Journal: Molecular Cell

Article Title: Functional landscape of SARS-CoV-2 cellular restriction

doi: 10.1016/j.molcel.2021.04.008

Figure Lengend Snippet: IFN-mediated restriction of SARS-CoV-2 relies on a limited subset of ISGs (A) Schematic representation of the gain-of-function screen to identify ISGs that inhibit SARS-CoV-2 replication. (B) Ranked log2FC of the percentage of infected cells (SARS-CoV-2 N + cells, blue shading) and normalized cell number (pink shading) after individual overexpression of 399 human ISGs and controls. Values are relative to the negative control CAT . Dashed lines illustrate cut offs for antiviral ISG hit calling strategy; the dotted blue line indicates log2FC infection = 4 × standard deviations (SDs) log2FC of CAT log2FC, and the dotted pink line indicates cell number = 70% of CAT . Controls are shown ( CAT , negative; LY6E , positive). (C) Correlation plots between screens. r, Pearson correlation coefficient. (D) 293T-ACE2 cells transduced with lentiviruses carrying each of the identified ISGs were infected with SARS-CoV-2 (MOI = 0.25) for 40 h prior to immunostaining for viral SARS-CoV-2 nucleoprotein (N). Data represent mean log2FC values (percentage of N + cells relative to parental control wells) from three independent experiments (n = 3). (E) Representative images are shown. Scale bars, 10 μm. (F) Calu-3 cells transduced with lentiviruses encoding the indicated ISGs were infected with SARS-CoV-2 (MOI =1.5) for 48 h prior to immunostaining for viral N protein. Data show mean ± SEM normalized infection (percentage of infected cells relative to parental control wells) from one representative experiment in quadruplicate (n = 4). (G) Differentiated HTBE cells stably expressing the indicating ISGs or negative control GFP were infected with SARS-CoV-2 (MOI = 1) on the apical side. At 18 h post-infection, supernatants were collected and the amount of SARS-CoV-2 focus-forming units per milliliter (FFU/mL) analyzed using Vero E6 cells. Data show mean ± SD and are representative from two sets of HTBE cells per ISG (n = 2). Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparison post hoc test (D) or one-way ANOVA with Dunnett’s post hoc test (F and G).

Article Snippet: HTBE (RNAseq) , ATCC , Cat# PCS-300-010.

Techniques: Infection, Over Expression, Negative Control, Transduction, Immunostaining, Control, Stable Transfection, Expressing, Comparison

Journal: Molecular Cell

Article Title: Functional landscape of SARS-CoV-2 cellular restriction

doi: 10.1016/j.molcel.2021.04.008

Figure Lengend Snippet:

Article Snippet: HTBE (RNAseq) , ATCC , Cat# PCS-300-010.

Techniques: Virus, Recombinant, Transfection, Reverse Transcription, SYBR Green Assay, Infection, Negative Control, Expressing, Software, Imaging

Journal: Journal of Inflammation (London, England)

Article Title: Is inflammation a consequence of extracellular hyperosmolarity?

doi: 10.1186/1476-9255-6-21

Figure Lengend Snippet: Interleukin-8 secretion induced by exposure to glycol-derived compounds . IL-8 secretion (ELISA, in pg/ml) of human T24 cells exposed during 48 hours to increasing osmolarities (300, 600, 900 mOsm) of different compounds: A) Mannitol (300 mOsm [141.23–287.79]; 600 mOsm [246.83–378.69]; 900 mOsm [703.31–1100.9]); B) Propylene Glycol (300 mOsm [141.23–287.79]; 600 mOsm [360.62–568.54]; 900 mOsm [682.24–1084.7]); C) Glycerol (300 mOsm [141.23–287.79]; 600 mOsm [373.93–590.49]; 900 mOsm [816.79–1141.7]). For each group, the horizontal bar is the median. Upper values represent the global p-values (Kruskal-Wallis test). The p-value given by the Tukey test above a stripchart indicates the result of the comparison of this group versus control. CI at 95% are given in parentheses (n = 5).

Article Snippet: The human colon, bladder and pulmonary cancer cell lines, HT-29, T24 and A549 (ATCC numbers HTB-38, HTB-4, and CCL-185, LGC Promochem, Molsheim, France) were cultured in Dulbecco's minimal essential medium (DMEM, Gibco) supplemented with 10% decomplemented fetal bovine serum (FBS, Eurobio, Les Ulis, France) and 1% non-essential amino acids, in a humid atmosphere 5% of CO 2 at 37°C.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Journal of Inflammation (London, England)

Article Title: Is inflammation a consequence of extracellular hyperosmolarity?

doi: 10.1186/1476-9255-6-21

Figure Lengend Snippet: Interleukin-6 secretion induced by exposure to glycol-derived compounds . IL-6 secretion (ELISA, in pg/ml) of human T24 cells exposed during 48 hours to increasing osmolarities (300, 600, 900 mOsm) of different compounds: A) Mannitol (300 mOsm [115.47–171.73]; 600 mOsm [108.12–199.79]; 900 mOsm [600.56–716.47]); B) Propylene Glycol (300 mOsm [115.47–171.73]; 600 mOsm [183.87–207.27]; 900 mOsm [214.75–484.27]); C) Glycerol (300 mOsm [115.47–171.73]; 600 mOsm [178.71–205.97]; 900 mOsm [373.47–587.57]). For each group, the horizontal bar is the median. Upper values represent the global p-values (Kruskal-Wallis test). The p-value given by the Tukey test above a stripchart indicates the result of the comparison of this group versus control. CI at 95% are given in parentheses (n = 5).

Article Snippet: The human colon, bladder and pulmonary cancer cell lines, HT-29, T24 and A549 (ATCC numbers HTB-38, HTB-4, and CCL-185, LGC Promochem, Molsheim, France) were cultured in Dulbecco's minimal essential medium (DMEM, Gibco) supplemented with 10% decomplemented fetal bovine serum (FBS, Eurobio, Les Ulis, France) and 1% non-essential amino acids, in a humid atmosphere 5% of CO 2 at 37°C.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Journal of Inflammation (London, England)

Article Title: Is inflammation a consequence of extracellular hyperosmolarity?

doi: 10.1186/1476-9255-6-21

Figure Lengend Snippet: TNF-α secretion induced by exposure to glycol-derived compounds . TNF-α secretion (ELISA, in pg/ml) of human T24 cells exposed during 48 hours to increasing osmolarities (300, 600, 900 mOsm) of different compounds: A) Mannitol (300 mOsm [69.40–174.15]; 600 mOsm [214.92–247.70]; 900 mOsm [464.06–829.81]); B) Propylene Glycol (300 mOsm [69.40–174.15]; 600 mOsm [90.364–236.51]; 900 mOsm [232.68–443.57]); C) Glycerol (300 mOsm [69.40–174.15]; 600 mOsm [140.36–351.25]; 900 mOsm [402.68–632.45]). For each group, the horizontal bar is the median. Upper values represent the global p-values (Kruskal-Wallis test). The p-value given by the Tukey test above a stripchart indicates the result of the comparison of this group versus control. CI at 95% are given in parentheses (n = 5).

Article Snippet: The human colon, bladder and pulmonary cancer cell lines, HT-29, T24 and A549 (ATCC numbers HTB-38, HTB-4, and CCL-185, LGC Promochem, Molsheim, France) were cultured in Dulbecco's minimal essential medium (DMEM, Gibco) supplemented with 10% decomplemented fetal bovine serum (FBS, Eurobio, Les Ulis, France) and 1% non-essential amino acids, in a humid atmosphere 5% of CO 2 at 37°C.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Journal of Inflammation (London, England)

Article Title: Is inflammation a consequence of extracellular hyperosmolarity?

doi: 10.1186/1476-9255-6-21

Figure Lengend Snippet: IL-1β secretion induced by exposure to glycol-derived compounds . IL-1β secretion (ELISA, in pg/ml) of human T24 cells exposed during 48 hours to increasing osmolarities (300, 600, 900 mOsm) of different compounds: A) Mannitol (300 mOsm [31.969–75.141]; 600 mOsm [59.164–80.941]; 900 mOsm [125.69–154.45]); B) Propylene Glycol (300 mOsm [31.969–75.141]; 600 mOsm [59.674–105.90]; 900 mOsm [97.915–195.67]); C) Glycerol (300 mOsm [31.969–75.141]; 600 mOsm [64.276–167.78]; 900 mOsm [66.748–251.01]). For each group, the horizontal bar is the median. Upper values represent the global p-values (Kruskal-Wallis test). The p-value given by the Tukey test above a stripchart indicates the result of the comparison of this group versus control. CI at 95% are given in parentheses (n = 5).

Article Snippet: The human colon, bladder and pulmonary cancer cell lines, HT-29, T24 and A549 (ATCC numbers HTB-38, HTB-4, and CCL-185, LGC Promochem, Molsheim, France) were cultured in Dulbecco's minimal essential medium (DMEM, Gibco) supplemented with 10% decomplemented fetal bovine serum (FBS, Eurobio, Les Ulis, France) and 1% non-essential amino acids, in a humid atmosphere 5% of CO 2 at 37°C.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Journal of Inflammation (London, England)

Article Title: Is inflammation a consequence of extracellular hyperosmolarity?

doi: 10.1186/1476-9255-6-21

Figure Lengend Snippet: Pro-inflammatory cytokines transcription following exposure of HT-29 and T24 cells to glycol-derived compounds . Cytokine transcription was analyzed by RNase Protection Assay performed on RNA from human HT-29 (A) or T24 (B) cells exposed for 24 hours to 900 mOsm Mannitol (Man), Propylene Glycol (Prop) or Glycerol (Gly) media as compared to controls (M: normal medium; C: isosmotic control). Hyperosmolarity increases the level of transcription of IL-8, IL-6, IL-1β and TNF-α. GAPDH transcription is not altered.

Article Snippet: The human colon, bladder and pulmonary cancer cell lines, HT-29, T24 and A549 (ATCC numbers HTB-38, HTB-4, and CCL-185, LGC Promochem, Molsheim, France) were cultured in Dulbecco's minimal essential medium (DMEM, Gibco) supplemented with 10% decomplemented fetal bovine serum (FBS, Eurobio, Les Ulis, France) and 1% non-essential amino acids, in a humid atmosphere 5% of CO 2 at 37°C.

Techniques: Derivative Assay, Rnase Protection Assay, Control

Journal:

Article Title: Transcription factor ZBP-89 is required for STAT1 constitutive expression

doi: 10.1093/nar/gkg929

Figure Lengend Snippet: Reduction of ZBP-89 expression selectively reduces STAT1 and STAT6 expression. (A) U2OS, MCF-7 and HeLa cells were transfected with ZBP-89 mutant siRNAs (Mut) or ZBP-89 siRNA (WT) for 48 h and then treated with 500 U/ml of IFNγ for another 20 h. Whole-cell extracts were prepared and the profiles of STAT proteins were detected using their specific antibodies. (B) Total RNA was isolated from MCF-7 cells and was used for RNase protection assay as described in Materials and Methods.

Article Snippet: The A549 (human non-small-cell lung cancer cell line), MCF-7 (human breast cancer cell line), HeLa (human cervical cancer cell line), U2OS (human ostrosarcoma cell line) and 293T cell lines were purchased from ATCC (Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS).

Techniques: Expressing, Transfection, Mutagenesis, Isolation, Rnase Protection Assay

Journal:

Article Title: Sensitivity of an Epstein-Barr Virus-Positive Tumor Line, Daudi, to Alpha Interferon Correlates with Expression of a GC-Rich Viral Transcript

doi:

Figure Lengend Snippet: Expression levels of EBV D-HIT and homologs in B-cell lines

Article Snippet: Superimposed on this pattern, for comparison purposes, are curves obtained for Daudi/ICRF (at 1 and 10 U), Raji (at 10 4 U), and P3HR1 (at 10 2 U), where the data are taken from the literature ( 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2 caption a7 Interferon sensitivities of Daudi/ATCC cells.

Techniques: Expressing

Journal:

Article Title: Sensitivity of an Epstein-Barr Virus-Positive Tumor Line, Daudi, to Alpha Interferon Correlates with Expression of a GC-Rich Viral Transcript

doi:

Figure Lengend Snippet: Interferon sensitivities of Daudi/ATCC cells. Cells were grown in the presence of various concentrations of IFN-α over a 28-day period, and the number of live cells (as a percentage of untreated cells) was plotted. The profile of Daudi/ATCC, showing viable cell counts as a percentage of cells grown in the absence of interferon (100%), is taken as a standard for medium IFN-α sensitivity. Superimposed on it are two curves from Daudi/ICRF cells (grown in the presence of 1 and 10 U of IFN-α/ml; high sensitivity), one from Raji cells (grown with 104 U; low sensitivity), and one from P3HR1 cells (102 U; high/medium sensitivity; data taken from the literature [2]), all EBV-positive lines.

Article Snippet: Superimposed on this pattern, for comparison purposes, are curves obtained for Daudi/ICRF (at 1 and 10 U), Raji (at 10 4 U), and P3HR1 (at 10 2 U), where the data are taken from the literature ( 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2 caption a7 Interferon sensitivities of Daudi/ATCC cells.

Techniques:

Journal:

Article Title: Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis

doi:

Figure Lengend Snippet: Reexpression of the human Smad4/DPC4 in the human pancreatic adenocarcinoma cell line Hs766T. (A) Northern blot analysis of total RNA from the parental cell line and stable transfectants hybridized with a human Smad4/DPC4 cDNA probe. Lane 2, Hs766T parental cell line; lanes 3–7, Smad4/DPC4 reconstituted clones D2, D4, D5, D8, and D9; lanes 8 and 9, negative control clones K3 and K6. (B) Western blot analysis for the human Smad4/DPC4 protein on total protein extracts. Lane 1, human pancreatic adenocarcinoma cell line Paca44 as control for endogenous Smad4/DPC4; lane 2, Hs766T parental cell line; lanes 3–7, Smad4/DPC4 reconstituted clones; lanes 8 and 9, negative control clones.

Article Snippet: Hs766T human pancreatic adenocarcinoma cells were obtained from the American Type Culture Collection.

Techniques: Northern Blot, Clone Assay, Negative Control, Western Blot, Control

Journal:

Article Title: Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis

doi:

Figure Lengend Snippet: Analysis of growth and TGF-β response in vitro and expression analysis of TGF-β receptors. (A) Growth of Smad4/DPC4-reexpressing cell clones and negative controls cultured in serum-supplemented growth medium. (B) Growth of Smad4/DPC4-reexpressing cells and negative control cells incubated in the absence (continuous bar) or presence (hatched bar) of TGF-β1 (5 ng/ml) under reduced serum concentrations. (A and B) Cells were plated in duplicate (A) or triplicate (B) for each time point, and results were confirmed in at least two independent experiments. Note that error bars for standard deviations are shown in B but are too narrow to be resolved. (C) Expression of TGF-βRI and TGF-βRII as analyzed by RNase protection assay. Lane 1, probe; lane 2, tRNA; lane 3, Hs766T parental cell line; lanes 4 and 5, Smad4/DPC4 negative control clones K3 and K6; lanes 6 and 7, Smad4/DPC4-reconstituted clones D5 and D8.

Article Snippet: Hs766T human pancreatic adenocarcinoma cells were obtained from the American Type Culture Collection.

Techniques: In Vitro, Expressing, Clone Assay, Cell Culture, Negative Control, Incubation, Rnase Protection Assay

Journal:

Article Title: Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis

doi:

Figure Lengend Snippet: Smad4/DPC4-mediated shifts in VEGF and TSP-1 expression levels. (A) Northern blot with total RNA was hybridized with a human VEGF cDNA probe, stripped, and rehybridized with a TSP-1-specific cDNA probe. Lane 1, Hs766T parental cell line; lanes 2 and 3, Smad4/DPC4-negative clones K3 and K6; lanes 4–6, Smad4/DPC4-reconstituted clones D4, D5, and D8. The difference in expression levels was confirmed with at least three independent RNA preparations each. Quantification of RNA levels repeatedly revealed a reduction of the VEGF steady-state mRNA level by a factor of two to three and induction of TSP-1 expression by a factor of approximately three in Smad4/DPC4-reexpressing clones grown in full medium or incubated in serum-free medium. (B) VEGF and TSP-1 Western blots with protein from conditioned media. Lanes 2 and 3, Smad4/DPC4-negative clones K3 and K6; lanes 5 and 6, Smad4/DPC4-reconstituted clones D5 and D8. The signals correspond to dimeric VEGF165 and trimeric TSP-1.

Article Snippet: Hs766T human pancreatic adenocarcinoma cells were obtained from the American Type Culture Collection.

Techniques: Expressing, Northern Blot, Clone Assay, Incubation, Western Blot

Journal:

Article Title: Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis

doi:

Figure Lengend Snippet: Vascularization of nude mouse tumors. CD31-immunostained section through a tumor derived from Smad4/DPC4-negative control clone K3 (A) and derived from Smad4/DPC4-positive clone D5 (B). Asterisks in A depict large vessels (diameter > 50 μm), and medium-sized vessels (diameter 10–50 μm) are indicated by arrows. (Bars = 100 μm.) (C) Quantification of cords, capillaries, and vessels in Smad4-negative and Smad4-positive Hs766T nude mouse tumors.

Article Snippet: Hs766T human pancreatic adenocarcinoma cells were obtained from the American Type Culture Collection.

Techniques: Derivative Assay, Negative Control

Journal:

Article Title: GLYCOGEN SYNTHASE KINASE-3 IS A NEGATIVE REGULATOR OF EXTRACELLULAR SIGNAL-REGULATED KINASE

doi: 10.1038/sj.onc.1209004

Figure Lengend Snippet: A. HT29, Caco-2, 293 or PC-3 cells were starved in serum free medium for 24 h followed by treatment with the GSK-3 inhibitor SB-216763 (10 μM), SB-415286 (30 μM), LiCl (20 mM) or vehicle control DMSO for 30 min and whole cell protein extracted for p-ERK1/2 and total ERK1/2 expression by Western blot. B. HT29 cells were starved in serum free medium for 8 h followed by treatment with various concentrations of SB-216763 for 30 min and whole cell protein extracted for p-ERK1/2 and total ERK1/2 expression by Western blot. C. HT29 cells were starved in serum free medium for 24 h followed by treatment with SB-216763 (10 μM) for various times and whole cell protein extracted for p-ERK1/2 and total ERK1/2 expression by Western blot. D. HT29 cells were starved in serum free medium for 24 h. Cells were pre-treated with the MEK-1 inhibitor U0126 or PD98059 for 30 min followed by combination treatment with GSK-3 inhibitor SB-216763 (10 μM) or vehicle control DMSO for an additional 30 min and whole cell protein extracted for p-ERK1/2 and total ERK1/2 expression by Western blot. E. HT29 cells were transfected with GSK-3α, GSK-3β or control siRNA. After a 48 h incubation, transfected cells were harvested and whole cell protein extracted for phosphorylation levels of ERK1/2 by Western blot using anti-p-ERK1/2 antibody; blots were stripped and reprobed with anti-ERK antibody (left). To confirm GSK-3 suppression, GSK-3α and GSK-3β expression was assessed by Western blot using anti-GSK-3α/β antibody; blots were stripped and reprobed with anti-actin antibody (right). Representative data from three separate experiments are shown here.

Article Snippet: Cell lines HT29 and Caco-2 (human colon cancer cell lines), PC-3 (human prostate cancer cell line), and 293 (human embryonic kidney cell line) were obtained from the American Type Culture Collection (ATCC).

Techniques: Control, Expressing, Western Blot, Transfection, Incubation, Phospho-proteomics

Journal:

Article Title: GLYCOGEN SYNTHASE KINASE-3 IS A NEGATIVE REGULATOR OF EXTRACELLULAR SIGNAL-REGULATED KINASE

doi: 10.1038/sj.onc.1209004

Figure Lengend Snippet: A, left. HT29 cells were starved in serum free medium for 24 h. Cells were pretreated with rottlerin for 30 min followed by treatment with the GSK-3 inhibitor SB-216763 (10 μM) for 30 min and whole cell protein extracted for p-ERK1/2 and total ERK1/2 expression by Western blot. Right. HT29 cells were starved in serum free medium for 24 h followed by treatment with SB-216763 (10 μM) for 30 min. Whole cell protein was prepared from control or SB-216763 treated cells followed by immunoprecipitation of PKCδ and PKCδ activity measured using MBP as substrate. B. HT29 cells transfected with control or PKCδ siRNA were starved in serum free medium for 24 h. Cells were treated with SB-216763 (10 μM) for 30 min and whole cell protein extracted for p-ERK1/2 and total ERK1/2 (left) or PKCδ, PKCα, actin (right) expression by Western blot. C. HT29 cells were transfected with control or GSK-3α and GSK-3β siRNA. After 24 h incubation, transfected cells were harvested and whole cell protein extracted for p-ERK1/2, total ERK1/2, GSK-3α, and GSK-3β expression by Western blot and PKCδ activity by kinase assay using MBP as substrate. Representative data from three separate experiments are shown here.

Article Snippet: Cell lines HT29 and Caco-2 (human colon cancer cell lines), PC-3 (human prostate cancer cell line), and 293 (human embryonic kidney cell line) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, Activity Assay, Transfection, Incubation, Kinase Assay

Journal:

Article Title: GLYCOGEN SYNTHASE KINASE-3 IS A NEGATIVE REGULATOR OF EXTRACELLULAR SIGNAL-REGULATED KINASE

doi: 10.1038/sj.onc.1209004

Figure Lengend Snippet: A. HT29 cells were starved in serum free medium for 24 h followed by pretreatment with or without the MEK inhibitor U0126 (10 μM) for 30 min and then treated with LiCl (20 mM), SB-216763 (10 μM), or SB-415286 (30 μM) alone or in combination for 4h. RNA was isolated and analyzed by Northern blotting. B. HT29 cells were transfected with a plasmid containing the human COX-2 promoter fragment linked to the luciferase reporter or empty vector. After a 24 h incubation, the transfected cells were treated with SB-216763 (10 μM) for an additional 24 h (left) or co-transfected. with GSK-3α, GSK-3β or control siRNA. After 48 h incubation, cells were harvested and luciferase activity measured in the crude cell lysates as described in the “Methods”. All results were normalized for transfection efficiency using the pRL-Tk-luc plasmid (Promega). C. HT29 cells were starved in serum free medium for 24 h followed by pretreatment with or without the MEK inhibitor U0126 (10 μM) for 30 min and then treated with LiCl (20 mM), SB-216763 (10 μM), or SB-415286 (30 μM) alone or in combination for 4h. Total RNA was isolated and IL-8 mRNA expression analyzed by RNase protection assay using the hCK-5 multi-probe (Pharmingen). The probe set includes cDNAs for L32 and GAPDH to control for RNA loading. D. HT29 cells were transfected with a plasmid containing the human IL-8 promoter fragment linked to the luciferase reporter or empty vector. After a 24 h incubation, the transfected cells were treated with SB-216763 (10 μM) for an additional 24 h and cells were harvested and luciferase activity measured in the crude cell lysates as described in the “Methods” section. Representative data from three separate experiments are shown here.

Article Snippet: Cell lines HT29 and Caco-2 (human colon cancer cell lines), PC-3 (human prostate cancer cell line), and 293 (human embryonic kidney cell line) were obtained from the American Type Culture Collection (ATCC).

Techniques: Isolation, Northern Blot, Transfection, Plasmid Preparation, Luciferase, Incubation, Control, Activity Assay, Expressing, Rnase Protection Assay

Journal: Journal of Biological Chemistry

Article Title: Cholesteryl Ester Transfer Protein Biosynthesis and Cellular Cholesterol Homeostasis Are Tightly Interconnected

doi: 10.1074/jbc.m103624200

Figure Lengend Snippet: FIG. 1. Triglyceride accumulation and CETP secretion in SW872 cells. SW872 cells grown to 100% confluence in DMEM/F-12 containing 10% fetal bovine serum were incubated with DMEM/F-12 6 400 mM oleate for up to 48 h. SW872 cells incubated without oleate (A) or with oleate for 24 h (B) or 48 h (C; 2.53 greater magnification compared with A and B) were stained with Oil Red O and hematoxylin/ eosin as described (55). D, perilipin A immunoblot. The proteins from cells treated (48 h) as described above were separated on 7.5% gels, transferred to polyvinylidene difluoride, and reacted with guinea pig anti-perilipin antiserum (see “Experimental Procedures”). Y1 cells (pos- itive control) are of mouse adrenal origin; HepG2 cells are negative control. E, inhibition of CETP activity in conditioned medium collected from SW872 cells or CETP purified from human plasma by excess TP2 monoclonal antibody (10 mg). Similar suppression was observed with 1 mg of TP2. E, time course of CETP secretion by SW872 cells. The values are the means 6 S.D. of duplicates. For details see “Experimental Procedures.”

Article Snippet: Materials—The human colon adenocarcinoma Caco-2 (American Type Culture Collection HTB-37), the human liposarcoma cell line SW872 (American Type Culture Collection HTB-92), and the hepatocarcinoma HepG2 (American Type Culture Collection HB-8065) were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Incubation, Staining, Western Blot, Control, Negative Control, Inhibition, Activity Assay, Purification, Clinical Proteomics

Journal: Journal of Biological Chemistry

Article Title: Cholesteryl Ester Transfer Protein Biosynthesis and Cellular Cholesterol Homeostasis Are Tightly Interconnected

doi: 10.1074/jbc.m103624200

Figure Lengend Snippet: FIG. 2. Cellular lipid status modu- lates CETP secretion. The effect of lipid delivery to cells on CETP secretion by SW872 and Caco-2 cells is shown in A and B, respectively. Confluent cells pretreated with medium containing 5% LPDS over- night were treated with serum-free me- dium containing oleic acid (500 mM), LDL (100 mg/ml), acetylated LDL (100 mg/ml), or 25-hydroxycholesterol (100 mM) for 24 h. This medium was removed and re- placed by DMEM/F-12 alone and incu- bated for 48 h. The conditioned medium was assayed for CETP activity. A, inset, Western blot of immunoprecipitated CETP present in the conditioned medium after the indicated treatment. C, cells pre- incubated in 5% LPDS were treated with- out (open squares) or with oleate (200 mM, closed squares) for the times indicated. This medium was removed, and DMEM/ F-12 was added for 48 h. This conditioned medium was assayed for CETP activity. Inset, rate of CETP synthesis (%kt/h) ver- sus the triglyceride content of cells, deter- mined by enzymatic assay. D, CETP ac- tivity secreted by SW872 cells after treatment for 24 h with medium contain- ing 5% LPDS, b-cyclodextrin (500 mM), or a mixture of both. Cells were pretreated with serum-containing medium overnight prior to the indicated treatment. Inset, Western blot of CETP in the conditioned medium after treatment as indicated. The values are the means 6 S.D. of duplicates and are representative of at least three experiments. Cydex., cyclodextrin; Ac- LDL, acetylated LDL; 25-OH Chol., 25-hydroxycholesterol.

Article Snippet: Materials—The human colon adenocarcinoma Caco-2 (American Type Culture Collection HTB-37), the human liposarcoma cell line SW872 (American Type Culture Collection HTB-92), and the hepatocarcinoma HepG2 (American Type Culture Collection HB-8065) were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Incubation, Enzymatic Assay

Journal: Journal of Biological Chemistry

Article Title: Cholesteryl Ester Transfer Protein Biosynthesis and Cellular Cholesterol Homeostasis Are Tightly Interconnected

doi: 10.1074/jbc.m103624200

Figure Lengend Snippet: FIG. 3. Cellular lipid status regulates CETP mRNA levels in SW872 cells. SW872 cells at 100% confluence were pretreated with medium containing 5% LPDS for 24 h and then subjected to the indi- cated treatment for an other 24 h. Total RNA was prepared after these treatments, 20-mg aliquots of RNA were separated on 8% SDS-poly- acrylamide-urea gels, transferred onto a membrane, and analyzed by RNase protection assay as described under “Experimental Procedures.” The bottom panel shows the densitometry values obtained for each band compared with actin. Ac-LDL, acetylated LDL; 25 OH-chol, 25-hydroxycholesterol.

Article Snippet: Materials—The human colon adenocarcinoma Caco-2 (American Type Culture Collection HTB-37), the human liposarcoma cell line SW872 (American Type Culture Collection HTB-92), and the hepatocarcinoma HepG2 (American Type Culture Collection HB-8065) were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Membrane, Rnase Protection Assay

Journal: Journal of Biological Chemistry

Article Title: Cholesteryl Ester Transfer Protein Biosynthesis and Cellular Cholesterol Homeostasis Are Tightly Interconnected

doi: 10.1074/jbc.m103624200

Figure Lengend Snippet: FIG. 4. Reduction of CETP secretion levels in SW872 cells by antisense oligonucleotides. SW872 cells at 70% confluence were transfected with Opti-MEM alone, LipofectAMINE, or LipofectAMINE mixed with 500 nM sense or antisense oligonucleotide (A) made against human CETP mRNA. These cells were incubated with fresh DMEM/ F-12 for 48 h. Collected medium was concentrated, and a 100-ml aliquot was used to measure CETP activity. The values are the means 6 S.D. of duplicates and are representative of at least three experiments. Oligo (A), oligonucleotide A.

Article Snippet: Materials—The human colon adenocarcinoma Caco-2 (American Type Culture Collection HTB-37), the human liposarcoma cell line SW872 (American Type Culture Collection HTB-92), and the hepatocarcinoma HepG2 (American Type Culture Collection HB-8065) were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Transfection, Incubation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Cholesteryl Ester Transfer Protein Biosynthesis and Cellular Cholesterol Homeostasis Are Tightly Interconnected

doi: 10.1074/jbc.m103624200

Figure Lengend Snippet: FIG. 5. Reduced CETP synthesis modifies cellular lipid metabolism in SW872 cells. SW872 cells untreated (none) or transfected with Lipo- fectAMINE containing 500 nM sense or antisense oligonucleotide A were incu- bated overnight in DMEM/F-12/5% LPDS. Subsequently, washed cells re- ceived the same oligonucleotide-contain- ing medium containing [14C]acetate (0.5 mCi/well) for 6 h. This medium was re- moved and replaced with DMEM/F-12 1 5% LPDS overnight. Label incorporated into free cholesterol (A), cholesteryl ester (B), and triglyceride (C) were determined as described under “Experimental Proce- dures.” The values are the means 6 S.D. of triplicate experiments.

Article Snippet: Materials—The human colon adenocarcinoma Caco-2 (American Type Culture Collection HTB-37), the human liposarcoma cell line SW872 (American Type Culture Collection HTB-92), and the hepatocarcinoma HepG2 (American Type Culture Collection HB-8065) were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Transfection

Journal: Journal of Biological Chemistry

Article Title: Cholesteryl Ester Transfer Protein Biosynthesis and Cellular Cholesterol Homeostasis Are Tightly Interconnected

doi: 10.1074/jbc.m103624200

Figure Lengend Snippet: FIG. 6. Effect of low CETP on cholesteryl ester content and efflux. SW872 cells at 70% confluence were treated with LipofectAMINE containing 500 nM oligonucleotides as indicated under “Experimental Procedures” and then incubated with [14C]acetate (0.5 mCi/well) for 3 days. During this incubation, oligonucleotides were added daily to the cells. The labeled CE content of cells after treatment with antisense oligonucleotide A (A) or antisense oligonucleotide B (B) is shown. In C, control cells were incubated with [3H]cholesterol for 3 days in medium containing 10% fetal bovine serum. Labeled cells were transfected with sense or antisense oligonucleotides (500 nM) as described under “Experimental Procedures.” After overnight incubation, cells received serum-free efflux medium containing 100 mg/ml HDL plus additional sense or antisense oligonucleotide. After 24 h, cellular lipids were extracted, and [3H]cholesteryl ester was determined. Hydrolysis was calculated from the decline in CE radioactivity at 24 h compared with that at t 5 0 (9.5 3 103 cpm/mg protein). The values are the means 6 S.D. of triplicate experiments. In D, cells were incubated as indicated for C except that radiolabeled cholesterol was not added. Following 24 h of treatment with the indicated oligonucleotide (t 5 0) and after incubation (10 h) with HDL (100 mg/ml), the cells were harvested, the lipids were extracted, and the cholesteryl esters were isolated by thin layer chromatography. The cholesteryl esters were transmethylated and quantitated by gas chromatography. The results are the means 6 S.D. of n 5 5 (sense) or 6 (antisense) replicates. The cholesteryl ester content of t 5 0 cells (after 24 h of treatment with oligonucleotide) was not significantly different between sense and antisense treatment.

Article Snippet: Materials—The human colon adenocarcinoma Caco-2 (American Type Culture Collection HTB-37), the human liposarcoma cell line SW872 (American Type Culture Collection HTB-92), and the hepatocarcinoma HepG2 (American Type Culture Collection HB-8065) were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Incubation, Labeling, Control, Transfection, Radioactivity, Isolation, Thin Layer Chromatography, Gas Chromatography