Structured Review

GE Healthcare fetal bovine serum fbs
Effects of masTF and hasTF in the scratch assay. bEnd.3 cells (A) and HRECs (B) were grown to confluence in six-well plates and serum-starved overnight in <t>DMEM</t> containing 2% <t>FBS.</t> Eight radial scratches per well were introduced (see Materials and Methods).
Fetal Bovine Serum Fbs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 2978 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/fetal bovine serum fbs/product/GE Healthcare
Average 96 stars, based on 2978 article reviews
Price from $9.99 to $1999.99
fetal bovine serum fbs - by Bioz Stars, 2020-07
96/100 stars

Images

1) Product Images from "Nonproteolytic Properties of Murine Alternatively Spliced Tissue Factor: Implications for Integrin-Mediated Signaling in Murine Models"

Article Title: Nonproteolytic Properties of Murine Alternatively Spliced Tissue Factor: Implications for Integrin-Mediated Signaling in Murine Models

Journal: Molecular Medicine

doi: 10.2119/molmed.2011.00416

Effects of masTF and hasTF in the scratch assay. bEnd.3 cells (A) and HRECs (B) were grown to confluence in six-well plates and serum-starved overnight in DMEM containing 2% FBS. Eight radial scratches per well were introduced (see Materials and Methods).
Figure Legend Snippet: Effects of masTF and hasTF in the scratch assay. bEnd.3 cells (A) and HRECs (B) were grown to confluence in six-well plates and serum-starved overnight in DMEM containing 2% FBS. Eight radial scratches per well were introduced (see Materials and Methods).

Techniques Used: Wound Healing Assay

2) Product Images from "A Functional Variant of IC53 Correlates with the Late Onset of Colorectal Cancer"

Article Title: A Functional Variant of IC53 Correlates with the Late Onset of Colorectal Cancer

Journal: Molecular Medicine

doi: 10.2119/molmed.2010.00192

Knockdown of IC53 blocked cell proliferation, migration and adhesion. (A) Stealth siRNA against IC53 eliminated IC53 protein expression. IC53 expression was analyzed in serum-starved HCT-116 cells. The cells were transfected with stealth siRNA against IC53 (siRNA) or a negative control (siRNA negative). The IC53 proteins were detected by using Western blot analysis with an anti-IC53 monoclonal antibody (1:1,000). An anti-GAPDH antibody was included in the analysis as a loading control. (B) Knockdown of IC53 inhibited HCT-116 cell proliferation. IC53-mediated cell proliferation was analyzed by using the MTT assay. The cells were maintained in DMEM containing 10% FBS and cultured in an incubator with 5% CO 2 at 37°C. The culture media were replaced every 48 h. HCT-116 cells transfected with stealth siRNA against IC53 showed a 64% decrease in proliferation compared with cells transfected with the negative control. (C) Knockdown of IC53 inhibited HCT-116 cell adhesion. HCT-116 cells were suspended in serum-free medium and seeded into collagen I (20 μg/mL)–coated 24-well plates. After incubating for 60 min at 37°C, the percentage of adhered cells was determined by using the colorimetric crystal violet assay. HCT-116 cells transfection with stealth siRNA against IC53 showed a 74% decrease in cell adhesion compared with cells transfected with the negative control. (D) Knockdown of IC53 inhibited HCT-116 cell migration. IC53-mediated cell migration was analyzed by the transwell assay. The cells were maintained in DMEM containing 10% FBS at 37°C. HCT-116 cells transfected with stealth siRNA against IC53 showed an 85% decrease in migration compared with cells transfected with the negative control. This figure represents 1 of 3 independent experiments, with quadruplicate samples in each experiment. The results represent the means ± SD in triplicate. ** P
Figure Legend Snippet: Knockdown of IC53 blocked cell proliferation, migration and adhesion. (A) Stealth siRNA against IC53 eliminated IC53 protein expression. IC53 expression was analyzed in serum-starved HCT-116 cells. The cells were transfected with stealth siRNA against IC53 (siRNA) or a negative control (siRNA negative). The IC53 proteins were detected by using Western blot analysis with an anti-IC53 monoclonal antibody (1:1,000). An anti-GAPDH antibody was included in the analysis as a loading control. (B) Knockdown of IC53 inhibited HCT-116 cell proliferation. IC53-mediated cell proliferation was analyzed by using the MTT assay. The cells were maintained in DMEM containing 10% FBS and cultured in an incubator with 5% CO 2 at 37°C. The culture media were replaced every 48 h. HCT-116 cells transfected with stealth siRNA against IC53 showed a 64% decrease in proliferation compared with cells transfected with the negative control. (C) Knockdown of IC53 inhibited HCT-116 cell adhesion. HCT-116 cells were suspended in serum-free medium and seeded into collagen I (20 μg/mL)–coated 24-well plates. After incubating for 60 min at 37°C, the percentage of adhered cells was determined by using the colorimetric crystal violet assay. HCT-116 cells transfection with stealth siRNA against IC53 showed a 74% decrease in cell adhesion compared with cells transfected with the negative control. (D) Knockdown of IC53 inhibited HCT-116 cell migration. IC53-mediated cell migration was analyzed by the transwell assay. The cells were maintained in DMEM containing 10% FBS at 37°C. HCT-116 cells transfected with stealth siRNA against IC53 showed an 85% decrease in migration compared with cells transfected with the negative control. This figure represents 1 of 3 independent experiments, with quadruplicate samples in each experiment. The results represent the means ± SD in triplicate. ** P

Techniques Used: Migration, Expressing, Transfection, Negative Control, Western Blot, MTT Assay, Cell Culture, Crystal Violet Assay, Transwell Assay

Overexpression of IC53 promoted proliferation, migration and adhesion of HCT-116 cells. (A) Stable transfection of IC53 plasmids increased IC53 expression. IC53 expression was analyzed in serum-starved HCT-116 cells by Western blot analysis with an anti-IC53 monoclonal antibody (1:1,000). The expression level of IC53 in the cells transfected with IC53 plasmids (HCT-116-IC53) was four-fold greater than that of the cells transfected with empty plasmid (HCT-116-A) or the untransfected control (HCT-116). An anti-GAPDH antibody was included in the analysis as a loading control. (B) Overexpression of IC53 induced HCT-116 cell proliferation. Cell proliferation was assayed by using the MTT assay. The cells were maintained in DMEM containing 10% FBS and 200 μg/mL G418 and cultured in 5% CO 2 at 37°C. The media were replaced every 48 h. Stable transfection of IC53 (HCT-116-IC53) markedly promoted HCT-116 cell proliferation 2.1-fold on day 3, 2.6-fold on day 5 and 1.97-fold on day 7 after plating, compared with either the untransfected (HCT-116) control or the empty vector control (HCT-116-A), as determined by the optical density (OD) measurements. (C) Overexpression of IC53 induced HCT-116 cell adhesion. HCT-116 cells were suspended in serum-free medium and seeded into collagen I (20 μg/mL)–coated 24-well plates. After incubation for 60 min at 37°C, the percentage of adhered cells was determined by using the colorimetric crystal violet assay. Cells stably transfected with the IC53 expression construct (HCT-116-IC53) dramatically promoted HCT-116 cell adhesion by 183% after 60 min compared with the untransfected control (HCT-116) or cells transfected with empty vector (HCT-116-A). (D) Overexpression of IC53 induced HCT-116 cell migration. Cell migration was analyzed by using the transwell assay. The cells were maintained in DMEM containing 10% FBS and 200 μg/mL G418 and cultured in 5% CO 2 at 37°C. Cells stably transfected with IC53 expression construct (HCT-116-IC53) migrated 180% or 300% more than their parental (HCT-116) or vector transfected cells (HCT-116-A), respectively. This figure represents 1 of 3 independent experiments, with quadruplicate samples in each experiment. The results represent the mean ± SD in triplicate. ** P
Figure Legend Snippet: Overexpression of IC53 promoted proliferation, migration and adhesion of HCT-116 cells. (A) Stable transfection of IC53 plasmids increased IC53 expression. IC53 expression was analyzed in serum-starved HCT-116 cells by Western blot analysis with an anti-IC53 monoclonal antibody (1:1,000). The expression level of IC53 in the cells transfected with IC53 plasmids (HCT-116-IC53) was four-fold greater than that of the cells transfected with empty plasmid (HCT-116-A) or the untransfected control (HCT-116). An anti-GAPDH antibody was included in the analysis as a loading control. (B) Overexpression of IC53 induced HCT-116 cell proliferation. Cell proliferation was assayed by using the MTT assay. The cells were maintained in DMEM containing 10% FBS and 200 μg/mL G418 and cultured in 5% CO 2 at 37°C. The media were replaced every 48 h. Stable transfection of IC53 (HCT-116-IC53) markedly promoted HCT-116 cell proliferation 2.1-fold on day 3, 2.6-fold on day 5 and 1.97-fold on day 7 after plating, compared with either the untransfected (HCT-116) control or the empty vector control (HCT-116-A), as determined by the optical density (OD) measurements. (C) Overexpression of IC53 induced HCT-116 cell adhesion. HCT-116 cells were suspended in serum-free medium and seeded into collagen I (20 μg/mL)–coated 24-well plates. After incubation for 60 min at 37°C, the percentage of adhered cells was determined by using the colorimetric crystal violet assay. Cells stably transfected with the IC53 expression construct (HCT-116-IC53) dramatically promoted HCT-116 cell adhesion by 183% after 60 min compared with the untransfected control (HCT-116) or cells transfected with empty vector (HCT-116-A). (D) Overexpression of IC53 induced HCT-116 cell migration. Cell migration was analyzed by using the transwell assay. The cells were maintained in DMEM containing 10% FBS and 200 μg/mL G418 and cultured in 5% CO 2 at 37°C. Cells stably transfected with IC53 expression construct (HCT-116-IC53) migrated 180% or 300% more than their parental (HCT-116) or vector transfected cells (HCT-116-A), respectively. This figure represents 1 of 3 independent experiments, with quadruplicate samples in each experiment. The results represent the mean ± SD in triplicate. ** P

