Journal: Cell Death & Disease

Article Title: Hepatokine ORM2 suppresses pathological ferritinophagy to prevent acute tissue injury

doi: 10.1038/s41419-026-08803-0

Figure Lengend Snippet: Orm2 expression in livers of wild-type (WT) mice at different times following 300 mg/kg APAP treatment, measured by qPCR ( A ) ( n = 3 (0 h), n = 5 (6 h), n = 4 (12 h and 24 h)) and western blot ( B ) ( n = 4 per group). Vinculin (VINC) was used as the loading control. C Representative gross liver and H&E images from WT and Orm2 -/- mice at 24 h post-APAP. Scale bar, 200 μm. Necrotic areas circled in white lines, with quantification of necrosis and areas of patchy hemorrhages shown below ( n = 4 per group). D Serum levels of ALT and AST in WT and Orm2 -/- mice at 24 h post-APAP. E Apoptosis measured by TUNEL staining from WT and Orm2 -/- mice at 24 h post-APAP. Scale bar, 200 μm. Quantification is on the right. F Survival curves of WT and Orm2 -/- mice treated with a lethal dose of APAP (750 mg/kg) ( n = 10 per group). Eight-week-old WT male mice were injected retro-orbitally with Ad- ORM2 or Ad- vector virus. After two weeks, WT and Orm2 -/- mice received 300 mg/kg APAP intraperitoneally. 24 h later, ORM2 protein and mRNA levels in liver tissue ( G ), liver morphology ( H ), H&E ( H ), serum ALT and AST levels ( I ), as well as TUNEL staining ( J ), were assessed ( n = 5 per group). Scale bars, 100 μm. All data are presented as mean ± SEM. In ( A ), data were analyzed by one-way ANOVA with Dunnett’s test. In ( F ), the survival curve was compared using the log-rank (Mantel–Cox) test, and the remaining data were analyzed by unpaired two-tailed Student’s t test. Exact p values are provided in the figure, *** p < 0.001, **** p < 0.0001. “n” refers to biological replicates. All experiments were performed in triplicates.

Article Snippet: Human ORM2 protein (MedChem Express, HY-P7490).

Techniques: Expressing, Western Blot, Control, TUNEL Assay, Staining, Injection, Plasmid Preparation, Virus, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Hepatokine ORM2 suppresses pathological ferritinophagy to prevent acute tissue injury

doi: 10.1038/s41419-026-08803-0

Figure Lengend Snippet: A Differentially enriched gene pathways from RNA-seq analysis of liver tissues in Orm2 -/- versus WT mice 24 h after APAP treatment ( n = 3 per group). B WT and Orm2 -/- mice were injected intraperitoneally with APAP (300 mg/kg). Liver samples were collected at the indicated time points for frozen section preparation. Sections were stained with FerroOrange to detect ferrous iron, with DAPI counterstaining. Mean FerroOrange fluorescence intensity is quantified below ( n = 3 per group, except 24 h: n = 4). Scale bars, 50 μm. C WT and Orm2 -/- mice were received APAP (300 mg/kg). Liver samples were collected at the indicated time points for frozen section preparation. Representative immunohistochemical staining for 4-HNE in liver sections, with mean intensity is quantified below ( n = 3 per group). Scale bars, 100 μm. D Measurement of malondialdehyde (MDA) levels in liver tissue from ( C ) ( n = 3 per group). E WT and Orm2 -/- mice were pre-treated with 200 mg/kg DFO or vehicle 2 h before APAP injection, with samples collected 24 h post-APAP. Representative H&E images of liver sections from mice, white dashed lines indicating cell death areas ( n = 4 per group). Scale bar, 100 μm. Damage areas were quantified on the right. F Serum ALT levels from mice in ( E ) were measured. G Primary hepatocytes isolated from WT and Orm2 -/- mice were treated with indicated concentrations of APAP and cell viability was measured using Cell Counting Kit 8 (CCK8). H Primary hepatocytes from WT and Orm2 -/- mice were treated with 5 μM APAP for the indicated times. LPO levels were measured. I Primary hepatocytes isolated from WT and Orm2 -/- mice were treated with 5 μM APAP in the presence of Fer-1 (2 μM) or DFO (50 μM), and cell viability was measured using CCK8. All data in this figure are represented as mean ± SEM. Data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test for comparisons between genotypes, and by one-way ANOVA with Dunnett’s test or unpaired t-test for comparisons between two or multiple experimental groups within the same genotype, respectively. Exact p values are provided in the figure, * p < 0.05, **** p < 0.0001, ns (no significance). All experiments were performed in triplicates.

Article Snippet: Human ORM2 protein (MedChem Express, HY-P7490).

Techniques: RNA Sequencing, Injection, Staining, Fluorescence, Immunohistochemical staining, Isolation, Cell Counting

Journal: Cell Death & Disease

Article Title: Hepatokine ORM2 suppresses pathological ferritinophagy to prevent acute tissue injury

doi: 10.1038/s41419-026-08803-0

Figure Lengend Snippet: A The schema illustrates primary hepatocytes isolated from WT and Orm2 -/- mice, then treated with FINO2 or RSL3, with cell survival, cell death, ferrous iron, and lipid peroxidation levels measured. B WT and Orm2 -/- primary hepatocytes were treated with indicated doses of FINO2 for 12 h, cell viability was measured using CCK8. C WT and Orm2 -/- primary hepatocytes were treated with 8 μM FINO2 for 12 h, and propidium iodide (PI) staining to assess cell death. Quantification on the right. Scale bar, 100 μm. D – E WT and Orm2 -/- primary hepatocytes were treated with 8 μM FINO2 for 3 h, Fe 2+ levels and lipid peroxidation were visualized using FerroOrange and C11-BODIPY staining respectively. Scale bar, 100 µm. F The schema illustrates HepG2 cells with ORM2 knockdown, cells were treated with FINO2 or RSL3, with cell survival, cell death, ferrous iron, and lipid peroxidation levels measured. G HepG2 cells with ORM2 knockdown were treated with FINO2 at the indicated concentrations for 12 h, and cell viability was assessed using CCK8. H HepG2 cells with ORM2 knockdown were treated with 8 μM FINO2 for 12 h, PI staining was measured. Quantification on the right. Scale bar, 100 μm. I – J HepG2 cells with ORM2 knockdown were treated with 8 μM FINO2 for 3 h, Fe 2+ levels and lipid peroxidation were visualized using FerroOrange and C11-BODIPY staining respectively. Scale bar, 100 μm. K Flag-ORM2 was overexpressed into HepG2 cells, cells were treated with FINO2 at indicated doses for 12 h. Cell viability was assessed using CCK8. L HepG2 cells with ORM2 overexpressing were treated with 8 μM FINO2 for 12 h, PI staining was measured. Quantification on the right. Scale bar, 100 μm. M – N HepG2 cells with ORM2 overexpressing were treated with 8 μM FINO2 for 3 h, Fe 2+ levels and lipid peroxidation were visualized using FerroOrange and C11-BODIPY staining, respectively. Scale bar, 100 μm. All data in this figure are represented as mean ± SEM. Data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test for comparisons between genotypes. Exact p values are provided in the figure. All experiments were performed in triplicates.

Article Snippet: Human ORM2 protein (MedChem Express, HY-P7490).

Techniques: Isolation, Staining, Knockdown

Journal: Cell Death & Disease

Article Title: Hepatokine ORM2 suppresses pathological ferritinophagy to prevent acute tissue injury

doi: 10.1038/s41419-026-08803-0

Figure Lengend Snippet: A HepG2 cells with shNC or sh ORM2 knockdown were treated with 8 μM FINO2 for the indicated time points, western blot to detect FTH1 and ORM2 protein expression. B HepG2 cells with shNC or sh ORM2 knockdown were treated with 8 μM FINO2 for 6 h. The intracellular ferrous ion (Fe 2+ ) levels were stained with FerroOrange (0.25 µmol/L) and analyzed by flow cytometry. C Primary hepatocytes were isolated from WT and Orm2 -/- mice, and cells were treated with 8 μM FINO2 and 10 μM Chloroquine (CQ). FTH1 protein expression was measured by western blot. D HepG2 cells with ORM2 knockdown or control were treated with 8 μM FINO2. Representative immunofluorescence images display ferritin (FTL) and lysosomes (LAMP2 positive foci) localization. Quantification of co-localization was analyzed using Image J (right panel). Scale bars, 5 μm. E WT and Orm2 -/- mice were injected with APAP. 24 h post-APAP, liver tissues were collected and lysed for FTL co-immunoprecipitation (co-IP), and TAX1BP1, NCOA4, FTL, and GAPDH antibodies were used for IB. F HEK293T cells were co-transfected with Flag-ORM2 and Myc-NCOA4 plasmids. Cells were treated with 8 μM FINO2 or vehicle for 3 h, followed by co-IP using Myc beads. Flag-ORM2 levels in the co-IP complexes were analyzed. G HEK293T cells were co-transfected with Flag-TAX1BP1 and HA-ORM2 . Cells were treated with 8 μM FINO2 or vehicle for 3 h, followed by co-IP using Flag beads. HA-ORM2 protein were measured. H HepG2 cells were overexpressed with Flag-ORM2 plasmid. Cells were treated with 4 μM RSL3. Representative immunofluorescence images of TAX1BP1 and FTL were detected, and the Pearson correlation for co-localization was quantified using ImageJ. Scale bars, 5 μm. I HEK293T cells were co-transfected with Flag-TAX1BP1 , Myc-NCOA4 , and increasing amounts of HA-ORM2 plasmids. Cells were then treated with 8 μM FINO2 plus 10 μM chloroquine (CQ) or DMSO vehicle. Co-IP were performed using Myc beads, and Flag, HA, Myc, and GAPDH antibodies were used for IB. J A model illustrates that ORM2 inhibits ferritin localization to lysosomes during ferroptosis by modulating NCOA4-TAX1BP1 interactions, reducing intracellular ferrous ions. All data in this figure are represented as mean ± SEM. In ( B ), data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test; in ( D ) and ( H ), by unpaired t -test. Exact p values are provided in the figure. All experiments were performed in triplicates.

