Journal: Oncotarget

Article Title: A novel pan-Nox inhibitor, APX-115, protects kidney injury in streptozotocin-induced diabetic mice: possible role of peroxisomal and mitochondrial biogenesis

doi: 10.18632/oncotarget.18540

Figure Lengend Snippet: Diabetes was induced in mice by intraperitoneal injection of STZ (50 mg/kg). Then APX-115 (60 mg/kg/day) or losartan (1.5 mg/kg/day) was administered orally for 12 weeks to diabetic mice. After 12 weeks, urine and blood samples were collected for analysis of ( A ) urinary albumin excretion, ( B ) albumin/creatinine ratio, ( C ) creatinine clearance rate, and ( D ) plasma cystatin C. ( E ) Kidneys were fixed in paraffin and cut into 3 μm sections that were subsequently stained with PAS reagent. Scale bar: 10 μm; original magnification: 630×. After PAS staining, ( F ) glomerular volume, ( G ) mesangial area, and ( H ) tuft area were analyzed using Image-Pro Plus 4.5.1. DM, STZ-induced diabetic mice. Data are presented as means ± SE of 10–12 mice/group; * p < 0.05 vs. control, † p < 0.05 vs. DM.

Article Snippet: A murine mesangial cell line (MES-13, cloned from mice transgenic for the early region of SV-40 virus, passage 25) was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Injection, Clinical Proteomics, Staining, Control

Journal: Oncotarget

Article Title: A novel pan-Nox inhibitor, APX-115, protects kidney injury in streptozotocin-induced diabetic mice: possible role of peroxisomal and mitochondrial biogenesis

doi: 10.18632/oncotarget.18540

Figure Lengend Snippet: ( A ) Plasma LPO, ( B ) urinary LPO, ( C ) kidney tissue LPO, ( D ) Nox1, ( E ) Nox2, and ( F ) Nox4 mRNA expression levels in kidneys were measured using real-time PCR. ( G and H ) Frozen kidney sections were stained with DHE at 5 µM (original magnification: 400×; scale bar: 20 μm). (A–H) Data are presented as means ± SE of 10–12 mice/group; * p < 0.05 vs. control, † p < 0.05 vs. DM. ( I ) Mesangial cells were incubated with or without APX-115 (1 µM) for 30 min and stimulated with or without 30 mM high glucose (HG) for 24 h followed by angII for 30 min. After that cells were incubated with 10 µM DCF-DA for 10 min and the fluorescence intensity was measured with a Zeiss vision system. Data are presented as means ± SE of at least 2 independent experiments; * p < 0.05 vs. control, † p < 0.05 vs. angII or angII+HG in DMSO.

Article Snippet: A murine mesangial cell line (MES-13, cloned from mice transgenic for the early region of SV-40 virus, passage 25) was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Clinical Proteomics, Expressing, Real-time Polymerase Chain Reaction, Staining, Control, Incubation, Fluorescence

Journal: Scientific Reports

Article Title: Pre-activation of mesenchymal stem cells with TNF-α, IL-1β and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury

doi: 10.1038/srep08718

Figure Lengend Snippet: (A) The experimental design is depicted for in vivo and in vitro experiments (drawn by Hao Chen using Coreldraw X5 and Photoshop CS3 software). For the in vivo experiments, adult Sprague-Dawley rats were not irradiated (control) or were irradiated (IR) with 14 Gy abdominal irradiation at day O, followed by a course of i.v. injection and continuous peritoneal infusion via Alzet pump with control medium (DMEM-F12) or conditioned medium from fibroblasts activated under radiation-induced inflammatory condition (FB-CM IEC-6(IR) ), non-activated MSCs (MSC-CM IEC-6(NOR) ), MSCs activated under radiation-induced inflammatory condition (MSC-CM IEC-6(IR) ) with or without neutralizing antibodies (NAs), MSCs activated by TNF-α, IL-1β and NO donor (MSC-CM TNF-α + IL-1β + NO ) and HO-1-knockdown MSCs activated by TNF-α, IL-1β and NO donor (HO1-KD-MSC-CM TNF-α + IL-1β + NO ). Rats were either sacrificed at 1, 3, 7 days for structural and functional examination or were monitored for survival status throughout the 14 day course of the experiment. For in vitro experiments, control rat intestinal epithelial IEC-6 cells or cells irradiated with 10 Gy were cultured for 7 days. In parallel, irradiated IEC-6 cells were co-cultured in six-well plates with unconcentrated MSC-CM IEC-6(NOR) or MSC-CM IEC-6(IR) with or without neutralizing antibodies, and the proliferation and apoptosis were evaluated every other day as shown. (B) Morphological features of BM-MSCs. (upper panel) Spindle-shaped morphology of primary BM-MSCs at day 3. (lower panel) More confluent BM-MSCs at passage 2. (C) Flow cytometric characterization of BM-MSCs. BM-MSCs (red) expressed CD29 (99.17 ± 1.36%) and CD44 (98.24 ± 1.01%) but not CD34 (2.79 ± 0.24%) and CD45 (1.69 ± 0.19%). (D) MSC-CM IEC-6(IR) improves the survival rates and lengthens the survival time of rats following 14 Gy abdominal irradiation. (left panel) Cumulative survival for irradiated rats infused with DMEM-F12, FB-CM IEC-6(IR) , MSC-CM IEC-6(NOR) or MSC-CM IEC-6(IR) was analyzed using the Kaplan-Meier method. The cumulated number of rats in each experimental group is presented in parenthesis. P -values were determined by log-rank testing. (right panel) Mean survival time. Data represent the mean ± SD. **, P < 0.05 versus IR + DMEM-F12 and IR + FB-CM IEC-6(IR) . ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) .

