Journal: Oncogenesis

Article Title: Targeting mitochondrial phosphatidylethanolamine alters mitochondrial metabolism and proliferation in hepatocellular carcinoma

doi: 10.1038/s41389-025-00593-y

Figure Lengend Snippet: A Images of representative liver dissected from 34-week-old mice (top) with a corresponding hematoxylin and eosin (H&E) stain (bottom). The inset H&E image is illustrating normal blood vessels (circular or oval areas that do not stain with H&E) in liver sections from LFD mice, or areas of HCC in WD + DEN/TAA treated mice, which also do not stain with H&E but are larger, irregularly shaped, and surrounded by nuclei (blue staining) ( n = 6 mice per group). B Serum ALT activity was elevated in WD + DEN/TAA groups. The male and female LFD groups and female WD + DEN/TAA group had an n = 3. The male WD + DEN/TAA group had an n = 4. C Serum AST activity was elevated in WD + DEN/TAA groups ( n = 3 per group). D Nuclei (Hoechst) and Ki-67 (proliferation marker, upregulated in HCC) fluorescent images of liver sections. Ki-67 staining was higher in WD + DEN/TAA treated liver sections ( n = 6 per group). E Relative fluorescent intensity of Ki-67 staining from images shown in D ( n = 6 per group). F PCA plot of mitochondrial lipids between non-tumor and tumor mitochondria ( n = 6 per group). G Heatmap of select lipids that were differentially altered between non-tumor and tumor mitochondria ( n = 6 per group). H The abundance of mitochondrial phospholipids was not different between non-tumor and tumor mitochondria ( n = 6 per group). The data are presented as means ± S.D.

Article Snippet: Between 12 and 20 μg of protein was resolved by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA), transferred onto nitrocellulose membrane or methanol-activated polyvinylidene fluoride (PVDF), normalized using Ponceau S staining solution (40000279, Thermo Fisher Scientific), cut to appropriate size, and blocked in 5% non-fat milk for 75 min. Blots were incubated overnight at 4 °C with total OXPHOS rodent WB antibody cocktail (ab110413, Abcam), voltage-dependent anion channel (VDAC, 4866, Cell Signaling Technology, Danvers, MA, USA), peroxin-5 (PEX5, 83020, Cell Signaling Technology), glucose transporter 1 (Glut 1, 12939, Cell Signaling Technology), transketolase (TKT, 64414, Cell Signaling Technology), phosphoenolpyruvate carboxykinase 1 (PCK1, 12940, Cell Signaling Technology), enolase 1 (ENO1, 3810, Cell Signaling Technology), aldolase a (ALDOA, 8060, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 2118S, Cell Signaling Technology), phosphoglycerate mutase (PGAM1, Cell Signaling Technology), superoxide dismutase 2 (SOD2, 13194, Cell Signaling Technology), dynamin-related protein 1 (DRP1, 8570, Cell Signaling Technology), mitochondrial fission factor (MFF, 84580, Cell Signaling Technology), optic atrophy 1 (OPA1, 80471, Cell Signaling Technology), mitofusin 1 (MFN1, 14739, Cell Signaling Technology), mitofusin 2 (MFN2, 9482, Cell Signaling Technology), Parkin (4211, Cell Signaling Technology), liver kinase B1 (LKB1, 3047, Cell Signaling Technology), phosphorylated LKB1 S428 (3482, Cell Signaling Technology), adenosine monophosphate activated protein kinase (AMPK, 2532, Cell Signaling Technology), phosphorylated AMPK (2535, Cell Signaling Technology), mammalian target of rapamycin (mTOR, 2983, Cell Signaling Technology), phosphorylated mTOR S2448 (2971, Cell Signaling Technology), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, 9452, Cell Signaling Technology), or phosphorylated 4E-BP1 S65 (9451, Cell Signaling Technology).