Article Snippet: Human ORM2 protein (MedChem Express, HY-P7490).

Techniques: Knockdown, Western Blot, Expressing, Staining, Flow Cytometry, Isolation, Control, Immunofluorescence, Injection, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Hepatokine ORM2 suppresses pathological ferritinophagy to prevent acute tissue injury

doi: 10.1038/s41419-026-08803-0

Figure Lengend Snippet: A WT mice were pre-injected intraperitoneally with recombinant ORM2 protein (rORM2, 7 mg/kg) or PBS for 2 h, followed by 300 mg/kg APAP. After 24 h, liver samples were collected for ORM2 expression analysis by western blot ( n = 4 and 3). B Representative liver morphology and H&E images of livers from mice in ( A ). Scale bar, 200 μm. C – F Serum ALT, AST, LPO, liver FTH1 protein and liver iron levels were measured from mice in ( A ), ( n = 4-3 per group). G WT mice received 7 mg/kg rORM2 or PBS 2 h before 750 mg/kg APAP, and survival curves were plotted, ( n = 12 per group). H WT mice were injected intraperitoneally with APAP (250 mg/kg) to induce liver injury. At 1.5- and 12-h post-APAP injection, mice received equal volumes of PBS, rORM2 (20 mg/kg), or NAC (300 mg/kg) as indicated. Serum samples were collected at 24 h after APAP, serum ALT and AST were measured ( n = 3 per group). I Representative H&E staining of liver sections from the indicated groups in ( H ). Scale bar, 100 μm. J WT mice received 7 mg/kg rORM2 2 h before performing liver ischemia-reperfusion (HIR). 12 h post-HIR, livers were collected for H&E, the white dashed lines indicated the damaged area ( n = 3 per group). Scale bar, 100 μm. K – L Serum ALT, AST, liver iron content, and liver MDA from mice in ( J ) were measured, ( n = 3 per group). All data in this figure are represented as mean ± SEM. In ( G ), the survival curve was compared using the log-rank (Mantel-Cox) test; In ( H ) data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test for comparisons between rORM2 and NAC groups, and by one-way ANOVA with Dunnett’s test for comparisons between APAP + PBS and rORM2+APAP groups; the remaining data were analyzed by unpaired two-tailed Student’s t test. Exact p values are provided in the figure. “n” refers to biological replicates. All experiments were performed in triplicates.

Article Snippet: Human ORM2 protein (MedChem Express, HY-P7490).

Techniques: Injection, Recombinant, Expressing, Western Blot, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Hepatokine ORM2 suppresses pathological ferritinophagy to prevent acute tissue injury

doi: 10.1038/s41419-026-08803-0

Figure Lengend Snippet: A Wild-type mice underwent sham or renal ischemia-reperfusion (KIR) surgery. Serum samples were collected at 12 h. ORM2 protein level of the serum was quantified relative to total protein using Ponceau S staining and western blot ( n = 4 per group). B Kidney ORM2 protein levels; liver and kidney Orm2 mRNA from ( A ) were measured by western blot and qPCR. C Wild-type mice underwent sham or renal ischemia-reperfusion (KIR) surgery, kidney samples were collected for H&E and ORM2 staining. H&E staining shows swelling (*) and nuclear condensation (→) of renal tubular epithelial cells ( n = 3 per group). Scale bars, 100 μm. D Sleeping beauty transposon PT3-Vector or PT3- Orm2-EGFP and SB100 transposase were delivered via hydrodynamic tail vein injection (HDT) in mice. One week later, mice underwent either sham or KIR surgery. EGFP-ORM2 expression in kidney sections was assessed by immunohistochemistry ( n = 3 per group). Scale bars, 50 μm. E WT and Orm2 -/- mice underwent KIR surgery, kidney samples were collected 12 h post-surgery. H&E staining shows swelling (*) and nuclear condensation (→) of renal tubular epithelial cells ( n = 3 (WT mice), n = 4 ( Orm2 -/- mice)). Scale bars, 100 μm. F Serum creatinine and blood urea nitrogen (BUN) levels of mice in ( E ) were measured 12 h post-KIR. G Lipid peroxidation (LPO) in kidney tissues of mice in ( E ) ( n = 3 per group). H WT and Orm2 -/- mice underwent intestinal ischemia-reperfusion (IIR) surgery, lung samples were collected 3 h post-surgery. Images show H&E staining of lung sections ( n = 3 per group). Scale bars, 50 μm. I Malondialdehyde (MDA) levels of lung samples from mice in ( H ) ( n = 3 per group). All data in this figure are represented as mean ± SEM. In ( I ), data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test; the remaining data were analyzed by unpaired two-tailed Student’s t test. Exact p values are provided in the figure. “n” refers to biological replicates. All experiments were performed in triplicates.

Article Snippet: Human ORM2 protein (MedChem Express, HY-P7490).

Techniques: Staining, Western Blot, Plasmid Preparation, Injection, Expressing, Immunohistochemistry, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Hepatokine ORM2 suppresses pathological ferritinophagy to prevent acute tissue injury

doi: 10.1038/s41419-026-08803-0

Figure Lengend Snippet: A Wild-type mice received 7 mg/kg recombinant mouse ORM2-Fc protein (rORM2) 2 h before KIR. Kidney sections stained with H&E and Prussian blue. Scale bars, 100 μm. B Urine creatinine and blood urea nitrogen (BUN) levels at 12 h from mouse urine samples in ( A ) ( n = 3 per group). C Wild-type mice received 7 mg/kg rORM2 2 h before intestinal I/R (IIR), lung samples were collected at 3 h post-surgery and stained for H&E and ORM2 ( n = 4 per group). Scale bars, 50 μm. D Iron and MDA levels of lung samples from ( C ) are presented ( n = 4 per group). E HEK293T cells were incubated with 1 μg/mL rORM2 protein with or without 1 μM RSL3 treatment for 6 h. Images show rORM2 and LAMP1 localization. Scale bars, 5 μm. F Western blot analysis of rORM2 levels in HEK293T cells after an 8-h incubation with the indicated concentrations of ORM2 recombinant protein in the media. G HEK293T cells were pretreated with 1 μg/mL rORM2 proteins for 2 h, followed by 8 μM FINO2 for 6 h. FerroOrange staining was used to assess ferrous ion levels. Scale bars, 100 μm. H HEK293T cells were treated with Amiloride (2 μM) for 2 h, then incubated with or without 1 μg/mL rORM2 for 2 h. Cells were then exposed to 8 μM FINO2 for 12 h, and cell viability was measured using CCK8 assay. I A model suggests that ischemia-reperfusion injuries increase hepatic ORM2 production and secretion. Circulating or recombinant ORM2 protein then mitigates the injury by inhibiting ferroptosis. All data in this figure are represented as mean ± SEM. In ( H ), data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test; the remaining data were analyzed by unpaired two-tailed Student’s t test. Exact p values are provided in the figure. “n” refers to biological replicates. All experiments were performed in triplicates.

Article Snippet: Human ORM2 protein (MedChem Express, HY-P7490).

Techniques: Recombinant, Staining, Incubation, Western Blot, CCK-8 Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

doi: 10.15252/embj.2018101433

Figure Lengend Snippet: See also Fig and Appendix Tables S4 and S5 . A Vacuolar proteolysis‐deficient pep4∆ and tul1∆ pep4∆ cells were labeled to saturation with heavy 13 C 6 15 N 2 ‐L‐lysine and then chased for 3 h in the presence of light L‐lysine, following quantitative proteome analysis by LC‐MS (two biological replicates). To analyze Dsc complex‐dependent protein turnover, the ratio of H/L ratios of tul1∆ pep4∆ over pep4∆ cells of 2511 proteins quantified in both strains was plotted, and proteins with a ratio of H/L ratios > 2 were selected. Red squares: regulated proteins with at least one predicted transmembrane (TM) domain; black triangles: regulated proteins without TM domains. B Schematic representation of the Dsc ubiquitin ligase complex (a top hit from the SGA screen) and the SPOTS complex (a putative Dsc substrate). SPT, serine palmitoyltransferase; LCB, long‐chain bases; SL, sphingolipids. C–F SDS–PAGE and Western blot analysis with the indicated antibodies (C, D) of total yeast lysates from WT cells and the indicated mutants; (E) of input and elution (with FLAG peptide) from denaturing FLAG‐Orm2 immunoprecipitations (IP) from WT cells and the indicated mutants. Control cells expressed untagged Orm2; (F) of input and elution (with FLAG peptide) from native FLAG‐Orm2 co‐immunoprecipitations from WT cells and the indicated mutants. The control strain expresses untagged Orm2.