Techniques Used: Over Expression, Migration, Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, MTT Assay, Cell Culture, Incubation, Crystal Violet Assay, Construct, Transwell Assay

3) Product Images from "Conditioned Media from Human Adipose Tissue-Derived Mesenchymal Stem Cells and Umbilical Cord-Derived Mesenchymal Stem Cells Efficiently Induced the Apoptosis and Differentiation in Human Glioma Cell Lines In Vitro "

Article Title: Conditioned Media from Human Adipose Tissue-Derived Mesenchymal Stem Cells and Umbilical Cord-Derived Mesenchymal Stem Cells Efficiently Induced the Apoptosis and Differentiation in Human Glioma Cell Lines In Vitro

Journal: BioMed Research International

doi: 10.1155/2014/109389

Matrigel invasion assay. U251 cells or U87 cells (induced or noninduced) were placed in transwells with 8 μ m pore size polycarbonate filters, precoated with Matrigel. The lower wells were filled with DMEM with 10% fetal bovine serum. The cells were incubated at 37°C for 24 hours. Nonmigratory cells on the upper surface of the transwells were removed and the migratory cells from the lower surface of the transwell were fixed and stained with 1% crystal violet. Cells were photographed and average cells per field were calculated. Experiments were done in triplicates on three times independently.
Figure Legend Snippet: Matrigel invasion assay. U251 cells or U87 cells (induced or noninduced) were placed in transwells with 8 μ m pore size polycarbonate filters, precoated with Matrigel. The lower wells were filled with DMEM with 10% fetal bovine serum. The cells were incubated at 37°C for 24 hours. Nonmigratory cells on the upper surface of the transwells were removed and the migratory cells from the lower surface of the transwell were fixed and stained with 1% crystal violet. Cells were photographed and average cells per field were calculated. Experiments were done in triplicates on three times independently.

Techniques Used: Invasion Assay, Incubation, Staining

4) Product Images from "Large-scale ex vivo generation of human neutrophils from cord blood CD34+ cells"

Article Title: Large-scale ex vivo generation of human neutrophils from cord blood CD34+ cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0180832

Optimization of culture conditions for ex vivo expansion of human neutrophils from human UCB CD34 + cells. (A) Absolute cell numbers were calculated on a particular day (e.g., 5×10 4 CD34 + cells seeded on day 0). In Stage 1, isolated CD34 + cells were cultured for six days in various medium formulas ( Table 1 ), absolute numbers of the total cell [A(i)] and CD34 + cell [A(ii)] were calculated on day 6. (B) Stage 2 started with the cells derived from group MNF+SFGM3T (IMDM +Nutrition Supplements + 100ng/ml SCF + 100 ng/ml Flt-3L + 50ng/ml G-CSF + 20ng/ml TPO + 25ng/ml IL-3 +15ng/mL GM-CSF) of Stage 1. Absolute numbers of total cells [B(i)], CD66b + cell [B(ii)] were calculated on day 9 with G-CSF ranging from 50 to 100 ng/ml and GM-CSF ranging from 5 to 20 ng/ml in MNF+SFGM3 medium (IMDM + nutrition supplements + FBS + 100 ng/ml SCF + 100 ng/ml Flt-3L + 75 ng/ml G-CSF + 15 ng/ml IL-3 + 10 ng/ml GM-CSF). (C) Stage 3 started with the cells derived from group MNF+SFGM3 of Stage 2. Absolute numbers of total cell [C(i)], CD66b + cell [C(ii)] and were calculated on day 15 in different medium formulas with G-CSF ranging from 100 to 500 ng/ml in MNF+SFG (IMDM + nutrition supplements + FBS + 100 ng/ml SCF + 100 ng/ml Flt-3L + 100 ng/ml G-CSF) medium. Results are presented as means ± SD of 6 independent experiments. One-way ANOVA followed by Dunnett’s multiple comparison tests was used for comparison among the various treatment groups.* P
Figure Legend Snippet: Optimization of culture conditions for ex vivo expansion of human neutrophils from human UCB CD34 + cells. (A) Absolute cell numbers were calculated on a particular day (e.g., 5×10 4 CD34 + cells seeded on day 0). In Stage 1, isolated CD34 + cells were cultured for six days in various medium formulas ( Table 1 ), absolute numbers of the total cell [A(i)] and CD34 + cell [A(ii)] were calculated on day 6. (B) Stage 2 started with the cells derived from group MNF+SFGM3T (IMDM +Nutrition Supplements + 100ng/ml SCF + 100 ng/ml Flt-3L + 50ng/ml G-CSF + 20ng/ml TPO + 25ng/ml IL-3 +15ng/mL GM-CSF) of Stage 1. Absolute numbers of total cells [B(i)], CD66b + cell [B(ii)] were calculated on day 9 with G-CSF ranging from 50 to 100 ng/ml and GM-CSF ranging from 5 to 20 ng/ml in MNF+SFGM3 medium (IMDM + nutrition supplements + FBS + 100 ng/ml SCF + 100 ng/ml Flt-3L + 75 ng/ml G-CSF + 15 ng/ml IL-3 + 10 ng/ml GM-CSF). (C) Stage 3 started with the cells derived from group MNF+SFGM3 of Stage 2. Absolute numbers of total cell [C(i)], CD66b + cell [C(ii)] and were calculated on day 15 in different medium formulas with G-CSF ranging from 100 to 500 ng/ml in MNF+SFG (IMDM + nutrition supplements + FBS + 100 ng/ml SCF + 100 ng/ml Flt-3L + 100 ng/ml G-CSF) medium. Results are presented as means ± SD of 6 independent experiments. One-way ANOVA followed by Dunnett’s multiple comparison tests was used for comparison among the various treatment groups.* P

Techniques Used: Ex Vivo, Isolation, Cell Culture, Derivative Assay

5) Product Images from "Marinobufagenin inhibits glioma growth through sodium pump α1 subunit and ERK signaling‐mediated mitochondrial apoptotic pathway"

Article Title: Marinobufagenin inhibits glioma growth through sodium pump α1 subunit and ERK signaling‐mediated mitochondrial apoptotic pathway

Journal: Cancer Medicine

doi: 10.1002/cam4.1469

Sodium pump α 1 subunit could mediate the ERK ‐targeted anticancer effect of MBG . (A) MBG inhibited ATP 1A1 protein expression in human glioma cells. U87 MG and U251 cells were treated with MBG , respectively, after 48 h, cells were harvested for ATP 1A1 level measurement by Western blotting. (B) Knocking down ATP 1A1 markedly changed the IC 50 values of MBG for glioma cells. U87 MG cells were treated with si‐ ATP 1A1 for 5 h to knockdown ATP 1A1 expression, then incubated in normal DMEM medium supplemented with 15% fetal bovine serum for another 24 h. After that, cell lines were seeded at 6 × 10 3 cells/well in 96‐well plates, and cells were allowed to adhere for overnight. Then, cells were treated with MBG at indicated doses. MTT assay was exerted to determine cell viability, thus to determine whether ATP 1A1 knockdown could hinder the anticancer effect of MBG in human glioma cells. (C) MBG ‐induced inhibition of ERK phosphorylation could be impeded by ATP 1A1 knockdown. Western blotting was used to determine the levels of ERK phosphorylation in U87 MG cells treated with MBG under ATP 1A1 knocking down. (D) Schematic diagram of an apoptotic cell death induced by MBG via the ERK signaling pathway. All values are denoted as the mean ± SD . n = 3. ** P
Figure Legend Snippet: Sodium pump α 1 subunit could mediate the ERK ‐targeted anticancer effect of MBG . (A) MBG inhibited ATP 1A1 protein expression in human glioma cells. U87 MG and U251 cells were treated with MBG , respectively, after 48 h, cells were harvested for ATP 1A1 level measurement by Western blotting. (B) Knocking down ATP 1A1 markedly changed the IC 50 values of MBG for glioma cells. U87 MG cells were treated with si‐ ATP 1A1 for 5 h to knockdown ATP 1A1 expression, then incubated in normal DMEM medium supplemented with 15% fetal bovine serum for another 24 h. After that, cell lines were seeded at 6 × 10 3 cells/well in 96‐well plates, and cells were allowed to adhere for overnight. Then, cells were treated with MBG at indicated doses. MTT assay was exerted to determine cell viability, thus to determine whether ATP 1A1 knockdown could hinder the anticancer effect of MBG in human glioma cells. (C) MBG ‐induced inhibition of ERK phosphorylation could be impeded by ATP 1A1 knockdown. Western blotting was used to determine the levels of ERK phosphorylation in U87 MG cells treated with MBG under ATP 1A1 knocking down. (D) Schematic diagram of an apoptotic cell death induced by MBG via the ERK signaling pathway. All values are denoted as the mean ± SD . n = 3. ** P

Techniques Used: Expressing, Western Blot, Incubation, MTT Assay, Inhibition

6) Product Images from "Inhibitory Effect of Loranthus parasiticus on IgE-Mediated Allergic Responses in RBL-2H3 Cells"

Article Title: Inhibitory Effect of Loranthus parasiticus on IgE-Mediated Allergic Responses in RBL-2H3 Cells