Techniques: Staining, Activity Assay, Marker

Journal: Oncogenesis

Article Title: Targeting mitochondrial phosphatidylethanolamine alters mitochondrial metabolism and proliferation in hepatocellular carcinoma

doi: 10.1038/s41389-025-00593-y

Figure Lengend Snippet: A Schematic created using BioRender illustrating PISD enzyme reaction with phosphatidylserine (PS) to form CO 2 and phosphatidylethanolamine (PE). B Schematic created using BioRender showing generation of shScrambled (shSCR) and shPISD lentivirus in 293T cells and subsequent infection and gene silencing in HEPA1-6 cells. C The shPISD lentivirus reduced PISD mRNA abundance ( n = 2 per group) and altered the abundance of various genes involved in phospholipid metabolism ( n = 5 per group). D PISD activity was reduced in shPISD treated cells ( n = 3 per group). E , F Thin layer chromatography images indicating shPISD treated cells had reduced mitochondrial PE content ( E ) and densitometry quantification of thin layer chromatography images presented in E ( n = 3 per group) ( F ). G A Seahorse mitochondrial stress test reveals shPISD treated cells have reduced FCCP stimulated respiration rates ( n = 10 per group). H shPISD treated cells have elevated proton leak, calculated by subtracting oxygen consumption rates (OCR) during basal conditions from OCR during oligomycin (oligo) treated conditions ( n = 10 per group). I , J Western blot image indicating shPISD treated cells have reduced complex I and IV protein abundance ( I ) and densitometry quantification of Western blot images shown in I ( n = 6 per group) ( J ). K 1- 14 C oleate oxidation is reduced in shPISD treated cells ( n = 6 per group). L 1- 14 C lignoceric acid oxidation is increased in shPISD treated cells ( n = 6 per group). M PCA plot of untargeted metabolomics data illustrating that shPISD treated cells have an altered metabolome compared to shSCR treated cells ( n = 3 per group). N Volcano plot of untargeted metabolomics data illustrating differences in metabolites between shSCR and shPISD treated cells ( n = 3 per group). O Heatmap of select lipid metabolism related metabolites that were differentially expressed between shSCR and shPISD treated cells ( n = 3 per group). The data are presented as means ± S.D.

Article Snippet: Between 12 and 20 μg of protein was resolved by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA), transferred onto nitrocellulose membrane or methanol-activated polyvinylidene fluoride (PVDF), normalized using Ponceau S staining solution (40000279, Thermo Fisher Scientific), cut to appropriate size, and blocked in 5% non-fat milk for 75 min. Blots were incubated overnight at 4 °C with total OXPHOS rodent WB antibody cocktail (ab110413, Abcam), voltage-dependent anion channel (VDAC, 4866, Cell Signaling Technology, Danvers, MA, USA), peroxin-5 (PEX5, 83020, Cell Signaling Technology), glucose transporter 1 (Glut 1, 12939, Cell Signaling Technology), transketolase (TKT, 64414, Cell Signaling Technology), phosphoenolpyruvate carboxykinase 1 (PCK1, 12940, Cell Signaling Technology), enolase 1 (ENO1, 3810, Cell Signaling Technology), aldolase a (ALDOA, 8060, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 2118S, Cell Signaling Technology), phosphoglycerate mutase (PGAM1, Cell Signaling Technology), superoxide dismutase 2 (SOD2, 13194, Cell Signaling Technology), dynamin-related protein 1 (DRP1, 8570, Cell Signaling Technology), mitochondrial fission factor (MFF, 84580, Cell Signaling Technology), optic atrophy 1 (OPA1, 80471, Cell Signaling Technology), mitofusin 1 (MFN1, 14739, Cell Signaling Technology), mitofusin 2 (MFN2, 9482, Cell Signaling Technology), Parkin (4211, Cell Signaling Technology), liver kinase B1 (LKB1, 3047, Cell Signaling Technology), phosphorylated LKB1 S428 (3482, Cell Signaling Technology), adenosine monophosphate activated protein kinase (AMPK, 2532, Cell Signaling Technology), phosphorylated AMPK (2535, Cell Signaling Technology), mammalian target of rapamycin (mTOR, 2983, Cell Signaling Technology), phosphorylated mTOR S2448 (2971, Cell Signaling Technology), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, 9452, Cell Signaling Technology), or phosphorylated 4E-BP1 S65 (9451, Cell Signaling Technology).