Article Snippet: TaqMan gene expression assays were from Thermo Fisher ( ORM2 : Sc04149509_s1; PGK1 : Sc04104844_s1).

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy, Ubiquitin Proteomics, SDS Page, Western Blot, Control

Journal: The EMBO Journal

Article Title: Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

doi: 10.15252/embj.2018101433

Figure Lengend Snippet: Gene Ontology (GO) analysis for cellular processes and components of 76 proteins that were stabilized in tul1∆ pep4∆ mutants relative to pep4∆ cells ( Appendix Tables S4 and S5 ). Only GO terms with more than twofold enrichment over the genome frequency are shown. Red GO terms were reported with false discovery rate (FDR) below 0.1; black GO terms with FDR above 0.1. Tul1 genetic interaction profile similarities (using http://thecellmap.org ) (Costanzo et al , ) (Pearson correlation coefficient, PCC = 0.10). Equal amounts of WT cells and indicated single or double mutants in serial dilutions were incubated on agar plates at the indicated temperatures. The dashed box indicates the tul1∆ tsc3∆ double mutant. Negative genetic interaction network of tul1∆ (Costanzo et al , ) (PCC = −0.3). Schematic presentation of Orm1 and Orm2 with Ypk1‐dependent phospho‐sites and the ubiquitination sites in Orm2 that were identified here. SDS–PAGE and Western blot of total cell lysates of the indicated strains demonstrating specificity of the anti‐Orm2 antibody. Quantification of ORM2 mRNA normalized to stable PGK1 mRNA from WT cells and tul1∆ mutants by qPCR ( n = 4). Data are presented as mean fold change from WT ± standard deviation. Statistical significance was assessed by Student's t ‐test. SDS–PAGE and Western blot analysis of total cell lysates of WT cells or the indicated mutants with the indicated antibodies.

Article Snippet: TaqMan gene expression assays were from Thermo Fisher ( ORM2 : Sc04149509_s1; PGK1 : Sc04104844_s1).

Techniques: Incubation, Mutagenesis, Ubiquitin Proteomics, SDS Page, Western Blot, Standard Deviation

Journal: The EMBO Journal

Article Title: Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

doi: 10.15252/embj.2018101433

Figure Lengend Snippet: See also Fig . Epifluorescence and phase‐contrast microscopy of living WT yeast cells and the indicated mutants expressing GFP‐Orm2 or GFP‐Orm2‐K25,33R (green) with dsRed‐HDEL (red). Arrowheads point to GFP‐Orm2 accumulations outside the endoplasmic reticulum (ER). Epifluorescence and phase‐contrast microscopy of tul1Δ mutants expressing GFP‐Orm2 (green) with Vps4‐mCherry (red, endosomes) or with Sec7‐mCherry (red, Golgi) or Vps4‐eGFP (green) with Sec7‐mCherry. Left panel: epifluorescence and phase‐contrast microscopy of the indicated mutants expressing GFP‐Orm2 (green) with mCherry‐Cps1 (red, MVB cargo). E indicates class E compartments; right panel: Equal amounts of the indicated mutants in serial dilutions were incubated on agar plates at the indicated temperatures. Data information: scale bars = 5 μm.

Article Snippet: TaqMan gene expression assays were from Thermo Fisher ( ORM2 : Sc04149509_s1; PGK1 : Sc04104844_s1).

Techniques: Microscopy, Expressing, Incubation

Journal: The EMBO Journal

Article Title: Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

doi: 10.15252/embj.2018101433

Figure Lengend Snippet: Single z planes from life cell confocal fluorescence microscopy of WT cells and tul1∆ mutants expressing GFP‐Orm2 and dsRed‐HDEL. Scale bars 5 μm. SDS–PAGE and Western blot analysis of total cell lysates of the indicated mutants with the indicated antibodies.

Article Snippet: TaqMan gene expression assays were from Thermo Fisher ( ORM2 : Sc04149509_s1; PGK1 : Sc04104844_s1).

Techniques: Fluorescence, Microscopy, Expressing, SDS Page, Western Blot

Journal: The EMBO Journal

Article Title: Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

doi: 10.15252/embj.2018101433

Figure Lengend Snippet: See also Fig . A–C WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 and 180 min at 26°C or at the non‐permissive temperature (37°C), where indicated. Total cell lysates were analyzed by SDS–PAGE and Western blotting with the indicated antibodies. (A) Graphs display the FLAG‐Orm2 protein levels determined by densitometric quantification of Western blots from cell lysates of WT cells and the indicated mutants 0, 90, and 180 min after the addition of CHX. Each experiment was repeated at least three times, FLAG‐Orm2 levels were normalized to Pgk1 loading controls, and each time point was related to t = 0 min (set to 1). Data are presented as mean ± standard deviation. (C) SEC13 WT and sec13‐4 cells were pre‐incubated for 30 min at 37°C before CHX was added. D Epifluorescence and phase‐contrast microscopy of living WT cells and cdc48‐ 3 mutants expressing GFP‐Orm2 incubated for 5 h at the non‐permissive temperature (37°C). E Epifluorescence and phase‐contrast microscopy of living tul1∆ and tul1∆ sec13‐4 mutants expressing GFP‐Orm2 (green) and dsRed‐HDEL (red) incubated at 26°C or shifted for 90 min to the non‐permissive temperature (37°C). Data information: scale bars 5 μm.

Article Snippet: TaqMan gene expression assays were from Thermo Fisher ( ORM2 : Sc04149509_s1; PGK1 : Sc04104844_s1).

Techniques: Blocking Assay, SDS Page, Western Blot, Standard Deviation, Incubation, Microscopy, Expressing

Journal: The EMBO Journal

Article Title: Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

doi: 10.15252/embj.2018101433

Figure Lengend Snippet: Cells were treated with cycloheximide (CHX) to block protein synthesis for the indicated times before cells were lysed. Total cell lysates were analyzed by SDS–PAGE and Western blot (A‐C,G) with the indicated antibodies. A, B WT cells or the indicated mutants expressing FLAG‐Orm2. C pdr5Δ mutants expressing FLAG‐Orm2 and Ub‐GFP were incubated with MG‐132 or vehicle (DMSO) 10 min prior to the addition of CHX. D Live‐cell epifluorescence and phase‐contrast microscopy of pep4Δ mutants expressing GFP‐Orm2. E SDS–PAGE and Western blot analysis of total cell lysates from WT and sec13‐4 cells incubated at the indicated temperatures. F Epifluorescence and phase‐contrast microscopy of sec13‐4 mutants expressing dsRed‐HDEL and GFP‐Orm2 (upper panels) or Vps4‐eGFP (lower panels) were incubated at 26°C or shifted for 90 min to the non‐permissive temperature (37°C). G WT cells or tul1∆ mutants expressing HA‐Orm1. H Epifluorescence and phase‐contrast microscopy of living WT cells and tul1Δ mutants expressing GFP‐Orm1. Data information: scale bars = 5 μm.

Article Snippet: TaqMan gene expression assays were from Thermo Fisher ( ORM2 : Sc04149509_s1; PGK1 : Sc04104844_s1).

Techniques: Blocking Assay, SDS Page, Western Blot, Expressing, Incubation, Microscopy

Journal: The EMBO Journal

Article Title: Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

doi: 10.15252/embj.2018101433

Figure Lengend Snippet: A WT ( pdr5∆) cells expressing FLAG‐Orm2 and Sec61‐GFP (integral ER membrane protein) treated with MG‐132 were fractionated into insoluble membrane (P100) and soluble cytoplasmic (S100) fractions and analyzed by SDS–PAGE and Western blot with the indicated antibodies. B, C WT cells, tul1Δ, and cdc48‐3 mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG‐132) as indicated and subsequently subjected to subcellular fractionation as in (A). S100 and P100 fractions were subjected to denaturing FLAG‐Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS–PAGE and Western blotting with the indicated antibodies. D P100 fractions from MG‐132‐treated WT ( pdr5∆ ) cells expressing FLAG‐Orm2 and Sec61‐GFP were extracted with Na 2 CO 3 solution at the indicated pH and subsequently fractionated by 100,000 × g centrifugation into soluble (S) and insoluble membrane (M) fractions, subjected to denaturing FLAG‐immunoprecipitations and analyzed by Western blotting with the indicated antibodies. The asterisk indicates reactivity of the secondary antibody with IgG heavy chains. E Schematic representation of Orm2 degradation by the EGAD pathway.

Article Snippet: TaqMan gene expression assays were from Thermo Fisher ( ORM2 : Sc04149509_s1; PGK1 : Sc04104844_s1).