Article Snippet: Cell lines, including nontransformed rat intestinal epithelial IEC-6 cells (CRL-1592, passage 13) and primary rat fibroblast (FB) cells (CRL-1213), were obtained from the American Type Culture Collection.

Techniques: In Vivo, In Vitro, Software, Irradiation, Control, Injection, Knockdown, Functional Assay, Cell Culture

Journal: Scientific Reports

Article Title: Pre-activation of mesenchymal stem cells with TNF-α, IL-1β and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury

doi: 10.1038/srep08718

Figure Lengend Snippet: (A) Changes of intestinal electrolyte transport were measured by short-circuit current analysis (I sc ) in response to electrical filed stimulation (EFS). (left panel) I sc response was stimulated by increasing frequencies of EFS ranging from 1 ~ 25 Hz. (right panel) I sc response was studied 1, 3, 7 days after irradiation. Data are expressed as the change in I sc and are the mean ± SD for n = 6 animals. **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . (B) D-Xylose absorption tests were performed at 1, 3, 7 days after irradiation. Values represent means ± SD of n = 3 animals, **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . (C) Intestinal permeability, measured as plasma level of D-lactate, was measured at 1, 3, 7 days after irradiation. Values represent means ± SD of n = 3 animals, **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . (D) Histological analysis by H&E staining. Scale bars 100 μm. (E) Morphometric evaluation of epithelium thickness. Each value represents the average of 20 independent measurements per animal (six animals per group). **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . (F) The morphological changes of intestinal tight junction after radiation. The structures between the adjoining cells were observed under transmission electron microscopy. Arrows, tight junctions; bars, 500 nm. IR + DMEM-F12, irradiated rats receiving DMEM-F12; IR + FB-CM IEC-6(IR) , irradiated rats receiving FB-CM IEC-6(IR) ; IR + MSC-CM IEC-6(NOR) , irradiated rats receiving MSC-CM IEC-6(NOR) ; IR + MSC-CM IEC-6(IR) , irradiated rats receiving MSC-CM IEC-6(IR) .

Article Snippet: Cell lines, including nontransformed rat intestinal epithelial IEC-6 cells (CRL-1592, passage 13) and primary rat fibroblast (FB) cells (CRL-1213), were obtained from the American Type Culture Collection.

Techniques: Irradiation, Permeability, Clinical Proteomics, Staining, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Pre-activation of mesenchymal stem cells with TNF-α, IL-1β and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury

doi: 10.1038/srep08718

Figure Lengend Snippet: (A) Apoptosis was assayed by TUNEL staining 3 days after irradiation. Scale bars 50 μm. (B) Quantification of TUNEL-positive cells. n = 3 in each group. The number of positive cells in 5 crypts was scored in 100 crypts per section and reported as mean ± SD. *, P < 0.05 versus IR + DMEM-F12, #, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . (C) Apoptosis of irradiated IEC-6 was assayed by TUNEL staining 3 days after radiation. Scale bars 25 μm. (D) Quantification of TUNEL-positive IEC-6 cells. Data are reported as means ± SD for 10 random fields per wells from four replicate wells per group. **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . FOV, field of view. (E) Apoptosis and cell death of IEC-6 were evaluated quantitatively by flow cytometry after PI/Annexin V staining. The right lower quadrant (RLQ) contains early apoptotic cells, and the upper right quadrant (RUQ) contains late apoptotic and necrotic cells. The proportions of cells in RLQ + RUQ were as follows: Control: 14.21%; IR + DMEM-F12: 53.15%; IR + MSC-CM IEC-6(NOR) : 45.26%; IR + MSC-CM IEC-6(IR) : 22.64%. (F) The percentage of total apoptotic cells and dead cells under each condition from panel E are shown. (G) The ratio of apoptosis IEC-6 cells was determined by PI/Annexin V staining at 1, 3, 5, 7 days after radiation. Data represent means ± SD of three independent experiments. **, P < 0.05 versus IR + DMEM-F12.

Article Snippet: Cell lines, including nontransformed rat intestinal epithelial IEC-6 cells (CRL-1592, passage 13) and primary rat fibroblast (FB) cells (CRL-1213), were obtained from the American Type Culture Collection.

Techniques: TUNEL Assay, Staining, Irradiation, Flow Cytometry, Control

Journal: Scientific Reports

Article Title: Pre-activation of mesenchymal stem cells with TNF-α, IL-1β and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury

doi: 10.1038/srep08718

Figure Lengend Snippet: (A) The proliferation of intestinal epithelial cells was examined by immunohistochemical staining with proliferating cell nuclear antigen (PCNA). Intestinal tissue samples were collected and analyzed 3 days after radiation. Scale bars 50 μm. (B) Quantification of PCNA-positive cells. n = 3 in each group. The number of positive cells in 5 crypts was scored in 100 crypts per section and reported as mean ± SD. *, P < 0.05 versus IR + DMEM-F12, #, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . (C) Immunohistochemical staining of IEC-6 cells with PCNA 3 days after radiation. Scale bars 50 μm. (D) Quantification of PCNA-positive cells for the treatment groups in panel C. Data are reported as mean ± SD for 10 random fields per wells from four replicate wells per group. **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . FOV, field of view. (E) The cell cycle status of IEC-6 cells following IR and/or co-culture with MSC-CM IEC-6(NOR) or MSC-CM IEC-6(IR) were detected by flow cytometry based on DNA content. (F) The percentage of S phase cells and G1 phase cells for each culture condition was determined. The proportions of cells in S phase were as follows: Control: 29.76%; IR + DMEM-F12: 9.86%; IR + MSC-CM IEC-6(NOR) : 10.91%; IR + MSC-CM IEC-6(IR) : 32.89%. (G) The ratio of S phase cells was examined 1, 3, 5, 7 days after radiation. Data represent mean ± SD of three independent experiments. **, P < 0.05 versus IR + DMEM-F12.

