Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet:

Article Snippet: Anti-mouse IgG (H + L)-HRP , Bio-Rad , RRID: AB_2617113.

Techniques: Cell-Signaling, Virus, Recombinant, Clinical Proteomics, Membrane, Staining, Cloning, Software

Journal: iScience

Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

doi: 10.1016/j.isci.2026.117658

Figure Lengend Snippet: Deletion of Pex19 impairs the growth of mESCs (A) Pex19 deficiency reduces the growth of mESCs. Equal numbers of WT and Pex19 KO 46C mESCs were seeded and cultured in ESGRO medium, and cell counts were analyzed on day 3 ( n = 3 biological replicates/group; data are presented as mean ± SEM; Student’s t test, ∗∗ p < 0.01). (B) Pex19 deficiency reduces the single-cell self-renewal capacity of mESCs. WT and Pex19 KO mESCs were cultured for colony formation for 5 days ( n = 3 biological replicates/group; data are presented as mean ± SEM, Student’s t test, ∗ p < 0.05). (C) Pex19 deficiency increases cell death and apoptosis of mESCs ( n = 3 biological replicates/group, Student’s t test, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (D) Adding back PEX19 restores the peroxisomal targeting of PEX14. Scale bars, 5 μm. (E–G) Adding back PEX19 rescues defective cell growth and elevated cell death in Pex19 KO mESCs. E and F, n = 3–4 biological replicates/group; data are presented as mean ± SEM, one-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, ∗∗∗q < 0.001, n.s., not significant). (H) Top enriched GSEA pathways in Pex19 KO vs. WT mESCs. Transcriptomes of WT and Pex19 KO 46C mESCs from the RNA-seq dataset were analyzed using GSEA as described in . NES, Normalized Enrichment Score; FDR, False Discovery Rate. See also .

Article Snippet: Mouse anti-PEX19 monoclonal antibody , Novus , NBP2-43544; RRID: AB_3307633.

Techniques: Cell Culture, Single Cell, Colony Assay, RNA Sequencing

Journal: iScience

Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

doi: 10.1016/j.isci.2026.117658

Figure Lengend Snippet: Deletion of Pex19 induces oxidative stress and impairs mitochondrial function in mESCs (A) Pex19 KO 46C mESCs have increased ROS. Levels of total cellular ROS were measured by FACS analysis of cell-permeable deep red dye ( n = 3 biological replicates/group; data are presented as mean ± SEM, Student’s t test, ∗∗∗∗ p < 0.0001). Scale bars, 5 μm. (B) The abundance and distribution of mitochondrial SOD2 in WT and Pex19 KO 46C mESCs. Scale bars, 5 μm. (C) NAC treatment rescues the defective mitochondrial membrane potential in Pex19 KO mESCs. WT and Pex19 KO 46C mESCs were treated with or without 2 mM NAC for 16 h and stained with MitoTracker, then subjected to FACS analysis ( n = 3–4 biological replicates/group, data are presented as mean ± SEM, two-way ANOVA, ∗∗q < 0.01). (D) WT and Pex19 KO 46C mESCs have comparable levels of an outer mitochondrial membrane protein, TOMM20. Total cell lysates were analyzed for the abundance of TOMM20 by immunoblotting. (E) Pex19 KO mESCs have impaired mitochondrial respiration. The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of WT and Pex19 KO 46C mESCs were analyzed by Seahorse XFe24 Analyzer ( n = 6 biological replicates/group; data are presented as mean ± SEM). Omy, oligomycin; FCCP, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; ROT and AA, Rotenone and antimycin A; 2-DG, 2-deoxy-glucose. (F) NAC treatment partially restores the defective mitochondrial respiration of Pex19 KO mESCs. The OCR of WT, Pex19 KO, and Pex19 KO 46C mESCs treated with 2 mM NAC for 16 h were analyzed by Seahorse XFe24 Analyzer ( n = 6 biological replicates/group; data are presented as mean ± SEM). (G) NAC treatment partially rescues the colony formation ability but not cell growth of Pex19 KO mESCs. WT and Pex19 KO 46C mESCs treated with or without 2 mM NAC were analyzed for their growth and colony formation ability ( n = 3 biological replicates/group; data are presented as mean ± SEM; two-way ANOVA, ∗∗q < 0.01, ∗∗∗q < 0.001, ∗∗∗∗q < 0.0001, n.s, not significant). (H) NAC treatment partially reduces cell death of Pex19 KO mESCs. WT and Pex19 KO 46C mESCs treated with or without 2 mM NAC for 24 h were analyzed by FACS ( n = 3 biological replicates/group; data are presented as mean ± SEM; two-way ANOVA; ∗∗q < 0.01, ∗∗∗q < 0.001, ∗∗∗∗q < 0.0001).

Article Snippet: Mouse anti-PEX19 monoclonal antibody , Novus , NBP2-43544; RRID: AB_3307633.