Techniques: Expressing, Membrane, SDS Page, Western Blot, Fractionation, Centrifugation

Journal: The EMBO Journal

Article Title: Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

doi: 10.15252/embj.2018101433

Figure Lengend Snippet: See also Fig . A The levels of two major ceramide (Cer) species in lipid extracts from WT (black), tul1Δ (dark gray), or Orm2‐K25,33R mutants (light gray) were measured using LC‐MS and quantified using spiked non‐yeast ceramides as internal standard. Data are normalized to Cer44:0;4 levels of WT cells (set to 1) and presented as mean ± standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t ‐test. P values are given above the respective bars. B Thin‐layer chromatography of sphingolipid extracts from [ 3 H]serine radiolabelled WT cells and tul1Δ mutants was analyzed by autoradiography. IPC, inositolphosphorylceramide; MIPC, mannosylinositolphosphorylceramide; MIP 2 C, mannosyldiinositolphosphorylceramide. X indicates an unknown lipid species that is insensitive to myriocin treatment. C SDS–PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti‐FLAG co‐immunoprecipitations from cells expressing FLAG‐Orm2‐WT or FLAG‐Orm2‐K25,33R. Control cells expressed untagged Orm2 WT. D, E SDS–PAGE or Phos‐tag SDS–PAGE and Western blot analysis with the indicated antibodies of total cell lysates (D) from WT cells expressing FLAG‐Orm2 or FLAG‐Orm2‐K25,33R; (E) from WT cells and tul1Δ mutants expressing FLAG‐Orm2, FLAG‐Orm2‐3A, or FLAG‐Orm2‐3D with or without myriocin treatment (1.5 μM) for 1 h. F Schematic representation of the regulation of SPT activity via TORC2‐Ypk1/2‐dependent phosphorylation of Orm proteins and EGAD‐dependent degradation of Orm2. Data information: (C, D) The asterisks label unspecific cross‐reactions of the respective antibodies.

Article Snippet: TaqMan gene expression assays were from Thermo Fisher ( ORM2 : Sc04149509_s1; PGK1 : Sc04104844_s1).

Techniques: Liquid Chromatography with Mass Spectroscopy, Standard Deviation, Thin Layer Chromatography, Autoradiography, SDS Page, Western Blot, Expressing, Control, Activity Assay, Phospho-proteomics

Journal: The EMBO Journal

Article Title: Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

doi: 10.15252/embj.2018101433

Figure Lengend Snippet: SDS–PAGE or Phos‐tag SDS–PAGE and Western blot analysis of total cell lysates from WT cells and tul1Δ mutants expressing FLAG‐Orm2. Right panel: densitometric quantification of phosphorylated forms of FLAG‐Orm2 or FLAG‐Orm2‐K25,33R from three independent experiments (mean ± standard deviation). SDS–PAGE or Phos‐tag SDS–PAGE and Western blot analysis of total cell lysates from WT cells and tul1Δ mutants expressing HA‐Orm1. Epifluorescence and phase‐contrast microscopy of living cdc48‐ 3 mutants expressing GFP‐Orm2‐3A (green) incubated for 5 h at the (non‐)permissive temperature (26°C, 37°C). Left panel: densitometric quantification of FLAG‐Orm2 protein levels (in WT and tul1∆ cells), and FLAG‐Orm2‐K25,33R or FLAG‐Orm2‐3A protein levels from at least three independent experiments. Right panel: densitometric quantification of FLAG‐Orm2‐3A protein levels (in WT and tul1∆ cells). Mean ± standard deviation normalized to the respective control (set to 1). Epifluorescence and phase‐contrast microscopy of cells expressing dsRed‐HDEL (red) and GFP‐Orm2‐K25,33R‐3A or GFP‐Orm2‐K25,33R‐3D (green). Arrowheads point to GFP‐Orm2 accumulations outside the endoplasmic reticulum (ER). Data information: scale bars = 5 μm.

Article Snippet: TaqMan gene expression assays were from Thermo Fisher ( ORM2 : Sc04149509_s1; PGK1 : Sc04104844_s1).

Techniques: SDS Page, Western Blot, Expressing, Standard Deviation, Microscopy, Incubation, Control

Journal: The EMBO Journal

Article Title: Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

doi: 10.15252/embj.2018101433

Figure Lengend Snippet: See also Fig . Left panel: In tor1‐1 avo3 ∆1274‐1430 cells, rapamycin specifically inhibits TORC2 but not TORC1 (Gaubitz et al , ). tor1‐1 avo3 ∆1274‐1430 cells were treated with cycloheximide (CHX) and rapamycin or vehicle (methanol) for the indicated times. Right panel: WT or Orm2 mutant cells were treated with CHX for the indicated times. The protein levels of FLAG‐Orm2 or of the indicated FLAG‐Orm2 mutants from total cell lysates were analyzed by SDS–PAGE and Western blotting with the indicated antibodies. Graphs display the FLAG‐Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0, 90, and 180 min after the addition of CHX. Each experiment was repeated at least three times. FLAG‐Orm2 levels were normalized to Pgk1 loading controls, and each time point was related to t = 0 min (set to 1). Data are presented as mean ± standard deviation. Denaturing immunoprecipitation (IP) of FLAG‐Orm2, FLAG‐Orm2‐3A, and FLAG‐Orm2‐3D. The control strain expresses untagged Orm2. Input and elutions (with FLAG peptides) were analyzed by SDS–PAGE and Western blot with the indicated antibodies. Epifluorescence and phase‐contrast microscopy of living tul1Δ mutants expressing GFP‐Orm2, GFP‐Orm2‐3A, or GFP‐Orm2‐3D (green) with dsRed‐HDEL (red). Arrowheads point to GFP‐Orm2 accumulations outside the endoplasmic reticulum (ER). Scale bars = 5 μm. SDS–PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti‐FLAG co‐immunoprecipitations from cells expressing FLAG‐Orm2‐K25,33R, FLAG‐Orm2‐K25,33R‐3A, or FLAG‐Orm2‐K25,33R‐3D. The control strain expresses untagged Orm2. The levels of the long‐chain bases (LCB) C18‐dihydrosphingosine (C18‐DHS) and C18‐phytosphingosine (C18‐PHS) and of two major ceramide (Cer) species in lipid extracts from WT, tul1∆ , Orm2‐K25,33R, Orm2‐3A, and Orm2‐3D mutants were measured using LC‐MS and quantified using spiked non‐yeast LCBs/ceramides as internal standard. Data are normalized to WT levels of the most abundant LCB/ceramide species (set to 1) and presented as mean ± standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t ‐test. P values are given above the respective bars. Model for the dual function of the Dsc complex in EGAD‐dependent proteasomal as well as ESCRT‐dependent lysosomal membrane protein degradation.

Article Snippet: TaqMan gene expression assays were from Thermo Fisher ( ORM2 : Sc04149509_s1; PGK1 : Sc04104844_s1).

Techniques: Mutagenesis, SDS Page, Western Blot, Standard Deviation, Immunoprecipitation, Control, Microscopy, Expressing, Liquid Chromatography with Mass Spectroscopy, Membrane

Journal: Scientific Reports

Article Title: Periodic heat waves-induced neuronal etiology in the elderly is mediated by gut-liver-brain axis: a transcriptome profiling approach

doi: 10.1038/s41598-024-60664-9

Figure Lengend Snippet: Heat stress augmented the secretion of hepatic ORM2 and its abundance in the hippocampus along with the frontal cortex, traversing through leaky Blood–Brain-Barrier (BBB). ( A ) ORM2 immunoreactivity was shown by immunohistochemistry in liver sections from Control and Heat Stress mouse groups; Images were taken at 20X magnification and displayed with a scale of 200 μm. ( B ) ORM2 immunoreactivity in both groups was measured as arbitrary light units from six separate microscopic fields and plotted along the ordinate. All p values were derived through an unpaired t-test, with the significance level set at p < 0.05. ( C ) IL-1β immunoreactivity was shown by immunohistochemistry in small intestinal sections from Control and Heat Stress mouse groups; Images were taken at 20X magnification and displayed with a scale of 100 μm. ( D ) IL-1β immunoreactivity in both groups was measured as arbitrary light units from six separate microscopic fields and plotted along the ordinate. All p values were derived through an unpaired t-test, with the significance level set at p < 0.05. ( E ) The bar graph was plotted as the difference in ratio value of the relative abundance of the gut microbial phyla Firmicutes and Bacteroidetes compared between control and heat stress groups. The p value was derived through an unpaired t-test, with the significance level set at p < 0.05. ( F ) A correlation analysis was conducted between the gut dysbiosis marker Firmicutes/Bacteroidetes ratio and the abundance of ORM2 in liver tissues. The correlation plot includes Pearson's linear regression depicted in red, along with a 95% confidence interval. ( G ) Claudin5 (red) and CD31 (green) were dual labeled through immunofluorescence staining with colocalization (yellow) observed at 60X (oil) magnification. The images were presented with a scale of 20 μm in the brain sections from both the Control and Heat Stress mouse groups. ( H ) Claudin5-CD31 colocalization (yellow) events in both groups were quantified as arbitrary fluorescent units from six distinct microscopic fields and plotted along the ordinate. All p values were derived through an unpaired t-test, with the significance level set at p < 0.05. ( I ) ORM2 immunoreactivity was shown by immunohistochemistry in brain sections (hippocampus and frontal cortex) from Control and Heat Stress mouse groups; Images were taken at 10X, 40X magnification displayed with the scale of 200 μm. ( J ) ORM2 immunoreactivity in both groups was measured as arbitrary light units from six separate microscopic fields and plotted along the ordinate. All p values were derived through an unpaired t-test, with the significance level set at p < 0.05.