Article Snippet: Cell lines, including nontransformed rat intestinal epithelial IEC-6 cells (CRL-1592, passage 13) and primary rat fibroblast (FB) cells (CRL-1213), were obtained from the American Type Culture Collection.

Techniques: Immunohistochemical staining, Staining, Co-Culture Assay, Flow Cytometry, Control

Journal: Scientific Reports

Article Title: Pre-activation of mesenchymal stem cells with TNF-α, IL-1β and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury

doi: 10.1038/srep08718

Figure Lengend Snippet: (A) Immunohistochemical staining with Lgr5. Intestinal tissue samples were collected and analyzed 3 days after radiation. Scale bars 25 μm. (B) Quantification of Lgr5-positive cells. n = 3 in each group. The number of positive cells in 5 crypts was scored in 100 crypts per section and reported as mean ± SD. **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . (C) The protein levels of Lgr5 in the jejunal mucosa of rats were detected by Western blot assays at 1, 3, 5, 7 days after radiation with β-actin as the internal control. The full-length blots are presented in . (D) Lgr5 mRNA expression in the jejunal mucosa of rats after radiation was evaluated by quantitative real-time RT-PCR. β-actin was used as a loading control. Values represent means ± SD; n = 3 in each group. *, P < 0.05 versus IR + DMEM-F12, #, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . (E) Immunohistochemical staining with Bmi1. Intestinal tissue samples were collected and analyzed 3 days after radiation. Scale bars 25 μm. (F) Quantification of Bmi1-positive cells. n = 3 in each group. The number of positive cells in 5 crypts was scored in 100 crypts per section and reported as mean ± SD. **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) .

Article Snippet: Cell lines, including nontransformed rat intestinal epithelial IEC-6 cells (CRL-1592, passage 13) and primary rat fibroblast (FB) cells (CRL-1213), were obtained from the American Type Culture Collection.

Techniques: Immunohistochemical staining, Staining, Western Blot, Control, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Pre-activation of mesenchymal stem cells with TNF-α, IL-1β and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury

doi: 10.1038/srep08718

Figure Lengend Snippet: (A) The levels of pro/anti-inflammatory cytokines in jejunal protein extracts 3 days after radiation were determined by enzyme-linked immunosorbent assay (ELISA). n = 4 rats/group. **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . (B) ELISA assays for serum pro/anti-inflammatory cytokines in rats of the 4 treatment groups are shown. Serum samples were collected 3 days after radiation. n = 4 rats/group, **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) . (C) The percentages of CD4 + Foxp3 + Treg cells in the CD4 + population of mesenteric lymph nodes (MLNs) were determined by flow cytometry (4–5 rats/group). Tissue samples were collected 3 days after radiation. **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM IEC-6(NOR) .

Article Snippet: Cell lines, including nontransformed rat intestinal epithelial IEC-6 cells (CRL-1592, passage 13) and primary rat fibroblast (FB) cells (CRL-1213), were obtained from the American Type Culture Collection.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Pre-activation of mesenchymal stem cells with TNF-α, IL-1β and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury

doi: 10.1038/srep08718

Figure Lengend Snippet: (A) The specific chemical composition of MSC-CM with or without inflammatory stimulation. (B) Identification of fifteen cytokines of MSC-CM IEC-6(IR) that are potentially beneficial to intestinal recovery. Fresh medium without cell culture (DMEM-F12) was used as a background control. (upper panel) Representative images of the cytokine antibody array are shown. The rectangles highlight cytokines higher in MSC-CM IEC-6(IR) than FB-CM IEC-6(IR) and MSC-CM IEC-6(NOR) (fold change > 2.0). Each sample consisted of a pool of unconcentrated conditioned medium from five different donor cells. Each measurement was duplicated reproducibly. (lower panel) The graphs show the relative intensity of the 15 cytokines. The expression in the control medium was arbitrarily set as 1.0. (C) The same unconcentrated conditioned medium was assayed by ELISA for six selected cytokines (bFGF, VEGF, IL-10, IGF-1, HGF and TGF-β1). (D) The same selected cytokines were further confirmed by Real-time RT-PCR. β-actin was used as a loading control. The results represent 3 independent experiments (mean ± SD), **, P < 0.05 versus MSC IEC-6(NOR) . (E) Neutralization of IGF-1 in MSC-CM IEC-6(IR) partially suppressed the beneficial effects of MSC-CM IEC-6(IR) on apoptosis (upper left panel) and proliferation (upper right panel) of irradiated IEC-6. The apoptosis and proliferation were evaluated by quantification of TUNEL-positive and PCNA-positive IEC-6 cells 3 days after radiation, respectively. Moreover, in vivo , the induction of CD4 + Foxp3 + Treg cells in MLNs (lower left panel) and the regulation of the pro-/anti-inflammatory cytokine balance in small intestine (lower right panel) were partially reversed by antibodies against IGF-1 3 days after radiation. n = 3 in each group. **, P < 0.05 versus IR + MSC-CM IEC-6(IR) . (F) Cumulative survival analyzed using the Kaplan-Meier method. P -values were determined by log-rank testing. (G) The effects of of IGF-1 on the apoptosis (left panel) and proliferation (right panel) of irradiated intestinal epithelial cells evaluated by quantification of TUNEL-positive and PCNA-positive cells in histological sections, respectively. *, P < 0.05 versus IR + DMEM-F12, #, P < 0.05 versus IR + MSC-CM IEC-6(IR) . (H) Cumulative survival analyzed using the Kaplan-Meier method. P -values were determined by log-rank testing.

