Fig 2: Identifying prognostic genes for developing a PEX5-dependent risk model. The six metabolic pathways in peroxisomes are depicted. The proteins with predicted PTS1 or PTS2 signals are denoted with red and blue fonts, respectively. The genes enclosed within the box represent the 11 signature genes selected by the LASSO model, as illustrated in panels (B,C) below. The 35 PTS1- or PTS2-containing genes in panel (A) were subjected to the LASSO coefficient analysis, using the TCGA dataset. The lines in different colors depict the coefficient distribution of each of the 35 genes. We selected the optimal parameter (lambda), as indicated by the first black dotted line, in the LASSO model in panel (B). (D) Forest plot of the 11 signature genes selected by the LASSO model. * p < 0.05; ** p < 0.01; *** p <0.001.

Fig 3: PEX5 KO inhibits the proliferation, migration, and invasion of U251 cells. (A) Detection of PEX5 in the control, sgPEX5-1, and sgPEX5-2 U251 cells (Original figures could be found in Supplementary File S1). (B) The control and sgPEX5-1 U251 cells were transfected with the GFP-PTS1 or GFP-PTS2 reporter constructs, or the indicated constructs. The cells were stained with GFP antibodies to enhance the fluorescent signal. Scale bars, 10 µm. U251 cells (control, sgPEX5-1, sgPEX5-2) were plated, and subjected to CCK8 (C), colony formation (D), flowcytometry (E), migration (F), and transwell (G) assays. Data are presented as the mean ± SD and representative of three independent experiments. ** p < 0.01; *** p < 0.001 (unpaired two-tailed Student’s t test). The yellow and blue asterisks in panel C indicated the p values for the sgPEX5-1 and sgPEX5-2 cells, respectively.

Fig 4: PEX5 KO inhibits the peroxisomal localization of the signature genes. (A) The control and sgPEX5-1 U251 cells were transfected with the signature gene-expressing constructs. The cells were stained with GFP antibodies to enhance the fluorescent signal. Scale bars, 10 µm. (B) The control and sgPEX5-1 U251 cells were co-stained with PMP70 and CAT antibodies. Scale bars, 10 µm. (C) The expression of signature genes in the control and PEX5 KO U251 cells detected by qPCR. Data are presented as the mean ± SD from three independent experiments. (D) Expression of the signature genes in the gliomas from low- and high-risk patients. **** p < 0.0001; ns, not significant.

Fig 5: Peroxisome content is reduced in PTEN KO HepG2 after OA:PA treatment. (A). Shown is the expression enrichment plot of genes of the peroxisome pathway in PTEN KO relative to wt cells as calculated from the RNA-Seq data. Note the flip of the signature after the treatment with OA:PA. (B). Representative confocal immunofluorescence images for PEX14 before and after treatment with OA:PA. (C). Quantification of fluorescence intensity shows the reduction in PEX14 expression after treatment. Statistical significance was determined by an unpaired two-tailed Student’s t-test (* p < 0.05). (D). Immunoblotting for PEX5 before and after treatment with OA:PA in wt and PTEN KO HepG2 cells. Shown is a representative result of three repetitions. (E). Analysis of RNA-Seq data for the mitochondrial β oxidation pathway following OA:PA treatment. In arrows (each gene marked in a different color) are genes whose expression is upregulated in wt cells and downregulated in PTEN KO cells. (F). Uptake of OA in wt and PERK KO HepG2 cells. Fluorescence was calculated for 20 cells and quantified. (G). Statistical significance was determined using an unpaired two-tailed Student’s t-test (* p < 0.05). (H). Representative images of lipid droplets using LipidSpot in wt and PTEN KO HepG2 cells after incubation with OA and OA:PA for 24 h. (I). Quantification of 20 cells for each treatment. Statistical significance was determined using an unpaired two-tailed Student’s t-test (* p < 0.05).