Journal: Communications Biology

Article Title: A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

doi: 10.1038/s42003-023-04739-9

Figure Lengend Snippet: a Schematic overview of the procedure used to identify compounds that reduce apoB produced by human iPSC-hepatocytes. b Graph showing post-drug: pre-drug ratios (∆ [apoB]) in the medium of apoB in compound-treated compared to DMSO-treated iPSC-hepatocytes (% DMSO). The blue dots represent each individual compound from the SC 3 library 10 K representative set. c Graph showing the z score of each individual compound (blue dots) on the post-drug: pre-drug apoB ratio. Red dot is DL-1. Red line indicates the z-score at a value of –2.75 ( z = –2.75).

Article Snippet: For drug-mediated CYP induction studies, MPCCs (7 days old) were exposed to serum-free culture medium containing rifampin (25 µM), omeprazole (50 µM), or dimethylsulfoxide (DMSO) alone (0.1% v/v) (Corning).

Techniques: Produced

Journal: Communications Biology

Article Title: A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

doi: 10.1038/s42003-023-04739-9

Figure Lengend Snippet: a , b Forty-six hits (SC3-01 through SC3-46) from the initial screen were tested in human iPSC-derived hepatocytes at a concentration of 2 µg/mL. a ApoB levels in the medium were determined by sandwich ELISA assay. Bar graph shows the effect on accumulation of apoB over 24 h in the culture medium determined by comparing the post-drug and pre-drug apoB ratio normalized to DMSO-treated controls. b Cell viability was determined by ATP-based luminescence assay. c , d Compounds with a significant reduction in apoB without affecting cell viability were further tested in hoFH patient-derived iPSC-hepatocytes. c Bar graph showing the ratio of apoB in post-drug and pre-drug culture medium relative to DMSO controls. d Graph shows average cell viability relative to controls. All data are shown as mean ± SD of four replicates ( n = 4). Each individual compound was compared to vehicle DMSO only. Statistical analyses were performed using Student’s t test; * p ≤ 0.05, p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: For drug-mediated CYP induction studies, MPCCs (7 days old) were exposed to serum-free culture medium containing rifampin (25 µM), omeprazole (50 µM), or dimethylsulfoxide (DMSO) alone (0.1% v/v) (Corning).

Techniques: Derivative Assay, Concentration Assay, Sandwich ELISA, Luminescence Assay

Journal: Communications Biology

Article Title: A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

doi: 10.1038/s42003-023-04739-9

Figure Lengend Snippet: a – e Human iPSC-hepatocytes were treated with compounds SC3-01, -06, -19, -21, and -23 from 0.03125 to 16 µg/ml for 24 h. Culture medium from pre-drug and post-drug treatment were collected for human-specific apoB ELISA. Post-drug apoB: pre-drug apoB ratio from each treatment was determined and compared to DMSO-treated cells. All data are shown as mean ± SD of three replicates ( n = 3). f Human iPSC-hepatocytes were treated with 2 µg/ml of compound SC3-01, -19, -21, and -23 for 24 h. Bar graph shows ALB , AFP , HNF4α , SLC10A1 , and ASGR1 mRNA levels that were determined by real-time qPCR. Fold change was calculated and normalized with vehicle only ( n = 3). Data are shown as mean ± SD. All statistical analysis was performed using Student’s t test or one-way ANOVA followed by Dunnett’s test as appropriate; * p ≤ 0.05, ** p ≤ 0.01. g Graph showing the impact on in vitro MTP activity of DL-21 (0.5 and 1.0 µg/ml) and the known MTP inhibitor CP346086 (50 and 100 nM) compared to vehicle (DMSO). h Immunoblots showing the steady-state level of apoB protein in human iPSC-hepatocytes treated with vehicle (DMSO) or 2 µg/ml SC3-01 or the MTP inhibitor SC3-21. HSP90 levels were used as a loading control. i Human iPSC-hepatocytes were treated with 2 µg/ml of SC3-01, SC3-21, or vehicle (DMSO) for 24 h. Bar graph shows normalized steady-state APOB mRNA levels determined by real-time qPCR ( n = 4).

Article Snippet: For drug-mediated CYP induction studies, MPCCs (7 days old) were exposed to serum-free culture medium containing rifampin (25 µM), omeprazole (50 µM), or dimethylsulfoxide (DMSO) alone (0.1% v/v) (Corning).