Journal: Mediators of Inflammation

doi: 10.1155/2016/8742562

Effect of LPE on phosphorylation or expression of rate-limiting enzymes in the arachidonate cascade. RBL-2H3 cells were seeded on a 6-well plate (5 × 10 5 cells/well) in MEM- α with 10% FBS at 37°C overnight and further incubated with DNP-IgE for 24 h. IgE-sensitized RBL-2H3 cells were preincubated with LPE (0 to 350 μ g/mL) prior to antigen challenge. The above cells were washed with 1x DPBS and lysed with cell lysis buffer. The expression of p-cPLA 2 , p-5-LO, COX-2, or β -actin was determined as described in Section 2 . Similar results were obtained in three independent experiments. ∗∗ P
Figure Legend Snippet: Effect of LPE on phosphorylation or expression of rate-limiting enzymes in the arachidonate cascade. RBL-2H3 cells were seeded on a 6-well plate (5 × 10 5 cells/well) in MEM- α with 10% FBS at 37°C overnight and further incubated with DNP-IgE for 24 h. IgE-sensitized RBL-2H3 cells were preincubated with LPE (0 to 350 μ g/mL) prior to antigen challenge. The above cells were washed with 1x DPBS and lysed with cell lysis buffer. The expression of p-cPLA 2 , p-5-LO, COX-2, or β -actin was determined as described in Section 2 . Similar results were obtained in three independent experiments. ∗∗ P

Techniques Used: Expressing, Incubation, Lysis

Effect of LPE on degranulation and cell viability in IgE-mediated RBL-2H3 cells. RBL-2H3 cells were seeded on a 24-well plate (1 × 10 5 cells/well) or a 96-well plate (1 × 10 4 cells/well) in MEM- α with 10% FBS at 37°C overnight and further incubated with DNP-IgE for 24 h. IgE-sensitized cells were preincubated with LPE (0 to 400 μ g/mL) for 1 h and then stimulated with DNP-HSA (0.1 μ g/mL) for 4 h. β -Hexosaminidase activity and cell viability were determined as described in Section 2 . Data are the mean ± SD values of triple or octuple determinations. ∗∗ P
Figure Legend Snippet: Effect of LPE on degranulation and cell viability in IgE-mediated RBL-2H3 cells. RBL-2H3 cells were seeded on a 24-well plate (1 × 10 5 cells/well) or a 96-well plate (1 × 10 4 cells/well) in MEM- α with 10% FBS at 37°C overnight and further incubated with DNP-IgE for 24 h. IgE-sensitized cells were preincubated with LPE (0 to 400 μ g/mL) for 1 h and then stimulated with DNP-HSA (0.1 μ g/mL) for 4 h. β -Hexosaminidase activity and cell viability were determined as described in Section 2 . Data are the mean ± SD values of triple or octuple determinations. ∗∗ P

Techniques Used: Incubation, Activity Assay

7) Product Images from "Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-?-Mediated and Non-Transforming Growth Factor-?-Mediated Transdifferentiation in Vitro"

Article Title: Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-?-Mediated and Non-Transforming Growth Factor-?-Mediated Transdifferentiation in Vitro

Journal: The American Journal of Pathology

doi:

Transdifferentiated cells exhibit increased migration in response to PDGF-BB. A: Av 17 cells cultured in growth medium ( hatched bars ) or in reduced medium ( solid bars ) for 6 days were tested for the ability to migrate toward EBM with 0.1% bovine serum albumin (control), EBM with 5% FBS (serum), 1 ng/ml bFGF, 10 ng/ml PDGF-BB, 50 ng/ml PDGF-BB, or 1 ng/ml TGF-β1. B: A neutralizing anti-PDGF-BB monoclonal antibody (mAb) and an isotype-matched IgG were tested for the ability to block the migration of av 17 cells, which were grown in reduced medium for 6 days, toward 10 ng/ml PDGF-BB. C: Av 15 cells cultured in growth medium ( hatched bars ) or in growth medium with 1 ng/ml TGF-β1 ( solid bars ) for 6 days were tested as in A . D: Checkerboard analysis was performed on av 17 cells grown in reduced medium for 6 days. PDGF-BB at 0, 1, 10, or 50 ng/ml was added to top and bottom wells as indicated, and cells allowed to migrate for 4 hours. The shaded boxes on the diagonal highlight the finding that PDGF-BB does not elicit random cell migration toward increasing concentrations of PDGF-BB in the upper and lower chambers.
Figure Legend Snippet: Transdifferentiated cells exhibit increased migration in response to PDGF-BB. A: Av 17 cells cultured in growth medium ( hatched bars ) or in reduced medium ( solid bars ) for 6 days were tested for the ability to migrate toward EBM with 0.1% bovine serum albumin (control), EBM with 5% FBS (serum), 1 ng/ml bFGF, 10 ng/ml PDGF-BB, 50 ng/ml PDGF-BB, or 1 ng/ml TGF-β1. B: A neutralizing anti-PDGF-BB monoclonal antibody (mAb) and an isotype-matched IgG were tested for the ability to block the migration of av 17 cells, which were grown in reduced medium for 6 days, toward 10 ng/ml PDGF-BB. C: Av 15 cells cultured in growth medium ( hatched bars ) or in growth medium with 1 ng/ml TGF-β1 ( solid bars ) for 6 days were tested as in A . D: Checkerboard analysis was performed on av 17 cells grown in reduced medium for 6 days. PDGF-BB at 0, 1, 10, or 50 ng/ml was added to top and bottom wells as indicated, and cells allowed to migrate for 4 hours. The shaded boxes on the diagonal highlight the finding that PDGF-BB does not elicit random cell migration toward increasing concentrations of PDGF-BB in the upper and lower chambers.

Techniques Used: Migration, Cell Culture, Blocking Assay

8) Product Images from "Sulforaphane Protects Astrocytes against Oxidative Stress and Delayed Death Caused by Oxygen and Glucose Deprivation"

Article Title: Sulforaphane Protects Astrocytes against Oxidative Stress and Delayed Death Caused by Oxygen and Glucose Deprivation

Journal: Glia

doi: 10.1002/glia.20793

Sulforaphane pre-treatment increases NRF2 mRNA and protein expression in rat cortical astrocytes A: Cortical rat astrocytes were pre-treated for 48h with SFP or vehicle and total RNA was extracted as described in Methods. The NRF2 mRNA fold change was calculated relative to control. Control is represented by cells maintained in DMEM/F12 medium supplemented with 10% FBS. NRF2 mRNA values were normalized to β-actin. The results are represented as mean ± SEM (n = 4). Data were analyzed using Student’s t-test. # p
Figure Legend Snippet: Sulforaphane pre-treatment increases NRF2 mRNA and protein expression in rat cortical astrocytes A: Cortical rat astrocytes were pre-treated for 48h with SFP or vehicle and total RNA was extracted as described in Methods. The NRF2 mRNA fold change was calculated relative to control. Control is represented by cells maintained in DMEM/F12 medium supplemented with 10% FBS. NRF2 mRNA values were normalized to β-actin. The results are represented as mean ± SEM (n = 4). Data were analyzed using Student’s t-test. # p

Techniques Used: Expressing

9) Product Images from "The Importance of the Prenyl Group in the Activities of Osthole in Enhancing Bone Formation and Inhibiting Bone Resorption In Vitro"

Article Title: The Importance of the Prenyl Group in the Activities of Osthole in Enhancing Bone Formation and Inhibiting Bone Resorption In Vitro

Journal: International Journal of Endocrinology

doi: 10.1155/2014/921954

Effects of treatment with 10 −5 M 7-methoxycoumarin or osthole or neither (control) on TRAP activity in cultured rabbit osteoclasts in α -MEM containing 15% FBS and 10 −8 M 1,25(OH) 2 D 3 . TRAP activity was expressed as the absorbance value at 530 nm. Data were shown as mean ± SD of triplicate cultures. * P
Figure Legend Snippet: Effects of treatment with 10 −5 M 7-methoxycoumarin or osthole or neither (control) on TRAP activity in cultured rabbit osteoclasts in α -MEM containing 15% FBS and 10 −8 M 1,25(OH) 2 D 3 . TRAP activity was expressed as the absorbance value at 530 nm. Data were shown as mean ± SD of triplicate cultures. * P

Techniques Used: Activity Assay, Cell Culture

Dose-dependent effects of 7-methoxycoumarin (a) and osthole (b) on the viability of rat bone marrow stromal cells (rBMSCs) and rat calverial osteoblasts (ROB). The cells were grown and treated with culture medium containing 3% FBS and indicated concentrations of 7-methoxycoumarin and osthole. The cell viability of rBMSCs and ROB were measured by MTT assay for 48 h. The data represented three experiments and were shown as the mean ± SD of sextuplicate wells. * P
Figure Legend Snippet: Dose-dependent effects of 7-methoxycoumarin (a) and osthole (b) on the viability of rat bone marrow stromal cells (rBMSCs) and rat calverial osteoblasts (ROB). The cells were grown and treated with culture medium containing 3% FBS and indicated concentrations of 7-methoxycoumarin and osthole. The cell viability of rBMSCs and ROB were measured by MTT assay for 48 h. The data represented three experiments and were shown as the mean ± SD of sextuplicate wells. * P

Techniques Used: MTT Assay

10) Product Images from "Acetylated Nanocellulose for Single-Component Bioinks and Cell Proliferation on 3D-Printed Scaffolds"

Article Title: Acetylated Nanocellulose for Single-Component Bioinks and Cell Proliferation on 3D-Printed Scaffolds

Journal: Biomacromolecules

doi: 10.1021/acs.biomac.9b00527

Fluorescence microscopy images of the cell populations seeded on nanocellulose samples in DMEM + 10% FBS on day 4: (a) CNF, (b) TOCNF, and (c) AceCNF. Cells were fixed with 4% PFA, and the nuclei of cells were stained with DAPI. The comparison shows a high population of H9C2 cells on the AceCNF 3D structure, almost as much as that on TOCNF, whereas a lower population is observed on CNF.
Figure Legend Snippet: Fluorescence microscopy images of the cell populations seeded on nanocellulose samples in DMEM + 10% FBS on day 4: (a) CNF, (b) TOCNF, and (c) AceCNF. Cells were fixed with 4% PFA, and the nuclei of cells were stained with DAPI. The comparison shows a high population of H9C2 cells on the AceCNF 3D structure, almost as much as that on TOCNF, whereas a lower population is observed on CNF.