Techniques: Infection, Activity Assay, Thin Layer Chromatography, Western Blot, Quantitative Proteomics

Journal: Oncogenesis

Article Title: Targeting mitochondrial phosphatidylethanolamine alters mitochondrial metabolism and proliferation in hepatocellular carcinoma

doi: 10.1038/s41389-025-00593-y

Figure Lengend Snippet: A Extracellular acidification rates (ECAR) during a Seahorse mitochondrial stress test were elevated in shPISD treated cells after the injection of glucose and oligomycin into the media ( n = 10 per group). B Percentage change in media lactate over a 24-h period revealed shPISD treated cells generate more lactate ( n = 6 per group). C 1- 14 C glucose oxidation was reduced in shPISD treated cells ( n = 12 per group). D 6- 14 C glucose oxidation was reduced in shPISD treated cells ( n = 12 per group). E Percentage decrease in media glucose concentration was greater in shPISD treated cells, indicating an increase in glucose uptake ( n = 6 per group). F 1- 14 C 2-deoxy-d-glucose uptake was greater in shPISD treated cells ( n = 12 for shSCR, n = 11 for shPISD). G, H Representative fluorescent images illustrating greater glucose uptake in shPISD treated cells ( G ) and quantification of fluorescent intensity of images presented in G ( n = 25 per group) ( H ). I, J Western blot images illustrating shPISD treated cells have increased GLUT 1 protein abundance, while other markers of glucose metabolism were unaltered ( I ) and densitometry quantification of Western blot images presented in I ( n = 6 per group) ( J ). K Heatmap of carbohydrate related metabolites that were significantly different between shSCR and shPISD treated cells ( n = 3 per group). L Number of metabolites in each KEGG classification that were significantly different between shSCR and shPISD treated cells. M Percentage of significantly different metabolites within each class. N Heatmap of significantly different glycerophospholipids between shSCR and shPISD treated cells ( n = 3 per group). The data are presented as means ± S.D.

Article Snippet: Between 12 and 20 μg of protein was resolved by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA), transferred onto nitrocellulose membrane or methanol-activated polyvinylidene fluoride (PVDF), normalized using Ponceau S staining solution (40000279, Thermo Fisher Scientific), cut to appropriate size, and blocked in 5% non-fat milk for 75 min. Blots were incubated overnight at 4 °C with total OXPHOS rodent WB antibody cocktail (ab110413, Abcam), voltage-dependent anion channel (VDAC, 4866, Cell Signaling Technology, Danvers, MA, USA), peroxin-5 (PEX5, 83020, Cell Signaling Technology), glucose transporter 1 (Glut 1, 12939, Cell Signaling Technology), transketolase (TKT, 64414, Cell Signaling Technology), phosphoenolpyruvate carboxykinase 1 (PCK1, 12940, Cell Signaling Technology), enolase 1 (ENO1, 3810, Cell Signaling Technology), aldolase a (ALDOA, 8060, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 2118S, Cell Signaling Technology), phosphoglycerate mutase (PGAM1, Cell Signaling Technology), superoxide dismutase 2 (SOD2, 13194, Cell Signaling Technology), dynamin-related protein 1 (DRP1, 8570, Cell Signaling Technology), mitochondrial fission factor (MFF, 84580, Cell Signaling Technology), optic atrophy 1 (OPA1, 80471, Cell Signaling Technology), mitofusin 1 (MFN1, 14739, Cell Signaling Technology), mitofusin 2 (MFN2, 9482, Cell Signaling Technology), Parkin (4211, Cell Signaling Technology), liver kinase B1 (LKB1, 3047, Cell Signaling Technology), phosphorylated LKB1 S428 (3482, Cell Signaling Technology), adenosine monophosphate activated protein kinase (AMPK, 2532, Cell Signaling Technology), phosphorylated AMPK (2535, Cell Signaling Technology), mammalian target of rapamycin (mTOR, 2983, Cell Signaling Technology), phosphorylated mTOR S2448 (2971, Cell Signaling Technology), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, 9452, Cell Signaling Technology), or phosphorylated 4E-BP1 S65 (9451, Cell Signaling Technology).