Techniques: Analysis, Membrane, Staining, Western Blot, Colony Assay

Journal: iScience

Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

doi: 10.1016/j.isci.2026.117658

Figure Lengend Snippet: Deletion of Pex19 impairs peroxisomal lipid metabolism and pluripotency in mESCs (A) Pex19 KO 46C mESCs have reduced AP staining. WT and Pex19 KO 46C mESCs were stained for AP activity using the fluorescent Alkaline Phosphatase Detection Kit. Scale bars, 100 μm. (B) The fraction of AP + colonies is reduced in Pex19 KO mESCs. WT and Pex19 KO 46C mESCs were stained for AP activity using the StemAb Alkaline Phosphatase Staining kit II, and the percentage of AP + colonies was quantified ( n = 15–18 random fields/group were analyzed; data are presented as mean ± SEM, two-way ANOVA, ∗∗∗∗q < 0.0001). Scale bars, 200 μm. (C and D) Pex19 KO mESCs have altered lipid metabolism. WT and Pex19 KO 46C mESCs were subject to a lipidomics analysis as described in . (C) Volcano plot of all annotated lipids with cutoffs of p < 0.05 and |FC|>2 (gray line and dotted lines). Red, significantly upregulated lipids; green, significantly downregulated lipids; Blue, all ether PE. (D) Heatmap of significantly changed lipids (cutoffs of p < 0.05 and |FC|>2). (E) Pex19 KO mESCs have an increased membrane fluidity. WT and Pex19 KO 46C mESCs were stained with 5 μM di-4-ANEPPDHQ for 30 min. The relative ordered fraction in each group was analyzed as described in ( n = 40 colonies/group; data are presented as violin plots; Student’s t test, ∗∗∗∗ p < 0.0001). (F) Treatment with plasmalogens partially restores the clumping morphology of Pex19 KO mESCs. WT and Pex19 KO 46C mESCs were treated with vehicle control (ethanol) or 30 μg/mL plasmalogens (PLs) for 2 days ( n = 25 WT ethanol, 25 WT PLs, 17 KO ethanol, and 19 KO PLs; random fields were analyzed; data are presented as mean ± SEM, two-way ANOVA, ∗∗∗q < 0.001, ∗∗∗∗q < 0.0001). Scale bars, 100 μm. (G) Treatment with plasmalogens partially rescues the AP activity in Pex19 KO mESCs. WT and Pex19 KO 46C mESCs were treated as in (F) and stained for AP activity ( n = 15–17 random fields/group were analyzed; data are presented as mean ± SEM, two-way ANOVA, ∗∗∗∗q < 0.0001). Scale bars, 100 μm. See also .

Article Snippet: Mouse anti-PEX19 monoclonal antibody , Novus , NBP2-43544; RRID: AB_3307633.

Techniques: Staining, Activity Assay, Analysis, Membrane, Morphology, Control

Journal: iScience

Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

doi: 10.1016/j.isci.2026.117658

Figure Lengend Snippet: Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .

Article Snippet: Mouse anti-PEX19 monoclonal antibody , Novus , NBP2-43544; RRID: AB_3307633.

Techniques: Blocking Assay, Staining, Analysis

Journal: iScience

Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

doi: 10.1016/j.isci.2026.117658

Figure Lengend Snippet: Deletion of Pex19 impairs mitotic spindle assembly in mESCs (A) PEX19, not peroxisomes, is enriched near the centrosomes in mitotic mESCs. Scale bars, 5 μm. (B) PEX19 is colocalized with acetyl-K40 α-tubulin at the spindle poles during mitosis. Scale bars, 10 μm. (C) PEX19 interacts with tubulins in mESCs. Total cell lysates from WT and Pex19 KO 46C mESCs were immunoprecipitated (IP) with anti-PEX19 antibodies. (D) Deletion of Pex19 reduces the abundance of α-tubulin and α-tubulin-K40ac in mESCs. WT and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and the abundance of tubulins was analyzed at 0 and 30 min after release into mitosis. (E) Deletion of Pex19 reduces the formation of mitotic spindles in mitotic mESCs. WT and Pex19 KO 46C mESCs were treated as in (D), and α-tubulin-K40ac was analyzed by immunofluorescence staining at 0, 15, and 30 min after release into mitosis. Scale bars, 10 μm. (F) Structural scheme of mouse PEX19 protein. The leucine triad (L18, L21, L22) and F29 in the N-terminal PEX3 binding motif are highlighted. (G) HA-tagged N-terminal PEX3 binding fragment of PEX19 (a.a. 1–49) is expressed in HEK293T cells. HEK293T cells overexpressing the HA-PEX19-N fragment were stained with anti-HA antibodies. Scale bars, 10 μm. (H) The N-terminal PEX3 binding motif of PEX19 interacts with α-tubulin in cells. Total cell lysates from HEK293T cells expressing the indicated HA-tagged PEX19 truncation fragments were analyzed for their interaction with endogenous α-tubulin by co-immunoprecipitation (IP-HA). Please note that HA-PEX19-N was too small to be detected in this immunoblot. See also .

Article Snippet: Mouse anti-PEX19 monoclonal antibody , Novus , NBP2-43544; RRID: AB_3307633.