Techniques Used: Fluorescence, Microscopy, Staining

(a) H9C2 viability of nanocellulose samples during 21 days in DMEM + FBS 10%, showing high biocompatibility and proliferation by an AlamarBlue assay. Note the positive control consisting of pure myoblast and Pyrex cylinders in DMEM + 10% FBS without the nanocellulose samples. (b) H9C2 proliferation of nanocellulose samples inside DMEM + FBS 10% for 21 days based on the fluorescence intensity obtained from the AlamarBlue assay.
Figure Legend Snippet: (a) H9C2 viability of nanocellulose samples during 21 days in DMEM + FBS 10%, showing high biocompatibility and proliferation by an AlamarBlue assay. Note the positive control consisting of pure myoblast and Pyrex cylinders in DMEM + 10% FBS without the nanocellulose samples. (b) H9C2 proliferation of nanocellulose samples inside DMEM + FBS 10% for 21 days based on the fluorescence intensity obtained from the AlamarBlue assay.

Techniques Used: Alamar Blue Assay, Positive Control, Fluorescence

11) Product Images from "High glucose downregulates myocardin expression in rat glomerular mesangial cells via the ERK signaling pathway"

Article Title: High glucose downregulates myocardin expression in rat glomerular mesangial cells via the ERK signaling pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.20498

High glucose decreased myocardin and SM α-actin mRNA levels Rat MCs were rendered quiescent in DMEM low glucose medium containing 0 % fetal bovine serum for 24 h and then treated with or without 30 mM D-glucose or mannitol for the indicated times. RNA was extracted for RT-qPCR analyses. (A) RT-qPCR data showing myocardin mRNA levels in rat glomerular MCs with or without 30 mM D-glucose treatment for different times. (B) RT-qPCR data showing SM α-actin mRNA levels in rat glomerular MCs with or without 30 mM D-glucose treatment for different times. (C) RT-qPCR data showing myocardin mRNA levels in rat glomerular MCs with or without 30 mM mannitol treatment for different times. (D) RT-qPCR data showing SM α-actin mRNA levels in rat glomerular MCs with or without 30 mM mannitol treatment for different times. RT-qPCR results were normalized to β-actin . * P
Figure Legend Snippet: High glucose decreased myocardin and SM α-actin mRNA levels Rat MCs were rendered quiescent in DMEM low glucose medium containing 0 % fetal bovine serum for 24 h and then treated with or without 30 mM D-glucose or mannitol for the indicated times. RNA was extracted for RT-qPCR analyses. (A) RT-qPCR data showing myocardin mRNA levels in rat glomerular MCs with or without 30 mM D-glucose treatment for different times. (B) RT-qPCR data showing SM α-actin mRNA levels in rat glomerular MCs with or without 30 mM D-glucose treatment for different times. (C) RT-qPCR data showing myocardin mRNA levels in rat glomerular MCs with or without 30 mM mannitol treatment for different times. (D) RT-qPCR data showing SM α-actin mRNA levels in rat glomerular MCs with or without 30 mM mannitol treatment for different times. RT-qPCR results were normalized to β-actin . * P

Techniques Used: Quantitative RT-PCR

High glucose decreased myocardin and SM α-actin protein levels in rat glomerular MCs Rat MCs were rendered quiescent in low-glucose DMEM medium containing 0 % fetal bovine serum for 24 h, and then treated with or without 30 mM D-glucose or mannitol. Protein was extracted for Western blot analyses. (A) Cumulative Western blot results showing myocardin protein levels with or without 30 mM D-glucose or mannitol for 24 h. Insert: Representative Western blots of myocardin and α-tubulin. (B) Cumulative Western blot results showing SM α-actin protein levels in rat glomerular MCs with or without 30 mM D-glucose or mannitol for 24 h. Insert: Representative Western blots of SM α-actin and α-tubulin. Western blots results were normalized to α-tubulin. * P
Figure Legend Snippet: High glucose decreased myocardin and SM α-actin protein levels in rat glomerular MCs Rat MCs were rendered quiescent in low-glucose DMEM medium containing 0 % fetal bovine serum for 24 h, and then treated with or without 30 mM D-glucose or mannitol. Protein was extracted for Western blot analyses. (A) Cumulative Western blot results showing myocardin protein levels with or without 30 mM D-glucose or mannitol for 24 h. Insert: Representative Western blots of myocardin and α-tubulin. (B) Cumulative Western blot results showing SM α-actin protein levels in rat glomerular MCs with or without 30 mM D-glucose or mannitol for 24 h. Insert: Representative Western blots of SM α-actin and α-tubulin. Western blots results were normalized to α-tubulin. * P

Techniques Used: Western Blot

L-glucose did not regulate myocardin and SM α-actin expression Rat MCs were rendered quiescent in low–glucose DMEM medium containing 0 % fetal bovine serum for 24 h and then treated with or without 30 mM D-glucose or L-glucose. Total RNA and protein were extracted for RT-qPCR and Western blot analyses, respectively. (A) RT-qPCR data showing myocardin mRNA levels with or without 30 mM L-glucose treatment for 24 h. (B) RT-qPCR data showing SM α-actin mRNA levels with or without 30 mM L-glucose treatment for 24 h. (C) Cumulative Western blot results showing myocardin protein levels with or without 30 mM L-glucose treatment for 24 h. Insert: Representative Western blots of myocardin and α-tubulin. (D) Cumulative Western blot results showing SM α-actin protein levels with or without 30 mM L-glucose treatment for 24 h in rat glomerular MCs. Insert: Representative Western blots of SM α-actin and α-tubulin. RT-qPCR results were normalized to β-actin, and the Western blot results were normalized to α-tubulin. * P
Figure Legend Snippet: L-glucose did not regulate myocardin and SM α-actin expression Rat MCs were rendered quiescent in low–glucose DMEM medium containing 0 % fetal bovine serum for 24 h and then treated with or without 30 mM D-glucose or L-glucose. Total RNA and protein were extracted for RT-qPCR and Western blot analyses, respectively. (A) RT-qPCR data showing myocardin mRNA levels with or without 30 mM L-glucose treatment for 24 h. (B) RT-qPCR data showing SM α-actin mRNA levels with or without 30 mM L-glucose treatment for 24 h. (C) Cumulative Western blot results showing myocardin protein levels with or without 30 mM L-glucose treatment for 24 h. Insert: Representative Western blots of myocardin and α-tubulin. (D) Cumulative Western blot results showing SM α-actin protein levels with or without 30 mM L-glucose treatment for 24 h in rat glomerular MCs. Insert: Representative Western blots of SM α-actin and α-tubulin. RT-qPCR results were normalized to β-actin, and the Western blot results were normalized to α-tubulin. * P

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

12) Product Images from "Basolateral delivery of the type I transforming growth factor beta receptor is mediated by a dominant-acting cytoplasmic motif"

Article Title: Basolateral delivery of the type I transforming growth factor beta receptor is mediated by a dominant-acting cytoplasmic motif

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E17-05-0334

In the absence of the VEED motif, type I TGFβRs are directly targeted to the apical membrane. (A) Polarized MDCK cells transiently transfected with either native wild-type TβRI or TβRIVEED/AAAA (TβRI-4X) for 16 h were Golgi blocked at 20°C for 3 h in serum-free DMEM. Following washing with cold PBS the apical and basal chambers were then treated with a dilute (0.05%) trypsin/PBS solution for the last 30 min of the Golgi block to remove cell surface proteins (0 min). After a PBS wash, prewarmed fresh 10% FBS/DMEM was added, and the plates were returned to 37°C and stained for the Myc-tagged type I TGFβR at the indicated times after release. (B) Quantitation of apical receptor expression as observed in A presented as arbitrary fluorescence units ± SEM of 25 cells from three independent experiments. (C) αIβII and αIVEED/AAAAβII (αI-4XβII) MDCK cell lines were polarized on 12-mm Transwell plates. Subsequent to Golgi block and trypsinization as described in A, newly expressed chimeric type I TGFβRs were visualized by immunofluorescence from 20 to 150 min after release. Cells were stained for TβRI using primary antibody to the external GM-CSF α chain and secondarily stained by Cy3 (red). Images are presented as perpendicular XZ cross-sectional images. Nuclei (blue) were stained with DAPI. (D) Quantitation as performed in B of 30 cells from three independent experiments.
Figure Legend Snippet: In the absence of the VEED motif, type I TGFβRs are directly targeted to the apical membrane. (A) Polarized MDCK cells transiently transfected with either native wild-type TβRI or TβRIVEED/AAAA (TβRI-4X) for 16 h were Golgi blocked at 20°C for 3 h in serum-free DMEM. Following washing with cold PBS the apical and basal chambers were then treated with a dilute (0.05%) trypsin/PBS solution for the last 30 min of the Golgi block to remove cell surface proteins (0 min). After a PBS wash, prewarmed fresh 10% FBS/DMEM was added, and the plates were returned to 37°C and stained for the Myc-tagged type I TGFβR at the indicated times after release. (B) Quantitation of apical receptor expression as observed in A presented as arbitrary fluorescence units ± SEM of 25 cells from three independent experiments. (C) αIβII and αIVEED/AAAAβII (αI-4XβII) MDCK cell lines were polarized on 12-mm Transwell plates. Subsequent to Golgi block and trypsinization as described in A, newly expressed chimeric type I TGFβRs were visualized by immunofluorescence from 20 to 150 min after release. Cells were stained for TβRI using primary antibody to the external GM-CSF α chain and secondarily stained by Cy3 (red). Images are presented as perpendicular XZ cross-sectional images. Nuclei (blue) were stained with DAPI. (D) Quantitation as performed in B of 30 cells from three independent experiments.

