Fig. 1.

Knockdown of PCBP1 increases ferroptosis sensitivity.

(A) The iron assay measured the total iron level in HN12 with transfection of vector, shPCBP1, or shPCBP1 plus PCBP1 cDNA plasmid (PCBP1res). The shPCBP1 #1 and #2 were prepared by inserting two different target sequences for PCBP1 into the shRNA vector. (B–G) Cell viability (B) and cell death (C–D), immunoblotting (E), lipid peroxidation (F), and labile iron pool (LIP) (G) assays in HN12 cells with vector, shPCBP1, or PCBP1res transfection. Cell viability and death were measured by using cell counting kit-8 (CCK-8) and SYTOX Green staining, respectively, after exposure to ferroptosis inducers, e.g., 10 μM erastin, 1 mM sulfasalazine (SAS), or cyst(e)ine deprivation (cys (−)) for 48 h. The SYTOX Green-stained cells were quantified using ImageJ software (C), and the mean positive fractions were compared with those of the control or different group (D). Lipid peroxidation was measured by adding 5 μM BODIPY C11 for 30 min, and LIP was measured using calcein acetoxymethyl ester and iron chelator, deferoxamine, in the cells after exposure to the ferroptosis inducers for 6 h. The error bars represent standard errors from three independent experiments. Original magnification, × 200. Scale bar, 50 μm. (H–J) Images of lipid peroxidation cells (H–I) and cellular iron amounts (J). Non-oxidized (red) and oxidized (green) were measured in cells exposed to DMSO or 10 μM erastin for 6 h by adding 5 μM BODIPY C11. Image quantification was performed using ImageJ software (H), and the ratios of oxidized cells to total cells were calculated (I). Iron levels were measured by iron assay after 10 μM erastin, 1 mM SAS, or cys (−) for 6 h. Original magnification, × 200. Scale bar, 50 μm. (K–L) mRNA (K) and protein (L) expression levels of PCBP1, PCBP2, DMT1, FTH1, FPN, and TFRC in HN12 cells were confirmed by RT-qPCR and immunoblotting, respectively. The error bars represent standard errors from three independent experiments. ns; no significance, *P < 0.05, **P < 0.01, ***P < 0.001 among different cells or treatments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)