Techniques: Enzyme-linked Immunosorbent Assay, In Vitro, Activity Assay, Western Blot

Journal: Communications Biology

Article Title: A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

doi: 10.1038/s42003-023-04739-9

Figure Lengend Snippet: a The structure and synthesis of the lead compound DL-1 (formerly SC3-01). N -phenyl-1,4-benzenediamine (1) was reacted with cyanuric chloride in the presence of anhydrous potassium carbonate to form the intermediate compound (2). The latter reacted with sodium hydrosulfide (NaSH) in dimethylformamide (DMF) to form compound (3), DL-1. b Human iPSC-hepatocytes from a healthy donor (K3) and hoFH patient (JD4) along with human primary hepatocytes and HepG2 cells were treated with 2 µg/ml of DL-1 and compared to vehicle-treated cells (DMSO; 100%) for 24 h. ApoB levels in the medium were determined by ELISA ( n = 3). c Graph showing the total cholesterol levels in the cell culture medium as determined by a cholesterol colorimetric assay. Pre:post treatment levels were compared to the DMSO-treated control group ( n = 7). d Human iPSC-hepatocytes were treated with DL-1 with concentrations ranging from 0.0325 to 48 µg/ml for 24 h. Albumin, apoA1, and apoB levels in the culture medium were determined by ELISA assay ( n = 3). e Cells were treated with 2 µg/ml of DL-1 from 8 to 48 h and pre-drug and post-drug medium was collected for analysis. The concentration of Albumin, apoA1, and apoB in the culture medium was determined by ELISA ( n = 3). Secreted protein levels were compared to DMSO-treated cells. Data are shown as mean ± SD. All statistical analysis was performed using Student’s t test or one-way ANOVA followed by Dunnett’s test as appropriate; * p < 0.05, ** p < 0.01.

Article Snippet: For drug-mediated CYP induction studies, MPCCs (7 days old) were exposed to serum-free culture medium containing rifampin (25 µM), omeprazole (50 µM), or dimethylsulfoxide (DMSO) alone (0.1% v/v) (Corning).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Colorimetric Assay, Concentration Assay

Journal: Communications Biology

Article Title: A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

doi: 10.1038/s42003-023-04739-9

Figure Lengend Snippet: Human iPSC-hepatocytes were treated with DL-1 (2 µg/mL), Lomitapide (20 nM), CP-346086 (20 nM), or vehicle (DMSO) for 72 h . a Hepatic neutral lipid was determined by staining with 2 µM of BODIPY 493/503 (green) for 15 mins and fixed with 4% PFA for microscopy. Nuclei were stained with DAPI (blue). Scale bar = 100 µm. b Bar graph representing the quantitative density measured by Keyence of neutral lipid staining. Fold change was determined by comparing to the DMSO-treated group. Data are shown as mean ± SD of five replicates ( n = 5). Statistical analyses were performed using one-way ANOVA followed by Dunnett’s test; *** p < 0.001.

Article Snippet: For drug-mediated CYP induction studies, MPCCs (7 days old) were exposed to serum-free culture medium containing rifampin (25 µM), omeprazole (50 µM), or dimethylsulfoxide (DMSO) alone (0.1% v/v) (Corning).

Techniques: Staining, Microscopy

Journal: Communications Biology

Article Title: A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

doi: 10.1038/s42003-023-04739-9

Figure Lengend Snippet: Compounds that were structurally similar to DL-1 that had higher than a 20% apoB reduction in the cluster compound activity assay were picked for dose-response analyses. Dose response curves of apoB levels in pre- and post-drug medium were determined by ELISA and post-drug: pre-drug apoB ratios were calculated and normalized using the DMSO-treated group. EC 50 for each compound was determined via Quest Graph™ EC 50 Calculator. Cell viability was determined using the CellTiter-Glo luminescence assay. Data presented as mean ± SD of four replicates ( n = 4). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For drug-mediated CYP induction studies, MPCCs (7 days old) were exposed to serum-free culture medium containing rifampin (25 µM), omeprazole (50 µM), or dimethylsulfoxide (DMSO) alone (0.1% v/v) (Corning).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Luminescence Assay

Journal: Communications Biology

Article Title: A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

doi: 10.1038/s42003-023-04739-9

Figure Lengend Snippet: a Graphs showing weight changes during the course of the experiment and serum levels of albumin, ALT, apoA1, apoB, Lp(a), total cholesterol, LDL/VLDL, and triglycerides in liver-humanized mice following 7 days of treatment with either vehicle (DMSO; n = 5) or DL-27 at 20 mg/kg/day ( n = 4). ELISA assays for albumin, apoA1, apoB, and Lp(a) were specific to human proteins and were normalized to human albumin levels to account for variation in the number of human hepatocytes present in each animal at the completion of dosing. Statistical analysis was performed using t tests; * p < 0.05, ** p < 0.01, *** p < 0.001. b Micrographs showing H&E and Oil Red O stained sections through livers of liver-humanized mice treated with vehicle (DMSO) or the triazine thiol DL-27. Quantification of Oil Red O across representative liver sections in each mouse used in a was determined using ImageJ and shown graphically as the cumulative area of Oil Red O staining. c Bar graphs showing the quantity of 12 different bile acids in the livers of the humanized mice treated with DMSO or DL-27. Levels were determined using LC/MS metabolomics and displayed as a log scale.