Techniques Used: Transfection, Blocking Assay, Staining, Quantitation Assay, Expressing, Fluorescence, Immunofluorescence

Apical mislocalized TGFβRs are signaling competent. (A) Type I TGFβR basolateral targeting motif has no effect on TGF-β-induced Smad3 signaling. Top: Chimeric receptor expressing MDCK lines expressing wild-type (αIβII #7) type I and type II receptors or a wild-type type II receptor and type I receptor mutated in the VEED basolateral targeting domain (αI-4XβII #2, #4, and #6) were treated in the absence (-) or presence of GM-CSF (GM, 100 ng/ml) or TGF-β (10 ng/ml) for 1 h before being processed for Western analysis using phospho (p) or total (t) Smad3 sera. Bottom: R1B Mv1Lu cells (do not express native TβRI; Boyd and Massagué, 1989 ) were transiently transfected with either native wild-type TβRI or TβRI mutated in the VEED domain (TβRI-4X) directing basolateral receptor delivery. Phospho and total Smad3 was determined following 1 h stimulation ± 10 ng/ml TGF-β. (B) MDCK cells stably expressing either a wild-type chimeric type I receptor (αI) and a type II receptor ( Murphy et al. , 2007 ) mutated such that it mislocalizes to the apical membrane (αIβIIA531G #43) or chimeric type I and type II receptors that both undergo apical trafficking (αI-4XβIIA531G #44) were polarized on transwell inserts and stained for the indicated proteins. Images are presented as perpendicular XZ confocal cross-sections, and nuclei were stained with DAPI. (C) Cartoon depicting the locale of native and chimeric TGFβRs based on the chimeric receptors immunostaining data from B. (D) Chimeric clones from B were cultured in six-well transwells for 72 h. Cells were serum starved with 0.1% FBS/DMEM for 16 h and then either left untreated (-) or stimulated with TGF-β (10 ng/ml) or GM-CSF (100 ng/ml) from apical (AP), basolateral (BL), or both sides (T) at 37°C for 1 h. Equivalent protein was processed by Western blotting for phospho (p) or total (t) Smad3. Blots for A and D are representative of three separate experiments. (E) Polarized MDCK clones from D as well as three additional clones (e.g., #21, #27, and #39) expressing apically targeting chimeric TβRI and TβRII were treated as in D for 3 h with either GM-CSF (100 ng/ml; left) or TGF-β (10 ng/ml; right) and processed by RT-PCR for expression of PAI-1. Data reflect mean ± SEM from three biological replicates for control αIβIIA531G (#43) and pooled replicates for each ( n = 8) of the αI-4XβIIA531G clones (#21, #27, #39, and #44).
Figure Legend Snippet: Apical mislocalized TGFβRs are signaling competent. (A) Type I TGFβR basolateral targeting motif has no effect on TGF-β-induced Smad3 signaling. Top: Chimeric receptor expressing MDCK lines expressing wild-type (αIβII #7) type I and type II receptors or a wild-type type II receptor and type I receptor mutated in the VEED basolateral targeting domain (αI-4XβII #2, #4, and #6) were treated in the absence (-) or presence of GM-CSF (GM, 100 ng/ml) or TGF-β (10 ng/ml) for 1 h before being processed for Western analysis using phospho (p) or total (t) Smad3 sera. Bottom: R1B Mv1Lu cells (do not express native TβRI; Boyd and Massagué, 1989 ) were transiently transfected with either native wild-type TβRI or TβRI mutated in the VEED domain (TβRI-4X) directing basolateral receptor delivery. Phospho and total Smad3 was determined following 1 h stimulation ± 10 ng/ml TGF-β. (B) MDCK cells stably expressing either a wild-type chimeric type I receptor (αI) and a type II receptor ( Murphy et al. , 2007 ) mutated such that it mislocalizes to the apical membrane (αIβIIA531G #43) or chimeric type I and type II receptors that both undergo apical trafficking (αI-4XβIIA531G #44) were polarized on transwell inserts and stained for the indicated proteins. Images are presented as perpendicular XZ confocal cross-sections, and nuclei were stained with DAPI. (C) Cartoon depicting the locale of native and chimeric TGFβRs based on the chimeric receptors immunostaining data from B. (D) Chimeric clones from B were cultured in six-well transwells for 72 h. Cells were serum starved with 0.1% FBS/DMEM for 16 h and then either left untreated (-) or stimulated with TGF-β (10 ng/ml) or GM-CSF (100 ng/ml) from apical (AP), basolateral (BL), or both sides (T) at 37°C for 1 h. Equivalent protein was processed by Western blotting for phospho (p) or total (t) Smad3. Blots for A and D are representative of three separate experiments. (E) Polarized MDCK clones from D as well as three additional clones (e.g., #21, #27, and #39) expressing apically targeting chimeric TβRI and TβRII were treated as in D for 3 h with either GM-CSF (100 ng/ml; left) or TGF-β (10 ng/ml; right) and processed by RT-PCR for expression of PAI-1. Data reflect mean ± SEM from three biological replicates for control αIβIIA531G (#43) and pooled replicates for each ( n = 8) of the αI-4XβIIA531G clones (#21, #27, #39, and #44).

Techniques Used: Expressing, Western Blot, Transfection, Stable Transfection, Staining, Immunostaining, Clone Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction

13) Product Images from "cAMP/Protein Kinase A Signaling Inhibits Dlx5 Expression via Activation of CREB and Subsequent C/EBPβ Induction in 3T3-L1 Preadipocytes"

Article Title: cAMP/Protein Kinase A Signaling Inhibits Dlx5 Expression via Activation of CREB and Subsequent C/EBPβ Induction in 3T3-L1 Preadipocytes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19103161

Activation of cAMP/PKA (protein kinase A) signaling inhibits distal-less homeobox 5 (Dlx5) expression in 3T3-L1 preadipocytes. ( A ) PKA activation suppresses Dlx5 expression during adipogenesis. Two-day post confluent 3T3-L1 cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) with indicated concentration of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1 µM dexamethasone (DEX), 10 µg/mL insulin, and 50 µM indomethacin (IND) for 24 h, and Dlx5 mRNA levels were determined by quantitative RT-PCR. ( B ) Dlx5 protein level was determined by Western blot. ( C ) Dlx5 mRNA level was determined in the cells treated with various concentrations of IBMX, forskolin, or 8-CPT-cAMP for 24 h. ( D ) Dlx5 protein level after 24 h treatment with 0.5 mM IBMX, 2 mM 8-CPT-cAMP (8-CPT), or 50 µM forskolin (FSK) was determined by Western blot. In IBMX-induced adipogenesis, inhibition of PKA activation by 50 µM H89 completely rescued Dlx5 mRNA expression ( E ) and protein ( F ) levels. Data represent the mean ± SD (standard deviation) (* p
Figure Legend Snippet: Activation of cAMP/PKA (protein kinase A) signaling inhibits distal-less homeobox 5 (Dlx5) expression in 3T3-L1 preadipocytes. ( A ) PKA activation suppresses Dlx5 expression during adipogenesis. Two-day post confluent 3T3-L1 cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) with indicated concentration of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1 µM dexamethasone (DEX), 10 µg/mL insulin, and 50 µM indomethacin (IND) for 24 h, and Dlx5 mRNA levels were determined by quantitative RT-PCR. ( B ) Dlx5 protein level was determined by Western blot. ( C ) Dlx5 mRNA level was determined in the cells treated with various concentrations of IBMX, forskolin, or 8-CPT-cAMP for 24 h. ( D ) Dlx5 protein level after 24 h treatment with 0.5 mM IBMX, 2 mM 8-CPT-cAMP (8-CPT), or 50 µM forskolin (FSK) was determined by Western blot. In IBMX-induced adipogenesis, inhibition of PKA activation by 50 µM H89 completely rescued Dlx5 mRNA expression ( E ) and protein ( F ) levels. Data represent the mean ± SD (standard deviation) (* p

Techniques Used: Activation Assay, Expressing, Incubation, Modification, Concentration Assay, Quantitative RT-PCR, Western Blot, Cycling Probe Technology, Inhibition, Standard Deviation

14) Product Images from "cAMP/Protein Kinase A Signaling Inhibits Dlx5 Expression via Activation of CREB and Subsequent C/EBPβ Induction in 3T3-L1 Preadipocytes"

Article Title: cAMP/Protein Kinase A Signaling Inhibits Dlx5 Expression via Activation of CREB and Subsequent C/EBPβ Induction in 3T3-L1 Preadipocytes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19103161

