www.broadinstitute.org/ctrp). Box and whisker plots indicate the 10th to 90th percentile range, and minimum and maximum values, each value of distributions are PCC on the y-axis. C PANC-1 cells were treated with TM at different doses for different amounts of time, and the expression changes in 4F2hc and GPX4 were determined by western blotting. The purple- and cyan- triangles represent high- and low- glycosylation, respectively. D The expression and localization of transmembrane protein 4F2hc in PANC-1 cells treated with or without PNGase F are determined by the confocal image on the left and flow cytometry on the right. E mRNA levels of the indicated gene in SLC3A2 knockdown PANC-1 cells were quantified by qRT–PCR. F Measurement of 4F2hc, NRF2, xCT (CST#12691, 35 kDa), DHODH, and GPX4 protein expression levels in PANC-1 cells with or without 4F2hc knockdown. G Cell viability of shSLC3A2 PANC-1 and MIA PaCa-2 cells treated with a range of RSL3 doses for 12 h. H Lipid ROS levels in shControl and shSLC3A2 PANC-1 cells treated with or without 0.8 μM RSL3 for 8 h. BODIPY™ 581/591 C11 staining was used for FACS analysis in FITC channel. I Extracellular glutamate levels in shControl and shSLC3A2 PANC-1 cells treated with or without 0.8 μM RSL3 for 12 h. J Intracellular GSH levels in shControl and shSLC3A2 PANC-1 cells treated with or without 0.8 μM RSL3 for 12 h. K, L Cell viability in shControl and shSLC3A2 PANC-1 and MIA PaCa-2 cells treated with or without 10 μg/ml TM or 0.8 μM RSL3 for 12 h. M Western blotting analyzing the protein expression of 4F2hc in SLC3A2 knockdown PANC-1 and MIA PaCa-2 cells transfected with wildtype (WT) 4F2hc, N365Q, and 4NQ plasmids, respectively. N, O Cell viability analysis of shSLC3A2 PANC-1 (N) and shSLC3A2 MIA PaCa-2 cells (O) transfected with WT, N365Q, and 4NQ plasmids, respectively, with or without 0.8 μM RSL3 treatment for 12 h. All data are shown as the mean ± SD. Statistical significance among the indicated groups was assessed by ANOVA analysis or unpaired Student’s t-test. At least three independent experiments except for B and D, 3 technical replicates for G. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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