Techniques: Injection, Concentration Assay, Western Blot, Quantitative Proteomics

Journal: Oncogenesis

Article Title: Targeting mitochondrial phosphatidylethanolamine alters mitochondrial metabolism and proliferation in hepatocellular carcinoma

doi: 10.1038/s41389-025-00593-y

Figure Lengend Snippet: A Representative images of MitoSOX staining illustrating greater mitochondrial superoxide content in shPISD treated cells. B Quantification of MitoSOX fluorescent intensity from images presented in A ( n = 30 per group). C Relative abundance (measured using metabolomics) of reduced glutathione (GSH) and glutathione disulfide (GSSG) in shSCR and shPISD treated cells ( n = 3 per group). D The relative protein abundance of superoxide dismutase 2 (SOD2) was reduced in shPISD treated cells ( n = 6 per group). E Western blot image of SOD2 protein abundance. F Western blot image showing protein abundances of markers of mitochondrial fission (DRP, MFF), fusion (OPA1, MFN1, MFN2), or mitophagy (Parkin) ( n = 3 per group). G Densitometry quantification of images shown in F ( n = 3 per group). H , I Representative fluorescent images of MitoTracker and LysoTracker colocalization. The white arrows are pointing to extracellular mitochondria stained green with MitoTracker. J Greater Manders colocalization coefficient of MitoTracker and LysoTracker indicates more mitophagy in shPISD treated cells ( n = 5 per group). K Number of extracellular mitochondria puncta was greater around shPISD treated cells ( n = 3 per group). The data are presented as means ± S.D.

Article Snippet: Between 12 and 20 μg of protein was resolved by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA), transferred onto nitrocellulose membrane or methanol-activated polyvinylidene fluoride (PVDF), normalized using Ponceau S staining solution (40000279, Thermo Fisher Scientific), cut to appropriate size, and blocked in 5% non-fat milk for 75 min. Blots were incubated overnight at 4 °C with total OXPHOS rodent WB antibody cocktail (ab110413, Abcam), voltage-dependent anion channel (VDAC, 4866, Cell Signaling Technology, Danvers, MA, USA), peroxin-5 (PEX5, 83020, Cell Signaling Technology), glucose transporter 1 (Glut 1, 12939, Cell Signaling Technology), transketolase (TKT, 64414, Cell Signaling Technology), phosphoenolpyruvate carboxykinase 1 (PCK1, 12940, Cell Signaling Technology), enolase 1 (ENO1, 3810, Cell Signaling Technology), aldolase a (ALDOA, 8060, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 2118S, Cell Signaling Technology), phosphoglycerate mutase (PGAM1, Cell Signaling Technology), superoxide dismutase 2 (SOD2, 13194, Cell Signaling Technology), dynamin-related protein 1 (DRP1, 8570, Cell Signaling Technology), mitochondrial fission factor (MFF, 84580, Cell Signaling Technology), optic atrophy 1 (OPA1, 80471, Cell Signaling Technology), mitofusin 1 (MFN1, 14739, Cell Signaling Technology), mitofusin 2 (MFN2, 9482, Cell Signaling Technology), Parkin (4211, Cell Signaling Technology), liver kinase B1 (LKB1, 3047, Cell Signaling Technology), phosphorylated LKB1 S428 (3482, Cell Signaling Technology), adenosine monophosphate activated protein kinase (AMPK, 2532, Cell Signaling Technology), phosphorylated AMPK (2535, Cell Signaling Technology), mammalian target of rapamycin (mTOR, 2983, Cell Signaling Technology), phosphorylated mTOR S2448 (2971, Cell Signaling Technology), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, 9452, Cell Signaling Technology), or phosphorylated 4E-BP1 S65 (9451, Cell Signaling Technology).

Techniques: Staining, Quantitative Proteomics, Western Blot