Article Snippet: The primary antibody against Orosomucoid 2 (ORM2) was purchased from Proteintech Group Inc (Rosemont, IL, USA).

Techniques: Immunohistochemistry, Control, Derivative Assay, Marker, Labeling, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Mitigation of non-alcoholic steatohepatitis via recombinant Orosomucoid 2, an acute phase protein modulating the Erk1/2-PPARγ-Cd36 pathway.

doi: 10.1016/j.celrep.2023.112697

Figure Lengend Snippet: Figure 2. ORM2 protein treatment ameliorated high-fat diet (HFD)-induced NAFLD and metabolic dysfunction in mice (A) Representative appearance pictures of mice (top) and liver (bottom) (n = 8/group). (B) Representative H&E and oil red O staining and quantified results (n = 6/group). Scale bars, 100 mm for H&E staining and 50 mm for oil red O staining. (C) Serum ALT, AST, total cholesterol (TC), and triglyceride (TG) levels were measured in mice at 24 weeks. n = 6 per group. (D) Liver TG and cholesterol contents (n = 6/per group). (E) Orm2 protein level in liver (top) and Orm2 in liver tissue were determined by ELISA (bottom). (F and G) OGTT (F) and insulin tolerance test (ITT; G) area under curve (AUC) shown. n = 8 per group. (H) qRT-PCR for the indicated metabolism genes expression organized for liver. Data are expressed as fold change in expression relative to control (CON) following the calculation of relative expression to Gapdh. Data are the mean ± SEM and were compared by ordinary one-way ANOVA followed by Tukey’s multiple comparisons test for (C)–(H); two-tailed unpaired Student’s t test for (B). An asterisk (*) indicates a significant difference between the CON group and the HFD group; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The hash symbol (#) indicates a significant difference between the ORM2-HFD group and the HFD group; #p < 0.05 and ##p < 0.01.

Article Snippet: The Orm2 heterozygote mice with a C57BL/6N background were purchased from Cyagen Bio (Guangzhou, China).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Control, Two Tailed Test

Journal: Cell reports

Article Title: Mitigation of non-alcoholic steatohepatitis via recombinant Orosomucoid 2, an acute phase protein modulating the Erk1/2-PPARγ-Cd36 pathway.

doi: 10.1016/j.celrep.2023.112697

Figure Lengend Snippet: Figure 3. ORM2 protein treatment ameliorated methionine and choline-deficient L-amino acid diet (MCD) induced NASH in mice (A) Representative appearance pictures of liver in CON (fed regular chow), MCD, and MCD-ORM2 (fed MCD) (n = 8/group). (B) Representative images of H&E staining (top), Picrosirius red staining (middle), and oil red O staining (bottom) of liver and quantitative results from ImageJ (n = 6/group). Scale bar: 50 mm. (C) Representative immunohistochemical image of Cd11b and Cd68 and quantitative results from ImageJ (n = 6/group).

Article Snippet: The Orm2 heterozygote mice with a C57BL/6N background were purchased from Cyagen Bio (Guangzhou, China).

Techniques: Staining, Immunohistochemical staining

Journal: Cell reports

Article Title: Mitigation of non-alcoholic steatohepatitis via recombinant Orosomucoid 2, an acute phase protein modulating the Erk1/2-PPARγ-Cd36 pathway.

doi: 10.1016/j.celrep.2023.112697

Figure Lengend Snippet: Figure 4. Orm2 knockout (KO) caused spontaneous weight gain and metabolic dysfunction in mice (A) Hepatic levels of Orm2 were analyzed by western blotting. n = 6 biologically independent mice per condition. (B) Representative appearance pictures of mice (top), epididymal white adipose (middle), and liver (bottom). (C) eWAT weight, BAT weight, and liver weight of WT and Orm2 KO mice during a regular chow diet for 20 weeks (n = 8/group). (D) Representative images of H&E (left) and oil red O (right) staining of liver. Scale bars, 100 mm for H&E and 50 mm for oil red O. (E) qRT-PCR for the indicated metabolism genes organized for liver. Data are expressed as fold change in expression relative to WT following the calculation of relative expression to Gapdh. (F) Intraperitoneal ITT performed at 18 weeks of age; glucose measurements over time after insulin was injected intraperitoneally (i.p.; left) and AUC (right) shown. n = 8 per group. All data are shown as mean ± SEM and were compared by two-tailed unpaired Student’s t test, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: The Orm2 heterozygote mice with a C57BL/6N background were purchased from Cyagen Bio (Guangzhou, China).

Techniques: Knock-Out, Western Blot, Staining, Quantitative RT-PCR, Expressing, Injection, Two Tailed Test

Journal: Cell reports

Article Title: Mitigation of non-alcoholic steatohepatitis via recombinant Orosomucoid 2, an acute phase protein modulating the Erk1/2-PPARγ-Cd36 pathway.

doi: 10.1016/j.celrep.2023.112697

Figure Lengend Snippet: Figure 5. Orm2 KO mice exhibit aggravated diet-induced NAFLD and NASH (A) Representative appearance pictures of mice (top), liver (middle), and epididymal white adipose (bottom). n = 8/group. (B) Body weight change in CON (fed regular chow), HFD, and KO-HFD (fed HFD). n = 8 per group. (C) Hepatic lipid (TG and TC) levels. n = 8 per group.

Article Snippet: The Orm2 heterozygote mice with a C57BL/6N background were purchased from Cyagen Bio (Guangzhou, China).

Techniques:

Journal: Cell reports

Article Title: Mitigation of non-alcoholic steatohepatitis via recombinant Orosomucoid 2, an acute phase protein modulating the Erk1/2-PPARγ-Cd36 pathway.

doi: 10.1016/j.celrep.2023.112697

Figure Lengend Snippet: Figure 6. Orm2 deletion activated the Erk1/2-PPARg-Cd36 signaling pathway in the mouse liver (A) Heatmap showing lipid metabolic process enriched in GO term in RNA-seq from liver. n = 3 per group. (B) Bubble plot showing three groups’ KEGG pathway enrichment in RNA-seq. (C) Heatmap showing changes in genes enriched in PPAR signaling pathway in RNA-seq.

Article Snippet: The Orm2 heterozygote mice with a C57BL/6N background were purchased from Cyagen Bio (Guangzhou, China).

Techniques: RNA Sequencing

Journal: Cell reports

Article Title: Mitigation of non-alcoholic steatohepatitis via recombinant Orosomucoid 2, an acute phase protein modulating the Erk1/2-PPARγ-Cd36 pathway.

doi: 10.1016/j.celrep.2023.112697

Figure Lengend Snippet: Figure 7. Orm2 inhibited fatty acid uptake by inhibiting Erk1/2-PPARg-Cd36 in hepatocytes (A) Orm2 knockdown efficiency and overexpression were measured by western blotting analysis. (B) Representative oil red O staining of AML12 cells with Orm2 downregulated or Orm2 overexpressed, treated with OA/PA mixture (OA 0.4 mM, PA 0.2 mM) for 24 h (n = 3 per group). (C) qRT-PCR for the indicated metabolism genes organized for AML12 cells with Orm2 downregulated or Orm2 overexpressed, treated with OA/PA mixture (OA 0.4 mM, PA 0.2 mM) for 24 h (n = 3 per group). Data are expressed as fold change in expression relative to CON following the calculation of relative expression to Gapdh.

Article Snippet: The Orm2 heterozygote mice with a C57BL/6N background were purchased from Cyagen Bio (Guangzhou, China).

Techniques: Knockdown, Over Expression, Western Blot, Staining, Quantitative RT-PCR, Expressing

Journal: Biomedicines

Article Title: Identification of Potential Drug Targets for Antiplatelet Therapy Specifically Targeting Platelets of Old Individuals through Proteomic Analysis

doi: 10.3390/biomedicines11112944

Figure Lengend Snippet: Expression of selected targets in young and aged mice platelets. ( A ) Expression of ApoH, Orm2, Rab1, cfh, and Azgp1/ZAG from proteomic analysis. ( B ) Western blot analysis of platelet activation markers (Fibronectin and CD42b), and selected targets of 14-month-old mice platelets (#1–7) and 24-month-old mice platelets (#1–9). Ponceau’s staining served as loading control. ( C ) Quantification of fibronectin, CD42b, ApoH, and Orm2 signal intensity. The p -values are analyzed by one-way ANOVA.

Article Snippet: We used the standard Western blot analysis protocols [ ] and specific individual antibodies and dilutions (fibronectin: Abcam ab2413 (1:1000), CD42b: Abcam ab183345 (1:1000), ApoH: Proteintech 66074 (1:1000), and Orm2: Abbexa abx101108 (1:1000).

Techniques: Expressing, Western Blot, Activation Assay, Staining, Control

Journal: Biomedicines

Article Title: Identification of Potential Drug Targets for Antiplatelet Therapy Specifically Targeting Platelets of Old Individuals through Proteomic Analysis

doi: 10.3390/biomedicines11112944

Figure Lengend Snippet: The expression level of Orm2 increased in aged mice platelets. ( A , B ). Confocal microscopy images using Orm2 and Rab11a specific Abs in 14- and 24-month-old mice platelets. ( C ). Quantification of Orm2 and Rab11a relative signal intensity. The p -values are analyzed using a one-way ANOVA.