Activation of cAMP/PKA (protein kinase A) signaling inhibits distal-less homeobox 5 (Dlx5) expression in 3T3-L1 preadipocytes. ( A ) PKA activation suppresses Dlx5 expression during adipogenesis. Two-day post confluent 3T3-L1 cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) with indicated concentration of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1 µM dexamethasone (DEX), 10 µg/mL insulin, and 50 µM indomethacin (IND) for 24 h, and Dlx5 mRNA levels were determined by quantitative RT-PCR. ( B ) Dlx5 protein level was determined by Western blot. ( C ) Dlx5 mRNA level was determined in the cells treated with various concentrations of IBMX, forskolin, or 8-CPT-cAMP for 24 h. ( D ) Dlx5 protein level after 24 h treatment with 0.5 mM IBMX, 2 mM 8-CPT-cAMP (8-CPT), or 50 µM forskolin (FSK) was determined by Western blot. In IBMX-induced adipogenesis, inhibition of PKA activation by 50 µM H89 completely rescued Dlx5 mRNA expression ( E ) and protein ( F ) levels. Data represent the mean ± SD (standard deviation) (* p
Figure Legend Snippet: Activation of cAMP/PKA (protein kinase A) signaling inhibits distal-less homeobox 5 (Dlx5) expression in 3T3-L1 preadipocytes. ( A ) PKA activation suppresses Dlx5 expression during adipogenesis. Two-day post confluent 3T3-L1 cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) with indicated concentration of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1 µM dexamethasone (DEX), 10 µg/mL insulin, and 50 µM indomethacin (IND) for 24 h, and Dlx5 mRNA levels were determined by quantitative RT-PCR. ( B ) Dlx5 protein level was determined by Western blot. ( C ) Dlx5 mRNA level was determined in the cells treated with various concentrations of IBMX, forskolin, or 8-CPT-cAMP for 24 h. ( D ) Dlx5 protein level after 24 h treatment with 0.5 mM IBMX, 2 mM 8-CPT-cAMP (8-CPT), or 50 µM forskolin (FSK) was determined by Western blot. In IBMX-induced adipogenesis, inhibition of PKA activation by 50 µM H89 completely rescued Dlx5 mRNA expression ( E ) and protein ( F ) levels. Data represent the mean ± SD (standard deviation) (* p

Techniques Used: Activation Assay, Expressing, Incubation, Modification, Concentration Assay, Quantitative RT-PCR, Western Blot, Cycling Probe Technology, Inhibition, Standard Deviation

15) Product Images from "An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation"

Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation

Journal: Oncotarget

doi: 10.18632/oncotarget.7502

EBNA3C residues 130-159 of EBV is critical for G1 to S phase progression A. Human PBMCs were grown for 12 h in RPMI medium containing 10% FBS (+serum) or 0.1% FBS (−serum). Further the cells were infected with BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus. Propidium iodide stained cells were analyzed by flow cytometry. B. and C. The bar diagram represents the change in cell cycle profile in G1 and S-G2M.
Figure Legend Snippet: EBNA3C residues 130-159 of EBV is critical for G1 to S phase progression A. Human PBMCs were grown for 12 h in RPMI medium containing 10% FBS (+serum) or 0.1% FBS (−serum). Further the cells were infected with BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus. Propidium iodide stained cells were analyzed by flow cytometry. B. and C. The bar diagram represents the change in cell cycle profile in G1 and S-G2M.

Techniques Used: Infection, Staining, Flow Cytometry, Cytometry

16) Product Images from "Differential Regulation of CXCL5 by FGF2 in Osteoblastic and Endothelial Niche Cells Supports Hematopoietic Stem Cell Migration"

Article Title: Differential Regulation of CXCL5 by FGF2 in Osteoblastic and Endothelial Niche Cells Supports Hematopoietic Stem Cell Migration

Journal: Stem Cells and Development

doi: 10.1089/scd.2012.0128

Altered expression of HSC niche-related genes and SDF-1 secretion by FGF2 in ELBOSs. (A) Confluent ELBOSs were cultured in 2% fetal bovine serum (FBS) medium (starvation conditions). The starved ELBOSs were treated with FGF2 (20 or 80 ng/mL) for
Figure Legend Snippet: Altered expression of HSC niche-related genes and SDF-1 secretion by FGF2 in ELBOSs. (A) Confluent ELBOSs were cultured in 2% fetal bovine serum (FBS) medium (starvation conditions). The starved ELBOSs were treated with FGF2 (20 or 80 ng/mL) for

Techniques Used: Expressing, Cell Culture

Altered expression of HSC niche-related genes and SDF-1 secretion by FGF2 in C166 cells. (A) Confluent C166 cells were cultured in 2% FBS medium for starvation. The starved C166 cells were treated with FGF2 (20 ng or 80 ng/mL) for 48 h.
Figure Legend Snippet: Altered expression of HSC niche-related genes and SDF-1 secretion by FGF2 in C166 cells. (A) Confluent C166 cells were cultured in 2% FBS medium for starvation. The starved C166 cells were treated with FGF2 (20 ng or 80 ng/mL) for 48 h.

Techniques Used: Expressing, Cell Culture

17) Product Images from "The Herbal Medicine KIOM-MA128 Inhibits the Antigen/IgE-Mediated Allergic Response in Vitro and in Vivo"

Article Title: The Herbal Medicine KIOM-MA128 Inhibits the Antigen/IgE-Mediated Allergic Response in Vitro and in Vivo

Journal: Molecules

doi: 10.3390/molecules21081015

Inhibitory effects of KIOM-MA128 on PGD 2 production and the activation of the arachidonate cascade. RBL-2H3 mast cells were seeded on a 6-well plate (5 × 10 5 cells/well) in MEM-α with 10% FBS and incubated overnight at 37 °C. The cells were further incubated with DNP-IgE (0.1 μg) for 24 h and then treated with KIOM-MA128 (0–2000 μg/mL). After 1 h, they were stimulated with DNP-Ag (0.1 μg/mL) for 4 h. The amounts of PGD 2 were determined as described in the Materials and Methods section. The data represent the mean ± SD values of three independent experiments. The cells were washed with 1× DPBS and lysed with cell lysis buffer. The levels of p-cPLA 2 , COX-2 and α-tubulin were determined using the procedure described in the Materials and Methods section. # p
Figure Legend Snippet: Inhibitory effects of KIOM-MA128 on PGD 2 production and the activation of the arachidonate cascade. RBL-2H3 mast cells were seeded on a 6-well plate (5 × 10 5 cells/well) in MEM-α with 10% FBS and incubated overnight at 37 °C. The cells were further incubated with DNP-IgE (0.1 μg) for 24 h and then treated with KIOM-MA128 (0–2000 μg/mL). After 1 h, they were stimulated with DNP-Ag (0.1 μg/mL) for 4 h. The amounts of PGD 2 were determined as described in the Materials and Methods section. The data represent the mean ± SD values of three independent experiments. The cells were washed with 1× DPBS and lysed with cell lysis buffer. The levels of p-cPLA 2 , COX-2 and α-tubulin were determined using the procedure described in the Materials and Methods section. # p

Techniques Used: Activation Assay, Incubation, Lysis

Effect of KIOM-MA128 on cell viability in IgE/Ag-activated RBL-2H3 mast cells. RBL-2H3 mast cells were seeded on a 96-well plate (1 × 10 4 cells/well) in MEM-α with 10% FBS and incubated overnight at 37 °C. The cells were further incubated with DNP-IgE (0.1 μg) for 24 h and then treated with KIOM-MA128 (0–2000 μg/mL). After 1 h, they were stimulated with DNP-Ag (0.1 μg/mL) for 4 h. Cell viability was determined using the procedure described in the Materials and Methods section. The data represent the mean ± SD values of three independent experiments.
Figure Legend Snippet: Effect of KIOM-MA128 on cell viability in IgE/Ag-activated RBL-2H3 mast cells. RBL-2H3 mast cells were seeded on a 96-well plate (1 × 10 4 cells/well) in MEM-α with 10% FBS and incubated overnight at 37 °C. The cells were further incubated with DNP-IgE (0.1 μg) for 24 h and then treated with KIOM-MA128 (0–2000 μg/mL). After 1 h, they were stimulated with DNP-Ag (0.1 μg/mL) for 4 h. Cell viability was determined using the procedure described in the Materials and Methods section. The data represent the mean ± SD values of three independent experiments.

Techniques Used: Incubation

18) Product Images from "Transcriptomic analysis of Campylobacter jejuni NCTC 11168 in response to epinephrine and norepinephrine"

Article Title: Transcriptomic analysis of Campylobacter jejuni NCTC 11168 in response to epinephrine and norepinephrine

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.00452

Growth response of C. jejuni NCTC 11168 to Epi or NE in iron-restricted MEMα medium containing 10% FBS . Results represent the mean plus the standard deviation (SD) of three independent experiments.
Figure Legend Snippet: Growth response of C. jejuni NCTC 11168 to Epi or NE in iron-restricted MEMα medium containing 10% FBS . Results represent the mean plus the standard deviation (SD) of three independent experiments.

Techniques Used: Standard Deviation

19) Product Images from "Conditioned Media from Human Adipose Tissue-Derived Mesenchymal Stem Cells and Umbilical Cord-Derived Mesenchymal Stem Cells Efficiently Induced the Apoptosis and Differentiation in Human Glioma Cell Lines In Vitro "

Article Title: Conditioned Media from Human Adipose Tissue-Derived Mesenchymal Stem Cells and Umbilical Cord-Derived Mesenchymal Stem Cells Efficiently Induced the Apoptosis and Differentiation in Human Glioma Cell Lines In Vitro

Journal: BioMed Research International

doi: 10.1155/2014/109389

Matrigel invasion assay. U251 cells or U87 cells (induced or noninduced) were placed in transwells with 8 μ m pore size polycarbonate filters, precoated with Matrigel. The lower wells were filled with DMEM with 10% fetal bovine serum. The cells were incubated at 37°C for 24 hours. Nonmigratory cells on the upper surface of the transwells were removed and the migratory cells from the lower surface of the transwell were fixed and stained with 1% crystal violet. Cells were photographed and average cells per field were calculated. Experiments were done in triplicates on three times independently.
Figure Legend Snippet: Matrigel invasion assay. U251 cells or U87 cells (induced or noninduced) were placed in transwells with 8 μ m pore size polycarbonate filters, precoated with Matrigel. The lower wells were filled with DMEM with 10% fetal bovine serum. The cells were incubated at 37°C for 24 hours. Nonmigratory cells on the upper surface of the transwells were removed and the migratory cells from the lower surface of the transwell were fixed and stained with 1% crystal violet. Cells were photographed and average cells per field were calculated. Experiments were done in triplicates on three times independently.