Techniques: Immunoprecipitation, Immunofluorescence, Staining, Binding Assay, Expressing, Co-Immunoprecipitation Assay, Western Blot

Journal: iScience

Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

doi: 10.1016/j.isci.2026.117658

Figure Lengend Snippet: Pex19 KO mESCs have reduced exit from pluripotency during the early stage of neural differentiation (A) Morphology of WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation. Scale barss, 100 μm. (B) Deletion of Pex19 delays the reduction of pluripotency genes during early neural differentiation. The expression of indicated genes was analyzed before and after three days of in vitro neural differentiation by qPCR ( n = 3 biological replicates/group; data are presented as mean ± SEM; two-way ANOVA, ∗∗∗q < 0.001, ∗∗∗∗q < 0.0001, n.s., not significant). (C) The protein level of Nanog in WT and Pex19 KO cells after three days of in vitro neural differentiation. Scale bars, 20 μm. (D) Adding back PEX19 restores the differentiation-induced reduction of pluripotency protein in Pex19 KO cells. The levels of indicated proteins were analyzed in WT, Pex19 KO (KO), and Pex19 KO + PEX19 (KO + PEX19) 46C mESCs before and after three days of in vitro neural differentiation by immunoblotting. (E) Treatment with DMSO partially increases G1 phase in Pex19 KO cells after three days of in vitro neural differentiation. WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (F) Adding back PEX19 or treatment with DMSO partially rescues the differentiation defects in Pex19 KO cells. The levels of indicated genes were analyzed in WT, Pex19 KO (KO), or Pex19 KO + PEX19 (KO + P19) 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO by qPCR ( n = 3 biological replicates/group, data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, ∗∗∗q < 0.001, and ∗∗∗∗q < 0.0001). (G) Treatment with DMSO partially restores the pluripotency exit defect in Pex19 KO cells. (H) Treatment with a low dose of nocodazole decreases the G1 population in WT cells after three days of in vitro neural differentiation. WT 46C mESCs before (D0) or after three days of in vitro neural differentiation with or without 12.5 ng/mL nocodazole were analyzed for G1 population as in (E) ( n = 3 biological replicates/group; data are presented as mean ± SEM; one-way ANOVA; ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (I) Treatment with a low dose of nocodazole delays the reduction of pluripotency genes in WT cells after three days of in vitro neural differentiation. The levels of indicated pluripotency genes were analyzed in WT 46C mESCs treated as in (H) ( n = 3 biological replicates/group; data are presented as mean ± SEM, one-way ANOVA, ∗ q < 0.05, ∗∗ q < 0.01, ∗∗∗ q < 0.001, ∗∗∗∗ q < 0.0001, n.s., not significant). See also .

Article Snippet: Mouse anti-PEX19 monoclonal antibody , Novus , NBP2-43544; RRID: AB_3307633.

Techniques: Morphology, In Vitro, Expressing, Western Blot, Staining, Analysis

Journal: iScience

Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

doi: 10.1016/j.isci.2026.117658

Figure Lengend Snippet: Deletion of Pex19 in mESCs impairs neural differentiation (A and B) Pex19 deficiency alters transcriptomic response of mESCs during the first week of in vitro neural differentiation. WT and Pex19 KO mESCs were subject to in vitro neural differentiation and their transcriptomes at D0, D4, and D7 during differentiation were analyzed by RNA-seq as described in ( n = 3 independent cell clones/genotype). (A) Heatmaps of 2052 differentially expressed protein-coding genes between Pex19 KO vs. WT cells at different time points. (B) Top enriched GO pathways in different gene clusters in (A). (C) Pex19 KO cells have reduced expression of SOX1-GFP at day 6 after in vitro neural differentiation. Scale bars, 300 μm. The percentage of SOX1-GFP positive cells at Day 6 were analyzed by FACS ( n = 5 biological replicates/genotype; data are presented as mean ± SEM; Student’s t test, ∗∗∗∗ p < 0.0001). (D) Pex19 KO cells have reduced expression of different neural markers during three weeks of in vitro neural differentiation. The mRNA levels of indicated genes were analyzed by qPCR ( n = 3 biological replicates/group; data are presented as mean ± SEM; Student’s t test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗, p < 0.001, and ∗∗∗∗ p < 0.0001). (E) Defective neural differentiation of Pex19 KO 46C mESCs at day 21 (D21). The abundance, shape, and distribution of neural morphology marker TUBB3 and neural maturation marker MAP2 were determined by IF staining. Scale bars, 100 μm. See also .

Article Snippet: Mouse anti-PEX19 monoclonal antibody , Novus , NBP2-43544; RRID: AB_3307633.

Techniques: In Vitro, RNA Sequencing, Clone Assay, Expressing, Morphology, Marker, Staining

Journal: iScience

Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

doi: 10.1016/j.isci.2026.117658

Figure Lengend Snippet:

Article Snippet: Mouse anti-PEX19 monoclonal antibody , Novus , NBP2-43544; RRID: AB_3307633.

Techniques: Cell-Signaling, Magnetic Beads, Virus, Recombinant, ROS Assay, Reverse Transcription, SYBR Green Assay, Staining, Plasmid Preparation, Flow Cytometry, Western Blot, Expressing, Control, Software