Article Snippet: Cell lines, including nontransformed rat intestinal epithelial IEC-6 cells (CRL-1592, passage 13) and primary rat fibroblast (FB) cells (CRL-1213), were obtained from the American Type Culture Collection.

Techniques: Cell Culture, Control, Ab Array, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Neutralization, Irradiation, TUNEL Assay, In Vivo

Journal: Scientific Reports

Article Title: Pre-activation of mesenchymal stem cells with TNF-α, IL-1β and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury

doi: 10.1038/srep08718

Figure Lengend Snippet: (A) TNF-α and IL-1β levels were assayed in the cultured medium of MSCs + non-irradiated/irradiated IEC-6 co-cultured system by ELISA. Nitric oxide (NO) in culture medium was assayed by a colorimetric assay for nitrite, a stable byproduct of NO. **, P < 0.05 versus control. (B) MSCs were supplemented with individual recombinant TNF-α (6 ng/ml), IL-1β (3 ng/ml), NO donor (SNP, sodium nitroprusside; 200 μmol/l) or combinations, and the concentration of IGF-1 in conditioned medium was then assessed. **, P > 0.05 versus MSC-CM IR . (C) MSCs + irradiated IEC-6 co-cultured system were supplemented with individual neutralizing antibodies for TNF-α (2 μg/ml), IL-1β (2 μg/ml), NO scavenger (PTIO; 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; 400 μmol/l) or combinations, and the concentration of IGF-1 in conditioned medium was then assessed. **, P < 0.05 versus MSC-CM IEC-6(IR) . MSCs were transfected with varying doses of HO-1 siRNA and nonspecific siRNA prior to TNF-α, IL-1β and NO donor stimulation, the protein levels of HO-1 (D), the secretion (E) and the mRNA expression (F) of IGF-1, bFGF, VEGF and TGF-β1 from MSCs were then detected. **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus MSC-CM TNF-α + IL-1β + NO . NS-KD-MSC-CM TNF-α + IL-1β + NO , conditioned medium from non-specific siRNA transfected MSCs activated by TNF-α, IL-1β and NO donor. Administration of HO1-KD-MSC-CM TNF-α + IL-1β + NO partially abolished the ability of MSC-CM TNF-α + IL-1β + NO to ameliorated the apoptosis (G) and proliferation (H) of irradiated intestinal epithelial cells which were evaluated by quantification of TUNEL-positive and PCNA-positive cells in histological sections, respectively. **, P < 0.05 versus IR + MSC-CM TNF-α + IL-1β + NO . The DNA-binding activities of NF-κB in nuclear extracts of intestinal tissue (I) were estimated by electrophoretic mobility shift assay (EMSA). The specificity of the DNA/protein was determined by competition reactions in which a 100-fold molar excess of unlabeled NF-κB oligonucleotide (specific competitor). (J) Improved survival of irradiated rats receiving MSC-CM TNF-α + IL-1β + NO (n = 28) was partially reversed in rats (n = 30) receiving HO1-KD-MSC-CM TNF-α + IL-1β + NO (IR + MSC-CM TNF-α + IL-1β + NO versus IR + HO1-KD-MSC-CM TNF-α + IL-1β + NO : P < 0.05).

Article Snippet: Cell lines, including nontransformed rat intestinal epithelial IEC-6 cells (CRL-1592, passage 13) and primary rat fibroblast (FB) cells (CRL-1213), were obtained from the American Type Culture Collection.

Techniques: Cell Culture, Irradiation, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Control, Recombinant, Concentration Assay, Transfection, Expressing, TUNEL Assay, Binding Assay, Electrophoretic Mobility Shift Assay

Journal:

Article Title: Persistent Infection of Human Oligodendrocytic and Neuroglial Cell Lines by Human Coronavirus 229E

doi:

Figure Lengend Snippet: Detection of HuCV antigens by indirect immunofluorescence on cells acutely infected by 229E strain, using 229E-specific MAb (5-11H.6). (A) H4 cells; (B) SK-N-SH cells; (C) U-373 MG cells; (D) U-87 MG cells; (E) GL-15 cells; (F) MO3.13 cells; (G) CHME-5 cells; (H) L-132 cells.

Article Snippet: Thus, we conclude that these isolated point mutations were probably induced by the RT-PCR and not by the persistent infection. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Cell line(s) Infection Position of point mutation a Codon change Amino acid change L-132, H4, and MO3.13 Acute and persistent 727 a b TGT→TTT C→F L-132 Persistent (passage 42) 571 a ACT→ATT T→I H4 Persistent (passage 48) MO3.13 Persistent (passage 40) 571 a ACT→ATT T→I 592 a GCA→GTA A→V Open in a separate window a Reference 55 . b Reference 28a ; ATCC strain.

Techniques: Immunofluorescence, Infection

Journal:

Article Title: Persistent Infection of Human Oligodendrocytic and Neuroglial Cell Lines by Human Coronavirus 229E

doi:

Figure Lengend Snippet: Susceptibility of neural cell lines to acute and persistent infection by HuCV 229E

Article Snippet: Thus, we conclude that these isolated point mutations were probably induced by the RT-PCR and not by the persistent infection. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Cell line(s) Infection Position of point mutation a Codon change Amino acid change L-132, H4, and MO3.13 Acute and persistent 727 a b TGT→TTT C→F L-132 Persistent (passage 42) 571 a ACT→ATT T→I H4 Persistent (passage 48) MO3.13 Persistent (passage 40) 571 a ACT→ATT T→I 592 a GCA→GTA A→V Open in a separate window a Reference 55 . b Reference 28a ; ATCC strain.

Techniques: Infection, Marker, Transfection

Journal:

Article Title: Persistent Infection of Human Oligodendrocytic and Neuroglial Cell Lines by Human Coronavirus 229E

doi:

Figure Lengend Snippet: Detection of HuCV antigens by indirect immunofluorescence on cells persistently infected by 229E strain, using 229E-specific MAb (5-11H.6). (A) H4 cells, passage 37; (B) MO3.13 cells, passage 40; (C) L-132 cells, passage 40.

Article Snippet: Thus, we conclude that these isolated point mutations were probably induced by the RT-PCR and not by the persistent infection. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Cell line(s) Infection Position of point mutation a Codon change Amino acid change L-132, H4, and MO3.13 Acute and persistent 727 a b TGT→TTT C→F L-132 Persistent (passage 42) 571 a ACT→ATT T→I H4 Persistent (passage 48) MO3.13 Persistent (passage 40) 571 a ACT→ATT T→I 592 a GCA→GTA A→V Open in a separate window a Reference 55 . b Reference 28a ; ATCC strain.

Techniques: Immunofluorescence, Infection

Journal:

Article Title: Persistent Infection of Human Oligodendrocytic and Neuroglial Cell Lines by Human Coronavirus 229E

doi:

Figure Lengend Snippet: Yield of infectious virions from persistent HuCV-229E infections of different cell lines. (A) H4 cells; (B) MO3.13 cells; (C) L-132 cells. Solid lines, supernatant (extracellular virus); dotted lines, cell lysate (intracellular virus).

Article Snippet: Thus, we conclude that these isolated point mutations were probably induced by the RT-PCR and not by the persistent infection. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Cell line(s) Infection Position of point mutation a Codon change Amino acid change L-132, H4, and MO3.13 Acute and persistent 727 a b TGT→TTT C→F L-132 Persistent (passage 42) 571 a ACT→ATT T→I H4 Persistent (passage 48) MO3.13 Persistent (passage 40) 571 a ACT→ATT T→I 592 a GCA→GTA A→V Open in a separate window a Reference 55 . b Reference 28a ; ATCC strain.

Techniques:

Journal:

Article Title: Persistent Infection of Human Oligodendrocytic and Neuroglial Cell Lines by Human Coronavirus 229E

doi:

Figure Lengend Snippet: Detection of the N protein gene by RT-PCR during persistent HuCV-229E infection of various cell lines. One tenth of the PCR amplicon obtained was loaded on a 1.5% (wt/vol) agarose gel for electrophoretic separation. (A) H4 cells; (B) MO3.13 cells; (C) L-132 cells. Lanes: N, noninfected cells; A, acutely infected cells; -, negative control for reverse transcriptase and for PCR; numbers, cell passages.

Article Snippet: Thus, we conclude that these isolated point mutations were probably induced by the RT-PCR and not by the persistent infection. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Cell line(s) Infection Position of point mutation a Codon change Amino acid change L-132, H4, and MO3.13 Acute and persistent 727 a b TGT→TTT C→F L-132 Persistent (passage 42) 571 a ACT→ATT T→I H4 Persistent (passage 48) MO3.13 Persistent (passage 40) 571 a ACT→ATT T→I 592 a GCA→GTA A→V Open in a separate window a Reference 55 . b Reference 28a ; ATCC strain.

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Amplification, Agarose Gel Electrophoresis, Negative Control

Journal:

Article Title: Persistent Infection of Human Oligodendrocytic and Neuroglial Cell Lines by Human Coronavirus 229E

doi:

Figure Lengend Snippet: Characterization of sequence changes detected in the S1 region of the surface glycoprotein during persistent HuCV-229E infection

Article Snippet: Thus, we conclude that these isolated point mutations were probably induced by the RT-PCR and not by the persistent infection. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Cell line(s) Infection Position of point mutation a Codon change Amino acid change L-132, H4, and MO3.13 Acute and persistent 727 a b TGT→TTT C→F L-132 Persistent (passage 42) 571 a ACT→ATT T→I H4 Persistent (passage 48) MO3.13 Persistent (passage 40) 571 a ACT→ATT T→I 592 a GCA→GTA A→V Open in a separate window a Reference 55 . b Reference 28a ; ATCC strain.

Techniques: Sequencing, Infection, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Epithelial Cholesterol Deficiency Attenuates Human Antigen R-linked Pro-inflammatory Stimulation via an SREBP2-linked Circuit *

doi: 10.1074/jbc.M116.723973

Figure Lengend Snippet: SREBP2 expression in the inflamed colon tissue. A, colonic tissue from 6-week-old female mice exposed to vehicle or DSS was subjected to IHC analysis for SREBP2 (n = 6–8, each group) (original magnification, ×200; black scale bar, 0.2 μm). The right panel shows quantitative analysis of tissue SREBP2 staining in control or DSS-treated mice. ***, significant difference from the control group (p < 0.001). B, enterocyte SREBP2 expression in colonic tissues from female mice exposed to vehicle or DSS. C, colonic tissue from female mice exposed to vehicle or DSS was subjected to Filipin staining (n = 15). The bottom box shows quantitative analysis of tissue Filipin staining in control or DSS-treated mice. *, significant difference from the control group (p < 0.05). D, total cholesterol (TC), HDL cholesterol (HDL-C), or LDL cholesterol (LDL-C) contents were measured in serum in female mice exposed to vehicle or DSS. Asterisks indicate a significant difference from the control group (**, p < 0.01; *, p < 0.05). E, the normal appearing regions and the inflamed lesions in colon tissues from patients with Crohn's disease (n = 8, each group) were stained for SREBP2 (original magnification, ×200; black scale bar, 0.2 μm). The right panel shows the relative amount of SREBP2 staining. ***, significant difference from the normally appearing parts (p < 0.001). F–I, HCT-8 cells transfected with the control vector or with shSREPB2 were treated with vehicle, TNF-α (10 ng/ml), or IL-1β (10 ng/ml) for 1 h. IL-8 (F and G) and CXCL-1 mRNA (H and I) mRNA were quantified. *, significant difference between two groups (p < 0.05). Error bars, S.D.

Article Snippet: Cell Culture Conditions and Reagents Intestinal cancer cell lines, including HCT-8 (passage 13), HCT-116 (passage 32), and intestine 407 (passage 10), were purchased from the ATCC (Manassas, VA).

Techniques: Expressing, Staining, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Epithelial Cholesterol Deficiency Attenuates Human Antigen R-linked Pro-inflammatory Stimulation via an SREBP2-linked Circuit *

doi: 10.1074/jbc.M116.723973

Figure Lengend Snippet: Cholesterol depletion-induced chemokine and SREBP2 expression in enterocytes. A, HCT-8 cells were treated with vehicle or 1 mm MβCD for the indicated times, and mRNA was quantified. * or #, significant differences from each 0 h control group (p < 0.05). B, HCT-8 cells were treated with vehicle or 20 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 4 h. Intracellular total cholesterol (TC), free cholesterol (FC), or cholesteryl ester (CE) contents were measured by a fluorometric assay. Different letters (a–c) over each bar represent significant differences between groups (p < 0.05). C, HCT-8 cells were treated with vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 4 h, and mRNA was quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). D, HCT-8 cells were treated with vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 24 h to assess IL-8 secretion using ELISA. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). E and F, intestine 407 (E) and HCT-116 (F) cells were treated with the vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 4 h, and mRNA was quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). G, the isolated mouse enterocytes were treated with vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 4 h, and mRNA was quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). H, HCT-8 cells were treated with the vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 1 h for protein analysis. I, HCT-8 cells were transfected with control or shSREBP2. Intracellular total cholesterol (TC), free cholesterol (FC), or cholesteryl ester (CE) contents were measured by a fluorometric assay. Asterisks indicate significant differences between control and shSREBP2-transfected cells (*, p < 0.05; **, p < 0.01). J, HCT-8 cells transfected with the control vector or shSREBP2 were exposed to the vehicle or 1 mm MβCD for 4 h. IL-8 and CXCL-1 mRNA were quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). K, HCT-8 cells transfected with the control vector or SREBP2-FLAG were exposed to the vehicle or 1 mm MβCD for 4 h. IL-8 and CXCL-1 mRNA were quantified. Different letters (a–c) over each bar represent significant differences between groups (p < 0.05). L, HCT-8 cells transfected with the control vector or SREBP2-FLAG were exposed to the vehicle or 1 mm MβCD for 24 h to assess IL-8 secretion using ELISA. Different letters (a–c) over each bar represent significant differences between groups (p < 0.05). A–J, all results are representative of three independent experiments. Error bars, S.D.

Article Snippet: Cell Culture Conditions and Reagents Intestinal cancer cell lines, including HCT-8 (passage 13), HCT-116 (passage 32), and intestine 407 (passage 10), were purchased from the ATCC (Manassas, VA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Isolation, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Epithelial Cholesterol Deficiency Attenuates Human Antigen R-linked Pro-inflammatory Stimulation via an SREBP2-linked Circuit *

doi: 10.1074/jbc.M116.723973

Figure Lengend Snippet: Effects of SREBP2 on PPARγ expression in cholesterol-depleted cells. A, HCT-8 cells transfected with control vector or shSREBP2 were treated with vehicle or 1 mm MβCD for 2 h, and mRNA was quantified. Different letters (a–c) over each bar represent significant differences between groups (p < 0.05). B, the control vector- or shSREBP2-expressing HCT-8 cells were transfected with 3× PPRE-TK luciferase plasmid and were treated with vehicle or 1 mm MβCD for 24 h for the luciferase assay. Different letters (a–c) over each bar represent significant differences between groups (p < 0.05). C, HCT-8 cells were treated with vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 2 h, and mRNA was quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). D and E, HCT-8 cells transfected with the control vector or PPARγ DN were treated with the vehicle or 1 mm MβCD for 4 h. IL-8 (D) and CXCL-1 (E) mRNA were quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). F and G, HCT-8 cells transfected with the control vector or PPARγ WT were treated with the vehicle or 1 mm MβCD for 4 h. IL-8 (F) and CXCL-1 (G) mRNA were quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). H and I, HCT-8 cells transfected with the control vector, shPXR (H), or shFXR (I) were treated with vehicle or 1 mm MβCD for 4 h, and mRNA was quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). Error bars, S.D.

Article Snippet: Cell Culture Conditions and Reagents Intestinal cancer cell lines, including HCT-8 (passage 13), HCT-116 (passage 32), and intestine 407 (passage 10), were purchased from the ATCC (Manassas, VA).

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Epithelial Cholesterol Deficiency Attenuates Human Antigen R-linked Pro-inflammatory Stimulation via an SREBP2-linked Circuit *

doi: 10.1074/jbc.M116.723973

Figure Lengend Snippet: Effects of SREBP2 on expression of EGR-1, an intermediator of PPARγ expression in cholesterol-depleted cells. A, HCT-8 cells were treated with vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 1 h for the protein analysis. B, HCT-8 cells were treated with vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 30 min, and mRNA was quantified. Different letters (a–c) over each bar represent significant differences between groups (p < 0.05). C, HCT-8 cells transfected with control vector or shEGR-1 were exposed to vehicle or 1 mm MβCD for 2 h, and mRNA was quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). D, HCT-8 cells transfected with the control vector or shEGR-1 were treated with vehicle or 1 mm MβCD for 2 h for the ChIP assay using anti-EGR-1 antibody. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). E, the control or shEGR-1-expressing HCT-8 cells were transfected with 3× PPRE-TK luciferase plasmid and were treated with vehicle or 1 mm MβCD for 24 h for the luciferase assay. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). F, HCT-8 cells transfected with the control vector or shSREBP2 were treated with vehicle or 1 mm MβCD for the indicated time for protein analysis. G, control or shSREBP2-expressing HCT-8 cells were transfected with human EGR-1 reporter plasmid and treated with vehicle or 1 mm MβCD for 6 h for the luciferase assay. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). H, the control vector- or shSREBP2-expressing HCT-8 cells were treated with vehicle or 1 mm MβCD for 30 min for the ChIP assay using anti-SREBP2 antibody. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). I and J, the control vector- or shEGR-1-expressing HCT-8 cells were treated with vehicle or 1 mm MβCD for 4 h. IL-8 (I) and CXCL-1 (J) mRNA were quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). All results are representative of three independent experiments. Error bars, S.D.

Article Snippet: Cell Culture Conditions and Reagents Intestinal cancer cell lines, including HCT-8 (passage 13), HCT-116 (passage 32), and intestine 407 (passage 10), were purchased from the ATCC (Manassas, VA).

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Epithelial Cholesterol Deficiency Attenuates Human Antigen R-linked Pro-inflammatory Stimulation via an SREBP2-linked Circuit *

doi: 10.1074/jbc.M116.723973

Figure Lengend Snippet: Roles of HuR in cholesterol depletion-mediated stabilization of pro-inflammatory chemokines. A and B, HCT-8 cells were exposed to vehicle or 1 mm MβCD for 4 h. Cellular transcription was then arrested by adding 5 μm actinomycin D. IL-8 (A) and CXCL-1 (B) mRNA were quantified. *, significant differences from the vehicle group at each indicated time (p < 0.05). C and D, the control vector- or shHuR-expressing HCT-8 cells were treated with vehicle or 1 mm MβCD for 4 h. IL-8 (C) and CXCL-1 (D) mRNA were quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). E, HCT-8 cells were treated with 1 mm MβCD for the indicated times. Cytosolic and nuclear fractions of cell lysates were subjected to Western blotting analysis. F, HCT-8 cells were treated with vehicle or 1 mm MβCD for 4 h. HuR protein was visualized with anti-HuR antibody (top). Bottom, relative quantitative values of cytosolic HuR. The ratio indicates the measured nuclear densities of HuR signal in the cytoplasm outside of the DAPI-stained area. ***, significant differences compared with the vehicle group (p < 0.001). G, HCT-8 cells were treated with vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 4 h. Cytosolic and nuclear proteins were analyzed. H and I, control vector- or shHuR-expressing HCT-8 cells were treated with 1 mm MβCD for 4 h. Cellular transcription was then arrested by adding 5 μm actinomycin D. IL-8 (H) and CXCL-1 (I) mRNA were quantified. *, significant difference from the vehicle group at each indicated time (p < 0.05). J, a plasmid containing a CMV promoter-linked reporter gene tagged with the 3′-UTR of the human IL-8 gene was constructed (top). The control or shHuR-expressing HCT-8 cells transfected with the reporter plasmid were treated with vehicle or 1 mm MβCD for 12 h, followed by measurement of luciferase activity (bottom). Different letters (a–c) over each bar represent significant differences between groups (p < 0.05). K and L, HCT-8 cells transfected with the control vector or shHuR were treated with vehicle or 1 mm MβCD for 4 h. RNA-bound HuR was immunoprecipitated, and the levels of HuR-bound IL-8 (K) and CXCL-1 (L) mRNA were quantified. Different letters (a–f) over each bar represent significant differences between groups (p < 0.05). n.s., not significant (A–L). All results are representative of three independent experiments. Error bars, S.D.

Article Snippet: Cell Culture Conditions and Reagents Intestinal cancer cell lines, including HCT-8 (passage 13), HCT-116 (passage 32), and intestine 407 (passage 10), were purchased from the ATCC (Manassas, VA).

Techniques: Plasmid Preparation, Expressing, Western Blot, Staining, Construct, Transfection, Luciferase, Activity Assay, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Epithelial Cholesterol Deficiency Attenuates Human Antigen R-linked Pro-inflammatory Stimulation via an SREBP2-linked Circuit *

doi: 10.1074/jbc.M116.723973

Figure Lengend Snippet: Effect of cholesterol-depleted activated SREBP2-linked signals on HuR translocation and subsequent binding to chemokine transcripts. A, the control vector-, shHuR-, shSREBP2-, shEGR-1- or PPARγ DN-transfected HCT-8 cells were treated with vehicle or 1 mm MβCD for 24 h. IL-8 secreted into the culture medium was quantified using ELISA. Different letters (a–h) over each bar represent significant differences between groups (p < 0.05). B, the control vector-, shSREBP2-, shEGR-1-, or PPARγ DN-transfected HCT-8 cells were treated with vehicle or 1 mm MβCD for 4 h. Cytosolic and nuclear fractions of cell lysates were subjected to Western blotting analysis. C, the control or PPARγ WT-transfected HCT-8 cells were treated with vehicle or 1 mm MβCD for 4 h. Cytosolic and nuclear fractions of cell lysates were subjected to Western blotting analysis. Cellular nuclear lysates were also immunoprecipitated with anti-FLAG antibody. The precipitated samples were subjected to Western blotting analysis. D and E, HCT-8 cells transfected with control vector, shPXR (D), or shFXR (E) were treated with vehicle or 1 mm MβCD for 4 h. Cytosolic and nuclear fractions of cell lysates were subjected to Western blotting analysis. F, the control, shSREBP2, shEGR-1, and PPARγ DN-transfected HCT-8 cells were treated with vehicle or 1 mm MβCD for 4 h. HuR protein and the nuclear regions were visualized using anti-HuR antibody and DAPI, respectively. The box graph shows the relative quantitative values of cytosolic HuR. The ratio is the measured densities of HuR signals outside of the DAPI-stained area. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). G and H, HCT-8 cells transfected with the control vector, shSREBP2, shEGR-1, or PPARγ DN were treated with vehicle or 1 mm MβCD for 4 h. RNA-bound HuR was immunoprecipitated, and the levels of HuR-bound transcripts of IL-8 (G) or CXCL-1 (H) mRNA were quantified. Different letters (a–g) over each bar represent significant differences between groups (p < 0.05). The box graph shows levels of cytosolic HuR protein that were analyzed using Western blotting analysis. All results are representative of three independent experiments. Error bars, S.D.

Article Snippet: Cell Culture Conditions and Reagents Intestinal cancer cell lines, including HCT-8 (passage 13), HCT-116 (passage 32), and intestine 407 (passage 10), were purchased from the ATCC (Manassas, VA).

Techniques: Translocation Assay, Binding Assay, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Immunoprecipitation, Staining

Journal: The Journal of Biological Chemistry

Article Title: Epithelial Cholesterol Deficiency Attenuates Human Antigen R-linked Pro-inflammatory Stimulation via an SREBP2-linked Circuit *

doi: 10.1074/jbc.M116.723973

Figure Lengend Snippet: Cholesterol depletion regulates HuR-mediated chemokine induction. A and B, HCT-8 cells were pre-exposed to vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 18 h. The cells were then treated with vehicle, 500 ng/ml DON, or 50 ng/ml ANS for 1 h. IL-8 (A) and CXCL-1 mRNA (B) mRNA were quantified. Different letters (a–f) over each bar represent significant differences between groups (p < 0.05). C and D, HCT-8 cells transfected with the control vector or shHuR were treated with vehicle, 500 ng/ml DON, or 50 ng/ml ANS for 1 h. IL-8 (C) and CXCL-1 (D) mRNA were quantified. Different letters (a–d) over each bar represent significant differences between groups (p < 0.05). E, HCT-8 cells were pretreated with vehicle or 1 mm MβCD in the absence or presence of 20 μg/ml cholesterol for 18 h. The cells were then treated with vehicle, 500 ng/ml DON, or 50 ng/ml ANS for 1 h. Cytosolic and nuclear fractions of cell lysate were subjected to Western blotting analysis. F and G, HCT-8 cells transfected with the control vector, shSREBP2, shEGR-1, or PPARγ DN were pre-exposed to vehicle or 1 mm MβCD for 18 h. The cells were then treated with vehicle, 500 ng/ml DON, or 50 ng/ml ANS for 1 h. IL-8 (F) and CXCL-1 (G) mRNA were quantified. Different letters (a–i) over each bar represent significant differences between groups (p < 0.05) (A–G). All results are representative of three independent experiments. Error bars, S.D.

Article Snippet: Cell Culture Conditions and Reagents Intestinal cancer cell lines, including HCT-8 (passage 13), HCT-116 (passage 32), and intestine 407 (passage 10), were purchased from the ATCC (Manassas, VA).

Techniques: Transfection, Plasmid Preparation, Western Blot