Article Snippet: We used the standard Western blot analysis protocols [ ] and specific individual antibodies and dilutions (fibronectin: Abcam ab2413 (1:1000), CD42b: Abcam ab183345 (1:1000), ApoH: Proteintech 66074 (1:1000), and Orm2: Abbexa abx101108 (1:1000).

Techniques: Expressing, Confocal Microscopy

Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids

Article Title: The ORMs Interact with Transmembrane Domain 1 of Lcb1 and Regulate Serine Palmitoyltransferase Oligomerization, Activity and Localization

doi: 10.1016/j.bbalip.2018.11.007

Figure Lengend Snippet: Strains used in this study

Article Snippet: The Orm1, Orm2 and Elo3 peptides and antibodies were produced by New England Peptide, Inc. Antibodies were affinity purified using CNBr-activated Sepharose 4B beads (GE Healthcare Life Sciences) to which the immunizing peptides were covalently coupled according to the manufacturer’s instructions.

Techniques:

Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids

Article Title: The ORMs Interact with Transmembrane Domain 1 of Lcb1 and Regulate Serine Palmitoyltransferase Oligomerization, Activity and Localization

doi: 10.1016/j.bbalip.2018.11.007

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: The Orm1, Orm2 and Elo3 peptides and antibodies were produced by New England Peptide, Inc. Antibodies were affinity purified using CNBr-activated Sepharose 4B beads (GE Healthcare Life Sciences) to which the immunizing peptides were covalently coupled according to the manufacturer’s instructions.

Techniques:

Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids

Article Title: The ORMs Interact with Transmembrane Domain 1 of Lcb1 and Regulate Serine Palmitoyltransferase Oligomerization, Activity and Localization

doi: 10.1016/j.bbalip.2018.11.007

Figure Lengend Snippet: A. Serial dilutions of cells with or without orm1 and ORM2 (ORMs) and expressing either full-length Lcb1 (WT) or Lcb1ΔTMD1 (ΔTMD1) were spotted onto YPD, or YPD + tunicamycin (1 μg/ml) and the plates were incubated at 37 °C (2 days), 26 °C (3 days), or 17 °C (7 days) as indicated. B. Total LCBs were extracted from the indicated strains (as in panel A) and quantified by HPLC as described in Materials and Methods. Results are the average ± SD of LCB levels from three independent colonies (*** p<0.001, n=3; N.S, not significant, n=3). Results are representative of multiple independent experiments. C. Twenty-five μg of microsomal protein from the samples used for LCB analysis (panel B) were separated by SDS-PAGE, transferred to nitrocellulose and Lcb1, Lcb2 and Tsc3 were detected by immunoblotting. Protein expression was quantitatively analyzed with Fiji ImageJ software (n=6). D. Wild-type (WT), ypk1Δypk2Δ, ypk1Δypk2Δ with a plasmid expressing Lcb1ΔTMD1, ypk1Δypk2Δorm1Δ. or ypk1Δypk2Δorm2Δ cells were spotted on plates containing YPD medium with or without PHS. Plates were incubated at 26° C for 3 days.

Article Snippet: The Orm1, Orm2 and Elo3 peptides and antibodies were produced by New England Peptide, Inc. Antibodies were affinity purified using CNBr-activated Sepharose 4B beads (GE Healthcare Life Sciences) to which the immunizing peptides were covalently coupled according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, SDS Page, Western Blot, Software, Plasmid Preparation

Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids

Article Title: The ORMs Interact with Transmembrane Domain 1 of Lcb1 and Regulate Serine Palmitoyltransferase Oligomerization, Activity and Localization

doi: 10.1016/j.bbalip.2018.11.007

Figure Lengend Snippet: A. Split-ubiquitin 2-hybrid interactions between yeast Lcb1-Cub or Lcb1ΔTMD1-Cub and Lcb2-Nub, Nub-Orm1, or Nub-Orm2 were assessed by growth on medium lacking histidine (HIS) and containing 50 mM 3-AT. Plates were incubated at 26 °C for 3 days. B. Cells expressing full-length Lcb1 or Lcb1ΔTMD1 were transformed with a plasmid expressing FLAG-Orm2. Solubilized microsomes were prepared, and following incubation with anti-FLAG beads adsorbed proteins were resolved by SDS-PAGE and visualized by immunoblotting with the indicated antibodies as described in Materials and Methods. Elo3, a component of the ER-associated fatty acid elongase complex, was used as a negative control. C. A glycosylation cassette was inserted after residue 4 (GC@4) into full-length Lcb1 or Lcb1 in which TMD1 had been replaced by the first TMD of FVT1 (TMD1FVT). Solubilized microsomes were resolved by SDS-PAGE before or after treatment with endoglycosidase H (Endo H) and Lcb1 was visualized by immunoblotting with an anti-Lcb1 antibody. D. SPT activity was measured in microsomes prepared from cells expressing full-length Lcb1, Lcb1ΔTMD1. or Lcb1-TMD1FVT. Results are the average ± SD from two independent colonies assayed in triplicate (* p< 0.05, *** p< 0.001, n=6). E. Microsomes were prepared from lcb1Δ mutant yeast cells expressing Lcb1-FLAG, Lcb1ΔTMD1-FLAG, or Lcb1-TMD1FVT-FLAG, solubilized, incubated with anti-FLAG beads and adsorbed proteins resolved by SDS-PAGE. Coimmunoprecipitated proteins were visualized by immunoblotting with the indicated antibodies. Elo3 was used as a negative control. F. Total LCB levels were determined from cells expressing wild-type Lcb1 or Lcb1-TMD1FVT with and without the ORM proteins as in Fig. 1B. The results are the average ± SD of three independent colonies for each strain (*** p<0.001, n=3; N.S, not significant, n=3). G. SPT activity was determined in microsomes prepared from yeast lcb1Δ mutant cells and expressing hLCB2a and ssSPTa with either hLCB1 or hLCB1TMD1yeast, a chimera in which TMD1 of hLCB1 was replaced with TMD1 from yeast Lcb1. SPT activity was measured in triplicate from microsomes prepared from three colonies of each strain and the results are the average ± SD; (N.S, not significant, n=9). H. Solubilized microsomes were prepared from yeast lcb1Δ mutant cells expressing FLAG-Orm2 and either yeast Lcb1 or hLCB1 TMD1yeast, hLCB2a, and ssSPTa. After adsorption on anti-FLAG beads, the interaction of Orm2 with the wild-type and chimeric proteins was assessed as in Fig. 2E.

Article Snippet: The Orm1, Orm2 and Elo3 peptides and antibodies were produced by New England Peptide, Inc. Antibodies were affinity purified using CNBr-activated Sepharose 4B beads (GE Healthcare Life Sciences) to which the immunizing peptides were covalently coupled according to the manufacturer’s instructions.

Techniques: Incubation, Expressing, Transformation Assay, Plasmid Preparation, SDS Page, Western Blot, Negative Control, Activity Assay, Mutagenesis, Adsorption

Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids

Article Title: The ORMs Interact with Transmembrane Domain 1 of Lcb1 and Regulate Serine Palmitoyltransferase Oligomerization, Activity and Localization

doi: 10.1016/j.bbalip.2018.11.007

Figure Lengend Snippet: A. Solubilized microsomes prepared from yeast lcb1Δ mutant cells expressing HA-Sac1 and either Lcb1-FLAG or Lcb1ΔTMD1 - FLAG were incubated with anti-FLAG antibodies and coimmunoprecipitation of HA-Sac1 and Orm2 was assessed. B. Lcb1- FLAG and HA-Sac1 were co-expressed in wild-type or tsc3Δ mutant cells and the coimmunoprecipitation of HA-Sac1 with Lcb1- FLAG was evaluated as in Panel A. C. Levels of free LCBs in wild-type, Sac1Δ, andorm1Δorm2Δ cells (seven independent colonies of each) were determined as described in Materials and Methods. Results are the average ± SD, *** p<0.001, n=7).

Article Snippet: The Orm1, Orm2 and Elo3 peptides and antibodies were produced by New England Peptide, Inc. Antibodies were affinity purified using CNBr-activated Sepharose 4B beads (GE Healthcare Life Sciences) to which the immunizing peptides were covalently coupled according to the manufacturer’s instructions.

