Fig 1: FTL and FTH were differentially regulated after erastin treatment. (A,B) FTL and FTH were measured by IB in PC9 cells treated with CHX (10 μg/ml, A) or erastin (10 μM, B) for indicated hours. The relative protein levels of FTL and FTH were shown as the ratios to GAPDH, and the “0h” points were arbitrarily set to 100%. (C) mRNA level of FTH and FTL in PC9 cells treated with erastin (10 μM) for indicated hours. (D) WT or TFCP2 (FOXA1)-binding motif mutant (Mut) FTL/FTH promoter Luc activity were measured in PC9 cells treated with erastin (10 μM) for indicated hours. (E) Enrichment of YAP at TFCP2 (FOXA1)-binding motif within the FTL or FTH promoter in PC9 cells was measured by ChIP-qPCR after erastin treatment (10 μM) for indicated hours. (F) Enrichment of YAP at TFCP2 (FOXA1)-binding motif within the FTL or FTH promoter in erastin (10 μM, 4 h) treated PC9 cells with or without NCOA4 knockdown was measured by ChIP-qPCR. (G) Enrichment of TFCP2 at TFCP2 (FOXA1)-binding motif within the FTL promoter in PC9 cells with or without NCOA4 knockdown was measured by ChIP-qPCR after erastin treatment (10 μM) for indicated hours. (H) FTL was measured by IB in indicated LUAD cells with or without erastin (10 μM, 24 h) treatment. (I) Correlation between induced cell death and remaining FTL level after erastin (10 μM, 24 h) treatment for 24 h. Induced cell death and remaining FTL level was calculated as fold or percentage to the ones treated with DMSO. (J) YAP, GLUD1, ALOX15, ACSL4 and TFRC expression were measured using IB in PC9 cells with erastin (10 μM) treatment for indicated hours. (K,L) YAP, GLUD1, ALOX15, ACSL4 and TFRC mRNA level (K) and α-KG concentration (L) were measured in PC9 cells with YAP overexpressed or knocked down. The data are shown as the mean ± SD from three biological replicates (including IB). **P < 0.01 indicates statistical significance. Data in (C–E,G) were analyzed using a two-way ANOVA test. Data in (F,K,L) were analyzed using student’s t-test. Data in I were analyzed using Spearman rank-correlation analysis.
Fig 2: Mitochondrial proteins and DNA are packaged into EVs. (a) MDA-MB-231 cells were transfected with siRNAs targeting Rab27a/b (siRab27), CD63 (siCD63), or nontargeting siRNA (siNT) and incubated for 5 d in the presence (LY95) or absence (Ctrl) of LY95 (0.3 µM). Oxygen consumption was determined using the Seahorse XFe96 Extracellular Flux Analyzer. Sequential treatment with oligomycin, FCCP, and rotenone/antimycin A (Rot+AntA) was performed as indicated. Values represent the mean ± SEM of three independent experiments. (b) Cells were incubated in the presence of LY95 (0.3 µM) or vehicle control (Ctrl) for 48 h. EVs were isolated from media by differential centrifugation and analyzed by Western blotting for VDAC, cyclophilin D, and GLUD1. (c) Mitochondria (mito.) were purified from cells and EVs. mtDNA was extracted from these preparations and analyzed by long-range PCR. The expected migration position of the mitochondrial nucleoid (∼16 × 106 D) is indicated. (d) EVs were purified by differential centrifugation and incubated in the presence and absence of DNase immobilized to agarose beads. mtDNA and nuclear DNA were determined by qPCR using primers complementary to sequences within the mitochondrial genes (COX1, CYTB, n = 5 independent experiments; or ND1, n = 3 independent experiments) or the nuclear β-globin genes. Values are mean ± SEM. *, P < 0.05; paired t test. (e) Cells were transfected with siRNAs targeting CD63 (siCD63) or a nontargeting siRNA (siNT). Cells were then incubated for 48 h in the absence (Ctrl; NT and siCD63) or presence of 0.3 µM LY95. EVs were purified from the medium by differential centrifugation, and mtDNA content of these was determined using qPCR with primers recognizing the mitochondrial COX1 gene. Values are mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.002; ***, P < 0.001; Mann-Whitney U test. (f) Media collected in the absence (Ctrl) and presence of 0.3 µM LY95 were subjected to differential centrifugation. Differential centrifugation pellets were overlaid with a sucrose density gradient (2–0.4 M sucrose) and centrifuged at 200,000 g overnight. Gradients were eluted, and fractions were collected for analysis by qPCR for the mitochondrial ND1 gene and by Western blotting for CD63. Values represent mean ± SEM, n = 5 independent experiments. *, P < 0.05; Mann-Whitney U test.
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