Techniques Used: Invasion Assay, Incubation, Staining

20) Product Images from "Selection, Analysis and Improvement of Anti-Angiogenesis Compounds Identified by an Anti-HIF-1α Screening and Validation System"

Article Title: Selection, Analysis and Improvement of Anti-Angiogenesis Compounds Identified by an Anti-HIF-1α Screening and Validation System

Journal: Journal of Cancer

doi: 10.7150/jca.15603

HMEC-based in vitro angiogenesis assay for measuring VEGF-mediated tubular network formation. HMEC cells were planted onto matrigel-coated wells and cultured in the basal media (BM) (i.e., RPMI-1640 medium with no FBS) or BM containing 15 ng/ml VEGF (BM+VEGF). (A) The tubular network formation was recorded at 8 h after planting HMEC cells onto the matrigel-coated wells in the indicated media. (B) The vessel crossing points of five randomly selected fields were counted 30 , 32 as angiogenic ability for quantitation comparison. *The p values of difference between the untreated control and the VEGF-treated samples are statistically significant (p
Figure Legend Snippet: HMEC-based in vitro angiogenesis assay for measuring VEGF-mediated tubular network formation. HMEC cells were planted onto matrigel-coated wells and cultured in the basal media (BM) (i.e., RPMI-1640 medium with no FBS) or BM containing 15 ng/ml VEGF (BM+VEGF). (A) The tubular network formation was recorded at 8 h after planting HMEC cells onto the matrigel-coated wells in the indicated media. (B) The vessel crossing points of five randomly selected fields were counted 30 , 32 as angiogenic ability for quantitation comparison. *The p values of difference between the untreated control and the VEGF-treated samples are statistically significant (p

Techniques Used: In Vitro, Angiogenesis Assay, Cell Culture, Quantitation Assay

21) Product Images from "Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo"

Article Title: Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

Journal: ISRN Oncology

doi: 10.1155/2013/518637

Effect of midkine overexpression on wild type cell survival. A midkine overexpressing SK-N-SH cell line (SK-N-SH HMK) was created as previously described [ 6 ]. (a) Midkine expression in the medium of wild type (WT), doxorubicin resistant (DoxR), and HMK cells were analyzed using western blot. SK-N-SH WT, DoxR, and HMK were cultured in 25 cm flasks to 80% confluence. Medium was then exchanged for 2 mL of low fetal bovine serum culture medium and cultured for an additional 48 hours. Cultured medium was then collected and frozen at −80°C prior to being subjected to western blot. The volume of medium loaded on the gel was normalized based on cell count in the flask prior to medium collection. Cell membranes were isolated through a well-established protocol. We have previously demonstrated that midkine transfected cells (HMK) have acquired some doxorubicin resistance [ 6 ]. The MTT cell survival assay was performed to determine if SK-H-SH HMK cells have acquired resistance to other chemotherapeutic drugs including (b) etoposide and (c) cisplatin. (d) SK-N-SH WT, DoxR, and HMK cells were treated with or without doxorubicin at 10 −7 M for 24 hours prior to collection of cell lysates. Proteins were extracted and probed using western blot for Pgp and β -actin.
Figure Legend Snippet: Effect of midkine overexpression on wild type cell survival. A midkine overexpressing SK-N-SH cell line (SK-N-SH HMK) was created as previously described [ 6 ]. (a) Midkine expression in the medium of wild type (WT), doxorubicin resistant (DoxR), and HMK cells were analyzed using western blot. SK-N-SH WT, DoxR, and HMK were cultured in 25 cm flasks to 80% confluence. Medium was then exchanged for 2 mL of low fetal bovine serum culture medium and cultured for an additional 48 hours. Cultured medium was then collected and frozen at −80°C prior to being subjected to western blot. The volume of medium loaded on the gel was normalized based on cell count in the flask prior to medium collection. Cell membranes were isolated through a well-established protocol. We have previously demonstrated that midkine transfected cells (HMK) have acquired some doxorubicin resistance [ 6 ]. The MTT cell survival assay was performed to determine if SK-H-SH HMK cells have acquired resistance to other chemotherapeutic drugs including (b) etoposide and (c) cisplatin. (d) SK-N-SH WT, DoxR, and HMK cells were treated with or without doxorubicin at 10 −7 M for 24 hours prior to collection of cell lysates. Proteins were extracted and probed using western blot for Pgp and β -actin.

Techniques Used: Over Expression, Expressing, Western Blot, Cell Culture, Cell Counting, Isolation, Transfection, MTT Assay, Clonogenic Cell Survival Assay

22) Product Images from "Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells"

Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.16-20610

Effects of cycloheximide on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 10 μM cycloheximide for 24 hours in DMEM containing 10% FBS without (Chx) or with 33% vitreous humor (V/Chx). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P
Figure Legend Snippet: Effects of cycloheximide on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 10 μM cycloheximide for 24 hours in DMEM containing 10% FBS without (Chx) or with 33% vitreous humor (V/Chx). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P

Techniques Used: Expressing, SDS Page, Purification, Western Blot, Imaging

Western blot analysis of ferritin H- and L-chains in the lysates of control (C) and vitreous-treated LEC (V). Confluent LEC were cultured in DMEM with 10% FBS with (V) or without (C) 33% (vol/vol) of vitreous humor for 24 or 96 hours. LEC lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Blot was reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 11; statistically different from corresponding C, * P
Figure Legend Snippet: Western blot analysis of ferritin H- and L-chains in the lysates of control (C) and vitreous-treated LEC (V). Confluent LEC were cultured in DMEM with 10% FBS with (V) or without (C) 33% (vol/vol) of vitreous humor for 24 or 96 hours. LEC lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Blot was reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 11; statistically different from corresponding C, * P

Techniques Used: Western Blot, Cell Culture, SDS Page, Purification, Imaging

( A ) Phase contrast image of control (C) and vitreous-treated LEC (V). LEC were treated for 96 hours with DMEM containing 1% FBS and 33% of vitreous humor. Control LEC were grown in DMEM containing 1% FBS without vitreous humor. ( B ) Western blot analysis of aquaporin 0 expression in treated (V) and control LEC (C). Lens lysates containing 50 μg proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membranes, and probed with rabbit anti-aquaporin 0 polyclonal antibodies. Canine lens was dissected and the section containing lens fibers was sonicated. Fifty micrograms of protein sample of sonicated lens fiber cells was used as a positive control (St). Blot was evaluated with ChemiDoc MP Imaging System.
Figure Legend Snippet: ( A ) Phase contrast image of control (C) and vitreous-treated LEC (V). LEC were treated for 96 hours with DMEM containing 1% FBS and 33% of vitreous humor. Control LEC were grown in DMEM containing 1% FBS without vitreous humor. ( B ) Western blot analysis of aquaporin 0 expression in treated (V) and control LEC (C). Lens lysates containing 50 μg proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membranes, and probed with rabbit anti-aquaporin 0 polyclonal antibodies. Canine lens was dissected and the section containing lens fibers was sonicated. Fifty micrograms of protein sample of sonicated lens fiber cells was used as a positive control (St). Blot was evaluated with ChemiDoc MP Imaging System.

Techniques Used: Western Blot, Expressing, SDS Page, Sonication, Positive Control, Imaging

Western blot analysis of TfR1 in control (C)- and vitreous-treated LEC (V). Confluent LEC were cultured in DMEM with 10% FBS with or without 33% (vol/vol) of vitreous humor for 24 or 96 hours. LEC lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blot was reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 9; statistically different from corresponding C, * P
Figure Legend Snippet: Western blot analysis of TfR1 in control (C)- and vitreous-treated LEC (V). Confluent LEC were cultured in DMEM with 10% FBS with or without 33% (vol/vol) of vitreous humor for 24 or 96 hours. LEC lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blot was reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 9; statistically different from corresponding C, * P

Techniques Used: Western Blot, Cell Culture, SDS Page, Purification, Imaging

Effects of actinomycin D on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 1 μg/1 mL actinomycin D for 24 hours in DMEM containing 10% FBS without (ActD) or with 33% vitreous humor (V/ActD). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P
Figure Legend Snippet: Effects of actinomycin D on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 1 μg/1 mL actinomycin D for 24 hours in DMEM containing 10% FBS without (ActD) or with 33% vitreous humor (V/ActD). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P

Techniques Used: Expressing, SDS Page, Purification, Western Blot, Imaging

Western blot analysis of ferritin H- and L-chains in the lysates of LEC treated with differentially processed fractions of vitreous humor. LEC were treated for 24 hours without (C) or with 33% of vitreous humor (V) and with equivalent amount of differentially processed vitreous fractions, in DMEM containing 10% FBS. Vf, vitreous humor filtered through glass wool; Va, acetone precipitate of Vf dissolved in complete DMEM; Vmf, Microcon 10 filtrate of Vf ; Vmr, Microcon 10 retentate of Vf; Vb, Vf boiled for 10 minutes. Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The blot shown is representative of two experiments.
Figure Legend Snippet: Western blot analysis of ferritin H- and L-chains in the lysates of LEC treated with differentially processed fractions of vitreous humor. LEC were treated for 24 hours without (C) or with 33% of vitreous humor (V) and with equivalent amount of differentially processed vitreous fractions, in DMEM containing 10% FBS. Vf, vitreous humor filtered through glass wool; Va, acetone precipitate of Vf dissolved in complete DMEM; Vmf, Microcon 10 filtrate of Vf ; Vmr, Microcon 10 retentate of Vf; Vb, Vf boiled for 10 minutes. Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The blot shown is representative of two experiments.