Techniques: Mutagenesis, Expressing, Incubation

Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids

Article Title: The ORMs Interact with Transmembrane Domain 1 of Lcb1 and Regulate Serine Palmitoyltransferase Oligomerization, Activity and Localization

doi: 10.1016/j.bbalip.2018.11.007

Figure Lengend Snippet: A. Yeast cells expressing Lcb2 containing a multimerizing version of GFP inserted between residues 82 and 83 (Lcb2GFP@82) were grown in YPD medium with and without LCBs (PHS, DHS, or 3-ketosphinganine (3-KDS) and the localization of Lcb2 was observed using fluorescence microscopy. The non-physiological LCB stearylamine (STA) served as a negative control. B. Lcb1-RFP was co-expressed with Lcb2GFP@82, and the localization of the proteins was compared by fluorescence microscopy. C. RFP-Orm2 and Lcb2GFP@82 were co-expressed and their localization visualized by fluorescence microscopy. D. PHS-induced relocalization of SPT in wild-type (-PHS, no bars, n>500 cells; +PHS, 51 ± 4% with bars, n=247 cells, 5 experiments) and orm1Δorm2Δ mutant cells (-PHS, no bars, n=93 cells; +PHS, no bars, n=187 cells, 3 experiments) expressing Lcb2GFP@82 was compared as in Panel A. E. PHS-induced relocalization of SPT in cells expressing Lcb2GFP@82 and the non-phosphorylatable Orm2-3A (-PHS, 49 ± 9% with bars, n=116 cells; +PHS, 52 ± 16% with bars, n=67 cells, 3 experiments) or the phosphomimetic Orm2-3D mutant (-PHS, no bars, n=80 cells; +PHS, no bars, n=118 cells, 3 experiments) was determined as in Panel A. F. PHS-induced relocalization of SPT in wild-type and sac1Δ cells (-PHS, no bars, n>100 cells; +PHS, 23 ± 10% with bars, n=99 cells, 4 experiments) expressing Lcb2GFP@82 was determined as in Panel A. Scale bar, 5 microns.

Article Snippet: The Orm1, Orm2 and Elo3 peptides and antibodies were produced by New England Peptide, Inc. Antibodies were affinity purified using CNBr-activated Sepharose 4B beads (GE Healthcare Life Sciences) to which the immunizing peptides were covalently coupled according to the manufacturer’s instructions.

Techniques: Expressing, Fluorescence, Microscopy, Negative Control, Mutagenesis

Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids

Article Title: The ORMs Interact with Transmembrane Domain 1 of Lcb1 and Regulate Serine Palmitoyltransferase Oligomerization, Activity and Localization

doi: 10.1016/j.bbalip.2018.11.007

Figure Lengend Snippet: A. Total LCB levels in wild-type, tsc3Δ, orm1Δorm2Δ, and orm1Δorm2Δtsc3Δ mutant cells were determined as described in Materials and Methods. Results are the average ± SD from three independent colonies of each strain, *** p<0.001, n=3; N.S, not significant, n=3. B. Microsomal protein extracts were prepared from wild-type (WT), tsc3Δ,orm1Δtsc3Δ and orm2Δtsc3Δ mutant cells, resolved by SDS-PAGE with or without the Phos-tag™ reagent, and the indicated proteins were detected by immunoblotting using antibodies to the proteins indicated on the right side of the panel. C. Plasmids co-expressing wild-type Orm1 and Orm2, the non-phosphorylatable Orm1-3 A and Orm2-3A, or the phosphomimetic Orm1-3D and Orm2-3D mutant proteins from divergent galactose inducible GAL1 and GAL10 promoters were introduced into an orm1Δorm2Δtsc3Δ mutant or an orm1Δorm2Δtsc3Δ mutant with LCB1 replaced by LCB1ΔTMD1. Growth of two independent colonies of each was assessed in the absence or presence of 2% (w/v) galactose. D. orm1Δorm2ΔLcb1Δ mutants co-expressing Lcb1-BirA and either wild-type Orm2, the non-phosphorylatable Orm2-3A, or the phosphomimetic Orm2-3D mutant proteins were grown with or without biotin as described in Materials and Methods. 5 μg of microsomal protein were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted for detection with anti-Lcb1, anti-Orm2 and streptavidin-HRP (Thermo Scientific). E. Microsomal proteins were prepared from orm1Δorm2ΔLcb1Δ mutants co-expressing Lcb1-FLAG and either wild-type Orm2, the non-phosphorylatable Orm2-3A, or the phosphomimetic Orm2-3D mutant proteins. Anti-FLAG pulldowns followed by anti-Lcb1, Lcb2 and Orm2 immunoblotting were performed as described in the Materials and Methods.

Article Snippet: The Orm1, Orm2 and Elo3 peptides and antibodies were produced by New England Peptide, Inc. Antibodies were affinity purified using CNBr-activated Sepharose 4B beads (GE Healthcare Life Sciences) to which the immunizing peptides were covalently coupled according to the manufacturer’s instructions.

Techniques: Mutagenesis, SDS Page, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Small Molecule Cjoc42 Improves Chemo-Sensitivity and Increases Levels of Tumor Suppressor Proteins in Hepatoblastoma Cells and in Mice by Inhibiting Oncogene Gankyrin

doi: 10.3389/fphar.2021.580722

Figure Lengend Snippet: Combination treatment of cancer cell lines with Cjoc42 and cisplatin leads to stronger inhibition of proliferation compared to monotherapy. (A) Proliferation assay in Huh6 cells treated with Cjoc42 alone, cisplatin alone, or a combination of Cjoc42 and cisplatin. D0 shows the number of cells at the time of plating. Statistically significant reduction in proliferation is seen when comparing control to Cjoc42 5 µM ( p < 0.0001), control to cisplatin 1 µg/mL ( p < 0.02), cisplatin 1 µg/mL to cisplatin + Cjoc42 1 µM ( p < 0.0001), and cisplatin 1 µg/mL to cisplatin + Cjoc42 5 µM ( p < 0.0001). (B) Microscope pictures at 20x magnification show a reduction in number and cluster of Huh6 cells when treated with cisplatin and increasing concentrations of Cjoc42. (C) QRT-PCR data in Huh6 treated cells shows an increase in apoptotic markers Bax and TP53 with concomitant treatment as well as an increase in mature hepatocyte markers ORM2 and CYP3A4 . (D) Proliferation assay in Hepa1c1c7 cells treated with Cjoc42 alone, cisplatin alone, or a combination of Cjoc42 and cisplatin. Statistically significant reduction in proliferation is seen when comparing control to cisplatin 1 µg/mL ( p < 0.0001), cisplatin 1 µg/mL to cisplatin + Cjoc42 1 µM ( p < 0.0001), cisplatin 1 µg/mL to cisplatin + Cjoc42 5 µM ( p < 0.0001), and cisplatin + Cjoc42 1 µM to cisplatin + Cjoc42 5 µM ( p < 0.008). (E) Left: Cytotoxicity Assay with cisplatin 1 μg/mL and increasing concentrations of Cjoc42 (1 μM, 5 μM, and 10 µM) in Huh6 cells. Right: Cytotoxicity Assay with cisplatin 1 µg/mL and increasing concentrations of Cjoc42 (1 μM, 5 μM, and 10 µM) in Hepa1c1c7 cells. A statistically significant difference was seen in cell viability between cisplatin 0.5 µg/mL or 1 µg/mL dose alone vs. in combination with Cjoc42 10 µM ( p < 0.01 and p < 0.04, respectively. (F) Western blot analysis of Huh6 cells treated with cisplatin 1 µg/mL and increasing concentrations of Cjoc42. (G) Western blot analysis of Hepa1c1c7 cells treated with cisplatin 1 µg/mL and increasing concentrations of Cjoc42. A statistically significant difference was seen in cell viability between cisplatin 0.5 µg/mL or 1 µg/mL dose alone vs. in combination with Cjoc42 10 µM ( p < 0.01 and p < 0.04, respectively). (H) A statistically significant difference in Bcl2 is seen comparing control cells to Cjoc42 5 µM ( p < 0.03). (I) TUNEL assay of Huh6 cells treated with Cjoc42 (5 µM), cisplatin (1 µg/mL), and with combination cisplatin plus Cjoc42. The upper panel shows typical microscope pictures of the same cells before staining. White arrow shows a big colony of untreated Huh6 cells, black arrows show small colonies in treated cells. Bottom images show the merge of the staining with Propidium Iodide/RNase A (red) and FITC-dUTP (green).

Article Snippet: The following probes were used: CUGBP1 (CELF1), Mm04279608_m1; C/EBPα, Mm01265914_s1; HNF4α, Mm01247712_m1; TP53, Mm01731290_g1; Rb1, Mm00485586_m1; cdc2(cdk1), Mm01149140_m; Caspase 6, Mm00438053_m1; ORM2, Mm04213463_g1; Human: BAX, Hs00180269_m1; TP53, Hs01034249_m1; ORM2, Hs01037491_m1; CYP3A4, Hs00604506_m1; Gankyrin (PSMD10), Hs01100439_g1.