Article Snippet: For drug-mediated CYP induction studies, MPCCs (7 days old) were exposed to serum-free culture medium containing rifampin (25 µM), omeprazole (50 µM), or dimethylsulfoxide (DMSO) alone (0.1% v/v) (Corning).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: Oncotarget

Article Title: Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting

doi: 10.18632/oncotarget.17480

Figure Lengend Snippet: ( A ) Workflow of GBM sphere formation and clonogenic survival assay. ( B ) Relative sphere formation and basal surviving fraction of GBM stem-like cells (GS-8), patient-derived GBM cultures (DK32, DK41) and GBM cell lines (U343MG, DD-T4) upon treatment with AIIB2/SP600125 (EC10, EC50) or controls (IgG, DMSO). ( C ) Relative sphere formation and clonogenic survival upon treatment with AIIB2/SP600125 (EC10, EC50) and X-ray irradiation (2, 4, 6 Gy) (controls are IgG and DMSO). (B, C) Results are mean +/− SEM ( n = 3–4, t -test). ( D ) Western blot analysis of β1 integrin, phospho-cJun (S63), cJun and β-actin of whole cell lysates from indicated GBM cells treated as indicated with AIIB2 (10 μg/ml), SP600125 (EC10), IgG control (10 μg/ml) and DMSO. Fold change is calculated by normalization to β-actin and IgG/DMSO controls according to representative blots.

Article Snippet: Non-specific IgG isotype antibodies (10 μg/ml) or DMSO (equal amount as inhibitors, < 0.1 % v/v) (AppliChem) were used as control.

Techniques: Clonogenic Cell Survival Assay, Derivative Assay, Irradiation, Western Blot

Journal: Oncotarget

Article Title: Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting

doi: 10.18632/oncotarget.17480

Figure Lengend Snippet: ( A ) Work flow of 3D invasion assays. ( B ) Representative images of indicated GBM cells invading into 3D type I collagen 72 h after treatment with AIIB2/SP600125 (EC50) or control IgG/DMSO. Scale bar, 50 μm. ( C ) Invasion distance of indicated treated GBM cell populations. Results are mean +/− SEM ( n = 3, t -test).

Article Snippet: Non-specific IgG isotype antibodies (10 μg/ml) or DMSO (equal amount as inhibitors, < 0.1 % v/v) (AppliChem) were used as control.

Techniques:

Journal: Oncotarget

Article Title: Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting

doi: 10.18632/oncotarget.17480

Figure Lengend Snippet: ( A ) Treatment scheme of mice bearing tumors of GS-8_GFP/fLuc stem-like cells. ( B ) Left panel shows representative image of a mouse brain with GS-8_GFP/fLuc tumor. Scale bar, 2 mm. Region 1 and 2 corresponding to middle and right panel magnifications show invading tumor cells (IC) and blood vessel (BV) of GS-8_GFP/fLuc tumors, respectively. Scale bar, 100 μm. ( C ) Representative images from GS-8_GFP/fLuc tumors showing β1 integrin (green), phospho-JNK (T183/Y185) (red) or merge with nuclei (DAPI, blue). Scale bar, 100 μm. ( D ) Gadolinium-enhanced (Gd) T1-weighted magnetic resonance images (MRI) from mice bearing GS-8_GFP/fLuc tumors 24 days after transplantation and β1 integrin/JNK inhibition without or in combination with radiochemotherapy (RCT). Dashed lines delineate tumors. Lower images show luminescence analyses of representative GS-8_GFP/fLuc tumors 24 days after transplantation and indicated treatment. ( E ) GS-8_GFP/fLuc tumor growth time to 100 fold radiance after indicated treatment. Data are mean +/− SEM (6 - 8 mice per group, one-way ANOVA). ( F ) Survival of GS-8_GFP/fLuc mice treated as indicated. Kaplan Meier analysis includes 6 mice IgG/DMSO, 8 mice AIIB2/SP600125, 6 mice IgG/DMSO+RCT, 6 mice AIIB2/SP600125+RCT (two-sided log rank test, p < 0.009: IgG/DMSO+RCT vs IgG/DMSO, p < 0.02: AIIB2/SP600125+RCT vs IgG/DMSO+RCT).

Article Snippet: Non-specific IgG isotype antibodies (10 μg/ml) or DMSO (equal amount as inhibitors, < 0.1 % v/v) (AppliChem) were used as control.

Techniques: Transplantation Assay, Inhibition

Journal: Oncotarget

Article Title: Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting

doi: 10.18632/oncotarget.17480

Figure Lengend Snippet: ( A ) Hierarchical clustering of altered phosphorylation sites (30% decrease, 50% increase, fold change) from phosphoproteome analysis of U343MG cells 1 h after indicated treatment with AIIB2, JNKi or AIIB2/JNKi (EC10) normalized to controls (percentage of phosphorylation site changes is shown on the right). ( B ) Percentage of phospho-sites showing 30% decreased and 50% increased phosphorylation upon indicated treatment among the 606 phosphorylations sited investigated in the phosphoproteome analysis. ( C ) Venn diagram analysis of altered phosphosites from (A). ( D ) Functional classification of altered proteins after treatment with AIIB2/SP600125 (EC10) in comparison to the IgG/DMSO controls. Enrichment of genes in signaling pathway: # p < 0.01 (Fisher's exact test). ( E ) Cytoscape-based molecular interaction network of β1 integrin and JNK isoforms (black) with altered (grey) and not altered (white) cell cycle associated proteins. ( F ) Fold change in phosphorylation of cell cycle associated proteins. Threshold of phosphosite changes (30% decrease and 50% increase) is marked in grey.

Article Snippet: Non-specific IgG isotype antibodies (10 μg/ml) or DMSO (equal amount as inhibitors, < 0.1 % v/v) (AppliChem) were used as control.

Techniques: Functional Assay

Journal: Oncotarget

Article Title: Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting

doi: 10.18632/oncotarget.17480

Figure Lengend Snippet: ( A ) Western blot analysis of indicated proteins from whole cell lysates of U343MG and GS-8 cells treated with AIIB2/SP600125 (EC10) or control IgG/DMSO without and with X-ray irradiation (6 Gy). Fold changes are calculated by normalization to β-actin and IgG/DMSO controls according to representative blots. ( B ) Immunofluorescence analysis of nuclei with 53BP1-positive foci after indicated treatments. Scale bar, 10 μm. ( C ) Quantification of the number of DSB per cell at the indicated time points after treatment with AIIB2/SP600125 (EC10) or control IgG/DMSO without and with X-ray irradiation. ( D ) Basal surviving fraction of U343MG cells upon treatment with AIIB2/SP600125 (EC10), LBH589 (EC50) or a combination thereof compared to control treatment (IgG/DMSO). ( E ) Clonogenic radiation survival of U343MG cells treated as described in (H). (C–E) Results are mean +/− SEM ( n = 3, t -test).

Article Snippet: Non-specific IgG isotype antibodies (10 μg/ml) or DMSO (equal amount as inhibitors, < 0.1 % v/v) (AppliChem) were used as control.

Techniques: Western Blot, Irradiation, Immunofluorescence

Journal: Oncotarget

Article Title: Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting

doi: 10.18632/oncotarget.17480

Figure Lengend Snippet: ( A ) Western blot analysis of indicated proteins and phosphorylation sites from whole cell lysates of U343MG cells treated with AIIB2/SP600125 (EC10) or control IgG/DMSO without and with X-ray irradiation (6 Gy). Representative blots are shown. ( B ) Quantification of ATM (S1981) and Cdc25A (S216) phosphorylation and p53 expression shown in (A). Fold changes are calculated by normalization to β-actin and IgG/DMSO controls. Results are mean +/− SEM ( n = 3, t -test). ( C ) Flow cytometric cell cycle analysis (BrdU, propidium iodide) of U343MG cells 24 h after treatment with AIIB2/SP600125 (EC10) or control IgG/DMSO without and with X-ray irradiation (6 Gy). Representative dot blots are shown. ( D ) Quantification of flow cytometric analysis from (C) showing distribution of treated GBM cells into G1/G0, S and G2/M phase of the cell cycle. Results are mean +/− SEM ( n = 3, t -test).

Article Snippet: Non-specific IgG isotype antibodies (10 μg/ml) or DMSO (equal amount as inhibitors, < 0.1 % v/v) (AppliChem) were used as control.

Techniques: Western Blot, Irradiation, Expressing, Cell Cycle Assay