cultured rat intestinal epithelial cell line iec-18  (DS Pharma Biomedical)

Journal: Metallomics : integrated biometal science

Article Title: Knockdown of copper-transporting ATPase 1 (Atp7a) impairs iron flux in fully-differentiated rat (IEC-6) and human (Caco-2) intestinal epithelial cells.

doi: 10.1039/c6mt00126b

Figure Lengend Snippet: Fig. 1 Atp7a knockdown impairs iron and copper homeostasis in IEC-6 cells. IEC-6 cells were transfected with a negative-control (Ctrl) (scrambled) or Atp7a-targeting, shRNA-expressing plasmids. 2 clonal knockdown (KD) cell populations were selected (KD1, KD2). Atp7a KD was verified at the mRNA (A) and protein (B) levels in fully-differentiated cells. A representative western blot is shown (B, inset). Intracellular iron and copper (C) concentrations were quantified by AAS in fully differentiated Ctrl and Atp7a KD IEC-6 cells. Values are means SDs. Data were analyzed by One-way ANOVA followed by Tukey’s post hoc analysis. **p o 0.01 and ****p o 0.0001, as compared to control values (A and B). Labeled means without a common letter differ (p o 0.05) (C). n = 3 independent experiments with 3 technical replicates per experiment.

Article Snippet: Cell culture and development of Atp7a knockdown cell lines Rat intestinal epithelial (IEC-6) cells (American Type Culture Collection [ATCC]; Manassas, VA; #CRL-1592) were cultured in DMEM (Corning; New York, NY) with 10% FBS (Sigma; St. Louis, MO), 10 U mL 1 insulin and 100 U mL 1 penicillin/ streptomycin (Corning) at 37 1C in a 5% CO2/95% O2 atmosphere.

Techniques: Knockdown, Transfection, Negative Control, shRNA, Expressing, Western Blot, Control, Labeling

Journal: Metallomics : integrated biometal science

Article Title: Knockdown of copper-transporting ATPase 1 (Atp7a) impairs iron flux in fully-differentiated rat (IEC-6) and human (Caco-2) intestinal epithelial cells.

doi: 10.1039/c6mt00126b

Figure Lengend Snippet: Fig. 2 Atp7a silencing impairs transepithelial iron flux in IEC-6 and Caco-2 cells. Ctrl and Atp7a KD IEC-6 and Caco-2 cells were grown on collagen- coated, transwell cell-culture inserts for 8 (IEC-6) or 21 (Caco-2) days. In some experiments, 200 mmol L1 deferroxamine (DFO) (or vehicle, PBS) was added to the apical and basolateral medium of cultured IEC-6 cells for the last 24 h to create iron-deficient conditions. Subsequently, 0.5 mmol L1

Article Snippet: Cell culture and development of Atp7a knockdown cell lines Rat intestinal epithelial (IEC-6) cells (American Type Culture Collection [ATCC]; Manassas, VA; #CRL-1592) were cultured in DMEM (Corning; New York, NY) with 10% FBS (Sigma; St. Louis, MO), 10 U mL 1 insulin and 100 U mL 1 penicillin/ streptomycin (Corning) at 37 1C in a 5% CO2/95% O2 atmosphere.

Techniques: Cell Culture

Journal: Metallomics : integrated biometal science

Article Title: Knockdown of copper-transporting ATPase 1 (Atp7a) impairs iron flux in fully-differentiated rat (IEC-6) and human (Caco-2) intestinal epithelial cells.

doi: 10.1039/c6mt00126b

Figure Lengend Snippet: Fig. 3 Atp7a KD alters expression of iron homeostasis-related genes. mRNA was purified from fully-differentiated Ctrl or Atp7a KD IEC-6 cells and SYBR- Green qRT-PCR was performed to quantify expression of iron and copper transport-related genes. 200 mmol L1 DFO was added to some wells for the last 24 hours to create an iron-deficient condition. Induction of transferrin receptor 1 (Tfr1) confirmed iron deficiency (F). Values are means SDs. Data were analyzed by One-way ANOVA followed by Tukey’s post hoc analysis. Labeled means without a common letter differ (p o 0.05) (A and C). *p o 0.05, **p o 0.01 and ****p o 0.0001, as compared to other groups (B, D–F). n = 3 independent experiments with 3 technical replicates per experiment.

Article Snippet: Cell culture and development of Atp7a knockdown cell lines Rat intestinal epithelial (IEC-6) cells (American Type Culture Collection [ATCC]; Manassas, VA; #CRL-1592) were cultured in DMEM (Corning; New York, NY) with 10% FBS (Sigma; St. Louis, MO), 10 U mL 1 insulin and 100 U mL 1 penicillin/ streptomycin (Corning) at 37 1C in a 5% CO2/95% O2 atmosphere.

Techniques: Expressing, Purification, SYBR Green Assay, Quantitative RT-PCR, Labeling

Journal: Metallomics : integrated biometal science

Article Title: Knockdown of copper-transporting ATPase 1 (Atp7a) impairs iron flux in fully-differentiated rat (IEC-6) and human (Caco-2) intestinal epithelial cells.

doi: 10.1039/c6mt00126b

Figure Lengend Snippet: Fig. 4 Atp7a silencing alters expression of iron transport-related proteins. Ctrl or Atp7a KD IEC-6 cells were grown for 8 days post-confluence and then total cell lysates were prepared for western blot analysis. a-Tubulin was used as an internal standard. Each blot was cut into strips and probed with different antibodies, since all proteins have distinct molecular weights (so a-tubulin is shown only once in panel A). The Fpn1 antibody detected several bands, which all decreased similarly in KD cells in a pilot experiment, but only the one of the correct m.w. (B54 kDa) is shown and quantified. Quantitative data from 3 independent experiments is shown in each panel, with a representative western blot shown as an inset. Values are means SDs. Data were analyzed by One-way ANOVA followed by Tukey’s post hoc analysis. *p o 0.05, and **p o 0.01, as compared to other groups (A–D). n = 3 independent experiments.

Article Snippet: Cell culture and development of Atp7a knockdown cell lines Rat intestinal epithelial (IEC-6) cells (American Type Culture Collection [ATCC]; Manassas, VA; #CRL-1592) were cultured in DMEM (Corning; New York, NY) with 10% FBS (Sigma; St. Louis, MO), 10 U mL 1 insulin and 100 U mL 1 penicillin/ streptomycin (Corning) at 37 1C in a 5% CO2/95% O2 atmosphere.

Techniques: Expressing, Western Blot

Journal: Metallomics : integrated biometal science

Article Title: Knockdown of copper-transporting ATPase 1 (Atp7a) impairs iron flux in fully-differentiated rat (IEC-6) and human (Caco-2) intestinal epithelial cells.

doi: 10.1039/c6mt00126b

Figure Lengend Snippet: Fig. 5 Atp7a KD causes transcriptional and posttranscriptional effects on the expression of iron transport-related genes. Total RNA, including mRNA and heteronuclear RNA (hnRNA), was harvested from fully-differentiated Ctrl or Atp7a KD IEC-6 cells and then gene expression levels were quantified by qRT- PCR (panels A, C and E). To quantify mRNA half-lives, cells were treated with actinomycin D (ActD) for 0, 6, 12 or 24 hours prior to cell harvest and then qRT-PCR was performed with gene-specific primers (panels B, D and F). Line fits were performed for data points for each gene and cell population. The mRNA half-life for each transcript is indicated with an ‘‘x’’, which has been placed at the approximate position where the starting mRNA levels were reduced by 50% (actual times [t1/2] are indicated as insets in each panel). Values are means SDs. Data were analyzed by One-way ANOVA followed by Tukey’s post hoc analysis. Labeled means without a common letter differ (p o 0.05) (A and C). ****p o 0.0001, as compared to other groups (E). n = 6 (mRNA and hnRNA quantification) or n = 3 (mRNA decay) independent experiments with 3 technical replicates per experiment.

Article Snippet: Cell culture and development of Atp7a knockdown cell lines Rat intestinal epithelial (IEC-6) cells (American Type Culture Collection [ATCC]; Manassas, VA; #CRL-1592) were cultured in DMEM (Corning; New York, NY) with 10% FBS (Sigma; St. Louis, MO), 10 U mL 1 insulin and 100 U mL 1 penicillin/ streptomycin (Corning) at 37 1C in a 5% CO2/95% O2 atmosphere.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Labeling

Journal: Metallomics : integrated biometal science

Article Title: Knockdown of copper-transporting ATPase 1 (Atp7a) impairs iron flux in fully-differentiated rat (IEC-6) and human (Caco-2) intestinal epithelial cells.

doi: 10.1039/c6mt00126b

Figure Lengend Snippet: Fig. 7 Atp7a KD increases membrane and cytosolic ferroxidase activity in IEC-6 cells. Membrane and cytosolic proteins were isolated from fully- differentiated Ctrl or Atp7a KD IEC-6 cells, and FOX activity was determined by an apo-transferrin-coupled assay from membrane (A) and cytosolic (B) proteins for 5–120 seconds. KD1 and KD2 were statistically identical. Asterisks indicate statistical differences from Ctrl values (*p o 0.05; ** p o 0.01) (One-way ANOVA followed by Tukey’s post hoc analysis). n = 3 independent experiments.

Article Snippet: Cell culture and development of Atp7a knockdown cell lines Rat intestinal epithelial (IEC-6) cells (American Type Culture Collection [ATCC]; Manassas, VA; #CRL-1592) were cultured in DMEM (Corning; New York, NY) with 10% FBS (Sigma; St. Louis, MO), 10 U mL 1 insulin and 100 U mL 1 penicillin/ streptomycin (Corning) at 37 1C in a 5% CO2/95% O2 atmosphere.

Techniques: Membrane, Activity Assay, Isolation

Journal: Metallomics : integrated biometal science

Article Title: Knockdown of copper-transporting ATPase 1 (Atp7a) impairs iron flux in fully-differentiated rat (IEC-6) and human (Caco-2) intestinal epithelial cells.

doi: 10.1039/c6mt00126b

Figure Lengend Snippet: Fig. 6 Atp7a KD enhances cell-surface ferrireductase activity in IEC-6 cells. Fully-differentiated Ctrl or Atp7a KD IEC-6 cells were treated with nitrotetrazolium blue (NTB). Color intensity, indicative of ferrireductase activity, was determined by spectrophotometric measurement of isopropanol eluates. Representative pictures are shown from Ctrl (A) or Atp7a KD (B and C) cells along with color intensity quantification of isopropanol eluates from all experiments (D). Values are means SDs (D). Data were analyzed by One-way ANOVA followed by Tukey’s post hoc analysis. ****p o 0.0001, as compared to the control value. n = 4 independent experiments.

Article Snippet: Cell culture and development of Atp7a knockdown cell lines Rat intestinal epithelial (IEC-6) cells (American Type Culture Collection [ATCC]; Manassas, VA; #CRL-1592) were cultured in DMEM (Corning; New York, NY) with 10% FBS (Sigma; St. Louis, MO), 10 U mL 1 insulin and 100 U mL 1 penicillin/ streptomycin (Corning) at 37 1C in a 5% CO2/95% O2 atmosphere.

Techniques: Activity Assay, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Cattle Bile Aggravates Diclofenac Sodium-Induced Small Intestinal Injury in Mice

doi: 10.1155/2011/315858

Figure Lengend Snippet: Cytotoxicity of bile acids for cultured intestinal epithelial IEC-18 cells in vitro. IEC-18 cells were treated for 24 hours with the bile acids found in the small intestine of mice administered with CB. The viability of treated cells was expressed as the percentage of absorbance of the cells treated with bile acids with respect to that of the untreated cells. Columns and bars represent the mean and SEM, respectively.

Article Snippet: The cytotoxicity of bile acids were compared using a cultured rat intestinal epithelial cell line, IEC-18 (DS Pharma Biomedical, Osaka, Japan) [ ].

Techniques: Cell Culture, In Vitro