Techniques: Inhibition, Proliferation Assay, Control, Microscopy, Quantitative RT-PCR, Cytotoxicity Assay, Western Blot, TUNEL Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Small Molecule Cjoc42 Improves Chemo-Sensitivity and Increases Levels of Tumor Suppressor Proteins in Hepatoblastoma Cells and in Mice by Inhibiting Oncogene Gankyrin

doi: 10.3389/fphar.2021.580722

Figure Lengend Snippet: A combination therapy of doxorubicin and Cjoc42 enhances cytotoxicity. (A) Cytotoxicity Assay with doxorubicin (0.05, 0.1, and 0.2 µg/mL) and increasing concentrations of Cjoc42 (1, 5, and 10 µM) in Huh6 cells. (B) Cytotoxicity Assay with doxorubicin (0.1, 0.2, and 0.5 µg/mL) and increasing concentrations of Cjoc42 (1, 5, and 10 µM) in Hepa1c1c7 cells. (C) Microscope pictures at 20x showing reduction in size and number of clustered Huh6 cells when treated with doxorubicin 0.05 µg/mL and increasing concentrations of Cjoc42 compared to Cjoc42 or doxorubicin alone. (D) QRT-PCR data with Hepa1c1c7 cells treated with 1 µM Cjoc42 alone, Doxorubicin 0.1 µg/mL alone, or a combination of both. There was a statistically significant increase in expression of the apoptotic marker Casp6 with combination treatment ( p < 0.05) and the mature hepatocyte marker ORM2 ( p < 0.05). (E) Western blotting in Huh6 cells treated with Cjoc42 alone, doxorubicin 0.05 µg/mL, or a combination of doxorubicin and Cjoc42. (F) Bcl2 expression was calculated as a ratio to β-actin. Bcl2 is significantly higher in cells treated with doxorubicin 0.1 µg/mL and Cjoc42 5 µM compared to doxorubicin 0.05 µg/mL alone ( p < 0.03). (G) Co-IP studies. Gank was immunoprecipitated from untreated Huh6 and from Huh6 cells treated with doxorubicin (0.05 µg/mL) and cjoc42 (5 µM). Tumor suppressor proteins were examined in these IPs. (H) Western blotting shows levels of tumor suppressor proteins in nuclear extracts of untreated and doxorubicin + cjoc42 treated Huh6 cells.

Article Snippet: The following probes were used: CUGBP1 (CELF1), Mm04279608_m1; C/EBPα, Mm01265914_s1; HNF4α, Mm01247712_m1; TP53, Mm01731290_g1; Rb1, Mm00485586_m1; cdc2(cdk1), Mm01149140_m; Caspase 6, Mm00438053_m1; ORM2, Mm04213463_g1; Human: BAX, Hs00180269_m1; TP53, Hs01034249_m1; ORM2, Hs01037491_m1; CYP3A4, Hs00604506_m1; Gankyrin (PSMD10), Hs01100439_g1.

Techniques: Cytotoxicity Assay, Microscopy, Quantitative RT-PCR, Expressing, Marker, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Frontiers in Pharmacology

Article Title: Small Molecule Cjoc42 Improves Chemo-Sensitivity and Increases Levels of Tumor Suppressor Proteins in Hepatoblastoma Cells and in Mice by Inhibiting Oncogene Gankyrin

doi: 10.3389/fphar.2021.580722

Figure Lengend Snippet: Combination treatment of cancer cell lines with Cjoc42 and cisplatin leads to stronger inhibition of proliferation compared to monotherapy. (A) Proliferation assay in Huh6 cells treated with Cjoc42 alone, cisplatin alone, or a combination of Cjoc42 and cisplatin. D0 shows the number of cells at the time of plating. Statistically significant reduction in proliferation is seen when comparing control to Cjoc42 5 µM ( p < 0.0001), control to cisplatin 1 µg/mL ( p < 0.02), cisplatin 1 µg/mL to cisplatin + Cjoc42 1 µM ( p < 0.0001), and cisplatin 1 µg/mL to cisplatin + Cjoc42 5 µM ( p < 0.0001). (B) Microscope pictures at 20x magnification show a reduction in number and cluster of Huh6 cells when treated with cisplatin and increasing concentrations of Cjoc42. (C) QRT-PCR data in Huh6 treated cells shows an increase in apoptotic markers Bax and TP53 with concomitant treatment as well as an increase in mature hepatocyte markers ORM2 and CYP3A4 . (D) Proliferation assay in Hepa1c1c7 cells treated with Cjoc42 alone, cisplatin alone, or a combination of Cjoc42 and cisplatin. Statistically significant reduction in proliferation is seen when comparing control to cisplatin 1 µg/mL ( p < 0.0001), cisplatin 1 µg/mL to cisplatin + Cjoc42 1 µM ( p < 0.0001), cisplatin 1 µg/mL to cisplatin + Cjoc42 5 µM ( p < 0.0001), and cisplatin + Cjoc42 1 µM to cisplatin + Cjoc42 5 µM ( p < 0.008). (E) Left: Cytotoxicity Assay with cisplatin 1 μg/mL and increasing concentrations of Cjoc42 (1 μM, 5 μM, and 10 µM) in Huh6 cells. Right: Cytotoxicity Assay with cisplatin 1 µg/mL and increasing concentrations of Cjoc42 (1 μM, 5 μM, and 10 µM) in Hepa1c1c7 cells. A statistically significant difference was seen in cell viability between cisplatin 0.5 µg/mL or 1 µg/mL dose alone vs. in combination with Cjoc42 10 µM ( p < 0.01 and p < 0.04, respectively. (F) Western blot analysis of Huh6 cells treated with cisplatin 1 µg/mL and increasing concentrations of Cjoc42. (G) Western blot analysis of Hepa1c1c7 cells treated with cisplatin 1 µg/mL and increasing concentrations of Cjoc42. A statistically significant difference was seen in cell viability between cisplatin 0.5 µg/mL or 1 µg/mL dose alone vs. in combination with Cjoc42 10 µM ( p < 0.01 and p < 0.04, respectively). (H) A statistically significant difference in Bcl2 is seen comparing control cells to Cjoc42 5 µM ( p < 0.03). (I) TUNEL assay of Huh6 cells treated with Cjoc42 (5 µM), cisplatin (1 µg/mL), and with combination cisplatin plus Cjoc42. The upper panel shows typical microscope pictures of the same cells before staining. White arrow shows a big colony of untreated Huh6 cells, black arrows show small colonies in treated cells. Bottom images show the merge of the staining with Propidium Iodide/RNase A (red) and FITC-dUTP (green).

Article Snippet: The following probes were used: CUGBP1 (CELF1), Mm04279608_m1; C/EBPα, Mm01265914_s1; HNF4α, Mm01247712_m1; TP53, Mm01731290_g1; Rb1, Mm00485586_m1; cdc2(cdk1), Mm01149140_m; Caspase 6, Mm00438053_m1; ORM2, Mm04213463_g1; Human: BAX, Hs00180269_m1; TP53, Hs01034249_m1; ORM2, Hs01037491_m1; CYP3A4, Hs00604506_m1; Gankyrin (PSMD10), Hs01100439_g1.

Techniques: Inhibition, Proliferation Assay, Control, Microscopy, Quantitative RT-PCR, Cytotoxicity Assay, Western Blot, TUNEL Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Small Molecule Cjoc42 Improves Chemo-Sensitivity and Increases Levels of Tumor Suppressor Proteins in Hepatoblastoma Cells and in Mice by Inhibiting Oncogene Gankyrin

doi: 10.3389/fphar.2021.580722

Figure Lengend Snippet: A combination therapy of doxorubicin and Cjoc42 enhances cytotoxicity. (A) Cytotoxicity Assay with doxorubicin (0.05, 0.1, and 0.2 µg/mL) and increasing concentrations of Cjoc42 (1, 5, and 10 µM) in Huh6 cells. (B) Cytotoxicity Assay with doxorubicin (0.1, 0.2, and 0.5 µg/mL) and increasing concentrations of Cjoc42 (1, 5, and 10 µM) in Hepa1c1c7 cells. (C) Microscope pictures at 20x showing reduction in size and number of clustered Huh6 cells when treated with doxorubicin 0.05 µg/mL and increasing concentrations of Cjoc42 compared to Cjoc42 or doxorubicin alone. (D) QRT-PCR data with Hepa1c1c7 cells treated with 1 µM Cjoc42 alone, Doxorubicin 0.1 µg/mL alone, or a combination of both. There was a statistically significant increase in expression of the apoptotic marker Casp6 with combination treatment ( p < 0.05) and the mature hepatocyte marker ORM2 ( p < 0.05). (E) Western blotting in Huh6 cells treated with Cjoc42 alone, doxorubicin 0.05 µg/mL, or a combination of doxorubicin and Cjoc42. (F) Bcl2 expression was calculated as a ratio to β-actin. Bcl2 is significantly higher in cells treated with doxorubicin 0.1 µg/mL and Cjoc42 5 µM compared to doxorubicin 0.05 µg/mL alone ( p < 0.03). (G) Co-IP studies. Gank was immunoprecipitated from untreated Huh6 and from Huh6 cells treated with doxorubicin (0.05 µg/mL) and cjoc42 (5 µM). Tumor suppressor proteins were examined in these IPs. (H) Western blotting shows levels of tumor suppressor proteins in nuclear extracts of untreated and doxorubicin + cjoc42 treated Huh6 cells.

Article Snippet: The following probes were used: CUGBP1 (CELF1), Mm04279608_m1; C/EBPα, Mm01265914_s1; HNF4α, Mm01247712_m1; TP53, Mm01731290_g1; Rb1, Mm00485586_m1; cdc2(cdk1), Mm01149140_m; Caspase 6, Mm00438053_m1; ORM2, Mm04213463_g1; Human: BAX, Hs00180269_m1; TP53, Hs01034249_m1; ORM2, Hs01037491_m1; CYP3A4, Hs00604506_m1; Gankyrin (PSMD10), Hs01100439_g1.

Techniques: Cytotoxicity Assay, Microscopy, Quantitative RT-PCR, Expressing, Marker, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation