10 research outputs found
Homology model of BSEP.
<p>The amino acids affected by the E135K, R1050C, and R1153H missense mutations are represented by the red spheres. The residues affected by the L1099LfsX38 mutation are indicated by the grayed protein backbone. The two Walker motifs are highlighted with the orange backbone spheres. The transmembrane region is comprised between the two horizontal segments.</p
(A) Liver biopsy in case 8 at 11 years of age shows preserved architecture, (HE; 10x). Liver biopsy in case 9 at 6 years of age shows (B) mild intralobular intrahepatic cholestasis (HE, 40x) and (C) fibrosis (Masson trichrome 10x); (D) IHC with anti-BSEP shows strong and diffuse staining at the canalicular edge as in control liver (not shown) (20x); (E) IHC with anti-cytokeratin 7 highlights reactive bile ductules and phenotypically modulated hepatocytes (10x).
<p>(A) Liver biopsy in case 8 at 11 years of age shows preserved architecture, (HE; 10x). Liver biopsy in case 9 at 6 years of age shows (B) mild intralobular intrahepatic cholestasis (HE, 40x) and (C) fibrosis (Masson trichrome 10x); (D) IHC with anti-BSEP shows strong and diffuse staining at the canalicular edge as in control liver (not shown) (20x); (E) IHC with anti-cytokeratin 7 highlights reactive bile ductules and phenotypically modulated hepatocytes (10x).</p
Model of BSEP and detailed view around the site of the R1050C mutation.
<p>Shown as sticks are the residues that stabilize, via non-covalent interactions, the short helix contiguous to the 659–728 disordered region (red dotted line). Hydrogen bonds and salt-bridges are indicated by black dots between residues. The schematic positions of the three most C-terminal residues of the disordered peptide are indicated. These include Lys 726, the residue found to be ubiquitinated in the mouse BSEP.</p
ATX-101 induces apoptosis in the MM cell line JJN-3.
<p>(A–C) Flow cytometric measurement of the apoptotic cell population by annexin V-Pacific Blue labeling. (A) JJN-3 cells treated with 6 µM ATX-101 and 0.5 µM melphalan alone or combined were incubated for 1, 2, and 3 days. Control cells were left unexposed. (B and C) JJN-3 cells treated with 6 and 10 µM ATX-101 were incubated for 1, 2, and 4 h. In addition to annexin V labeling, cells were stained with DRAQ5 for DNA profile. (C) The histograms show the cell cycle distribution of live (blue) and apoptotic (pink) cells after 1 h of ATX-101 treatments. (A–C) show data from representative experiments out of three. (D) Flow cytometric measurement of caspase 8, 9, and 3/7 activity by Fluorescent Labeled Inhibitor of Caspases (FLICA) assay. JJN-3 cells were left unexposed and exposed to 8 µM ATX-101 for 2 and 4 h before the FLICA probe was added for staining. The FLICA probe binds irreversible only to the activated caspase and labels apoptotic cells. Data is from four independent experiments for caspase 8 activity and three independent experiments for caspase 9 and 3/7 activity (mean ± SD, ** P < 0.01, Student’s t-test).</p
APIM and PIP-box peptides have overlapping binding site on PCNA.
<p>(A) Protein sequence and structural model of PCNA (PDB entry 1vym) with M40 highlighted in red and the center loop (CL) in yellow (upper panel). Live cell (HeLa) confocal fluorescence images of CFP-PCNA wild type (WT) and CFP-PCNA M40 mutants. Bar, 5 µm (lower panel). (B) Normalized FRET (N<sub>FRET</sub>) measurements between WT and mutated CFP-PCNA M40/APIM-YFP (light grey diamonds, PCNA WT−/PCNA M40A−/PCNA M40N−/PCNA M40R−/PCNA M40S- APIM) and WT and mutated CFP-PCNA M40/PIP-YFP (dark grey diamonds, PCNA WT−/PCNA M40A−/PCNA M40N−/PCNA M40R/PCNA M40S- PIP). CFP/YFP (vectors only) was used as background control (open diamonds). Data is from three independent experiments (mean ± SEM, n = 72–214). P-values were calculated by the unpaired Student’s t-test.</p
ATX-101, a cell-penetrating APIM-peptide, targets PCNA.
<p>(A) Confocal fluorescence image of live HeLa cells 2 minutes after addition of fluorescently tagged ATX-101. Bar, 5 µm. (B) Cell growth measured by MTT assay of HeLa cells stably expressing YFP and APIM-(hABH2 <sub>1–7</sub> F4W)-YFP unexposed (♦ and×, respectively) and after continuous exposure to 0.5 µM cisplatin (▴ and •, respectively) (left panel) and parental HeLa cells unexposed (♦) and after continuous exposure to 8 µM ATX-101 (×), 0.5 µM cisplatin (▴), and combination of ATX-101 and cisplatin (•) (right panel). Data is from one representative experiment out of at least three. (C) Normalized FRET (N<sub>FRET</sub>) measurements in HeLa cells between CFP-PCNA and APIM-YFP without and in the presence of ATX-101. The cells were treated with 8 µM ATX-101 8 h after transient transfection and incubated for 16 h before the N<sub>FRET</sub> measurements. CFP/YFP (vectors only) was used as background control. Data is from three independent experiments (mean ± SEM, n = 36–40). P-value was calculated by the unpaired Student’s t-test. (D) Cell growth measured by MTT assay of HeLa cells unexposed (♦) and after continuous exposure to 8 µM ATX-A (—), 8 µM ATX-101 (×), 0.5 µM cisplatin (▴), and combination of ATX-A or ATX-101 and cisplatin (▪ and •, respectively). The confocal image shows fluorescently tagged ATX-A in HeLa cells as in (A). Bar, 5 µm. Data is from one representative experiment out of three.</p
ATX-101 inhibits cell growth of cancer cell lines.
<p>(A) Cell growth after ATX-101 addition in different cell lines measured by MTT assay. K562 (chronic myelogenous leukemia), CCRF-CEM (T-lymphoblast, acute lymphocytic leukemia), RPMI-8226 and JJN-3 (MM), HeLa (cervical cancer), PC3 and DU145 (prostate cancer), H460 (non-small cell lung carcinoma), HCT116 (colorectal carcinoma), A549 (non-small cell lung carcinoma), U2OS (osteosarcoma) and HaCaT (spontaneously immortalized keratinocyte) cells were left unexposed (♦) and exposed to 4, 6, 8, 10, and/or 12 µM of ATX-101 (▪, ▴, ×, —, and •, respectively). (B and C) Cell growth measured by MTT assay of the MM cell lines RPMI-8226 and JJN-3, respectively, unexposed (♦) and after continuous exposure to 6 or 4 µM of ATX-101 (×), 2 or 0.5 µM melphalan (▴), and combination of ATX-101 and melphalan (•). (A–C) Data is normalized to cell growth from untreated cells on day 1 and from one representative experiment out of at least three.</p
ATX-101 induces cancer cell specific apoptosis.
<p>Flow cytometric measurement of the apoptotic cell population by annexin V-Pacific Blue labeling. JJN-3 cells were treated with 4 and 8 µM ATX-101 for 2 h (left panel), and U937 cells were treated for 24 h (right panel). Lymphocytes freshly isolated from buffy coats (from blood donors) treated in parallel with JJN-3 and U937 are included as controls. Data is from representative experiments out of two.</p
Additional file 1: Table S1. of A mild form of adenylosuccinate lyase deficiency in absence of typical brain MRI features diagnosed by whole exome sequencing
Whole exome sequencing data output. (DOCX 12 kb