Techniques Used: Western Blot, SDS Page, Purification, Imaging

Effects of treatment of LEC with vitreous humor collected from dogs 7 to 10 years old with (Vcat) or without (V) age-related cataract on expression of ferritin chains and TfR1. LEC were treated with 33% vitreous humor in DMEM containing 10% FBS for 96 hours. Cells nontreated with vitreous humors were used as controls (C). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains, and purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 7; statistically different from corresponding V, * P
Figure Legend Snippet: Effects of treatment of LEC with vitreous humor collected from dogs 7 to 10 years old with (Vcat) or without (V) age-related cataract on expression of ferritin chains and TfR1. LEC were treated with 33% vitreous humor in DMEM containing 10% FBS for 96 hours. Cells nontreated with vitreous humors were used as controls (C). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains, and purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 7; statistically different from corresponding V, * P

Techniques Used: Expressing, SDS Page, Purification, Western Blot, Imaging

23) Product Images from "Peroxisome Proliferator-activated Receptor ? Ligands Inhibit Transforming Growth Factor-?-induced, Hyaluronan-dependent, T Cell Adhesion to Orbital Fibroblasts *"

Article Title: Peroxisome Proliferator-activated Receptor ? Ligands Inhibit Transforming Growth Factor-?-induced, Hyaluronan-dependent, T Cell Adhesion to Orbital Fibroblasts *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.179317

TGF-β1 induces human peripheral blood T cell adhesion to orbital fibroblasts through HA-CD44 interaction. A , peripheral blood mononuclear cells were incubated with CD3/CD28 beads in RPMI 1640 with 10% FBS medium at 37 °C for 2 days. After that, recombinant IL-2 (50 units/ml) was added to the culture and incubated for several days according to the cell number. After T cell expansion, the expression of CD3 and CD44 on the cell surface was examined by flow-cytometry. More than 99% of the enriched cells express CD3 and CD44. B , enriched T cells were fluorescently labeled by incubation with calcein-AM. After labeling, some T cells were incubated with 40 μg/ml monoclonal CD44 antibody. Confluent orbital fibroblasts were cultured in reduced serum for 3 days and treated with 2 ng/ml TGF-β1 for 24 h. In some cultures, orbital fibroblasts were treated with 100 milliunits/ml HA'ase for 1 h. T cells were added and allowed to adhere for 90 min at 4 °C. Plates were washed three times, and fluorescence was measured at 535 nm. White bars , fibroblast vehicle control; black bars , fibroblast treated with TGF-β for 24 h. There was a significant increase in T cell adhesion to TGF-β1-treated orbital fibroblasts (***, p
Figure Legend Snippet: TGF-β1 induces human peripheral blood T cell adhesion to orbital fibroblasts through HA-CD44 interaction. A , peripheral blood mononuclear cells were incubated with CD3/CD28 beads in RPMI 1640 with 10% FBS medium at 37 °C for 2 days. After that, recombinant IL-2 (50 units/ml) was added to the culture and incubated for several days according to the cell number. After T cell expansion, the expression of CD3 and CD44 on the cell surface was examined by flow-cytometry. More than 99% of the enriched cells express CD3 and CD44. B , enriched T cells were fluorescently labeled by incubation with calcein-AM. After labeling, some T cells were incubated with 40 μg/ml monoclonal CD44 antibody. Confluent orbital fibroblasts were cultured in reduced serum for 3 days and treated with 2 ng/ml TGF-β1 for 24 h. In some cultures, orbital fibroblasts were treated with 100 milliunits/ml HA'ase for 1 h. T cells were added and allowed to adhere for 90 min at 4 °C. Plates were washed three times, and fluorescence was measured at 535 nm. White bars , fibroblast vehicle control; black bars , fibroblast treated with TGF-β for 24 h. There was a significant increase in T cell adhesion to TGF-β1-treated orbital fibroblasts (***, p

Techniques Used: Incubation, Recombinant, Expressing, Flow Cytometry, Cytometry, Labeling, Cell Culture, Fluorescence

TGF-β-induced HA production by orbital fibroblasts and T cell-fibroblast adhesion is dependent upon HAS2 expression. A , orbital fibroblasts were transfected with siRNA for HAS1 , HAS2 , or a scramble control ( SC ) siRNA as described under “Experimental Procedures.” The cells were serum-starved in RPMI 1640 with 0.5% FBS and treated with 2 ng/ml TGF-β1 for 6 h, and HAS expression levels were analyzed by qRT-PCR. The mRNA for HAS1 and HAS2 in the TGF-β-treated SC siRNA samples was normalized to 100 for comparison of gene expression. Each siRNA reduced its target mRNA expression selectively and significantly (up to 80%). For HA detection, HAS1 or HAS2 siRNA-transfected orbital fibroblasts were exposed to 2 ng/ml TGF-β1 for 24 h, and secreted HA and pericellular HA was analyzed by HA ELISA. Knockdown of HAS2 , but not HAS1 , significantly reduced both secreted HA and pericellular HA in human orbital fibroblasts. **, p
Figure Legend Snippet: TGF-β-induced HA production by orbital fibroblasts and T cell-fibroblast adhesion is dependent upon HAS2 expression. A , orbital fibroblasts were transfected with siRNA for HAS1 , HAS2 , or a scramble control ( SC ) siRNA as described under “Experimental Procedures.” The cells were serum-starved in RPMI 1640 with 0.5% FBS and treated with 2 ng/ml TGF-β1 for 6 h, and HAS expression levels were analyzed by qRT-PCR. The mRNA for HAS1 and HAS2 in the TGF-β-treated SC siRNA samples was normalized to 100 for comparison of gene expression. Each siRNA reduced its target mRNA expression selectively and significantly (up to 80%). For HA detection, HAS1 or HAS2 siRNA-transfected orbital fibroblasts were exposed to 2 ng/ml TGF-β1 for 24 h, and secreted HA and pericellular HA was analyzed by HA ELISA. Knockdown of HAS2 , but not HAS1 , significantly reduced both secreted HA and pericellular HA in human orbital fibroblasts. **, p

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Pio and Rosi do not influence human orbital fibroblast viability. Confluent strains of human orbital fibroblasts were cultured in RPMI 1640 with 0.5% FBS for 3 days before treatment with different concentrations of Pio or Rosi with or without 2 ng/ml TGF-β1 for 24 h. Viability was measured by XTT assay. Results shown are representative of three independent experiments and are the mean ± S.E. ( n = 8). No significant differences were observed with any treatment (analysis of variance). V , vehicle control.
Figure Legend Snippet: Pio and Rosi do not influence human orbital fibroblast viability. Confluent strains of human orbital fibroblasts were cultured in RPMI 1640 with 0.5% FBS for 3 days before treatment with different concentrations of Pio or Rosi with or without 2 ng/ml TGF-β1 for 24 h. Viability was measured by XTT assay. Results shown are representative of three independent experiments and are the mean ± S.E. ( n = 8). No significant differences were observed with any treatment (analysis of variance). V , vehicle control.

Techniques Used: Cell Culture, XTT Assay

24) Product Images from "Wogonin Attenuates Isoprenaline-Induced Myocardial Hypertrophy in Mice by Suppressing the PI3K/Akt Pathway"

Article Title: Wogonin Attenuates Isoprenaline-Induced Myocardial Hypertrophy in Mice by Suppressing the PI3K/Akt Pathway

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00896

Wogonin reverses isoprenaline-induced hypertrophy in RA-differentiated H9c2 cells. H9c2 cells were cultured in DMEM containing 1% FBS and 1 μM RA for 5 days before incubating with wogonin (1 and 10 μM) with/without isoprenaline (10 μM) for a further 24 h. (A) The cardiac-specific genes, Tnnc1 and Tnnt2 ( n = 3), and (B) myocardial cell-specific hypertrophic markers, ANP and BNP ( n = 5), were determined with RT-qPCR and normalized to β-actin. Data are expressed as fold-change relative to the level of control cells. (C) H9c2 cell shape was demonstrated by the immunofluorescence with α-SMA antibody. (D) Quantitation of average cell surface area ( n = 8). Data are presented as mean ± SEM; ∗ p
Figure Legend Snippet: Wogonin reverses isoprenaline-induced hypertrophy in RA-differentiated H9c2 cells. H9c2 cells were cultured in DMEM containing 1% FBS and 1 μM RA for 5 days before incubating with wogonin (1 and 10 μM) with/without isoprenaline (10 μM) for a further 24 h. (A) The cardiac-specific genes, Tnnc1 and Tnnt2 ( n = 3), and (B) myocardial cell-specific hypertrophic markers, ANP and BNP ( n = 5), were determined with RT-qPCR and normalized to β-actin. Data are expressed as fold-change relative to the level of control cells. (C) H9c2 cell shape was demonstrated by the immunofluorescence with α-SMA antibody. (D) Quantitation of average cell surface area ( n = 8). Data are presented as mean ± SEM; ∗ p

Techniques Used: Cell Culture, Aqueous Normal-phase Chromatography, Quantitative RT-PCR, Immunofluorescence, Quantitation Assay

Wogonin inhibits the Akt signaling pathway initiated by isoprenaline treatment. H9c2 cells were cultured in DMEM containing 1% FBS and 1 μM RA for 5-day differentiation. (A) Differentiated H9c2 cells were treated with isoprenaline (10 μM) and/or wogonin (10 μM) with indicated dose for 24 h. Cell extracts were blotted by antibodies against pAkt, Akt, pCREB, CREB, pJNK, JNK, pERK1/2, ERK1/2, pP38 and P38. (B) Quantitation of phosphoprotein to total protein ( n = 3). (C) Differentiated H9c2 cells, pretreated with wogonin (10 μM) for 15 min, then were incubated with DBcAMP (10 μM) for another 15 min. Cell extracts were blotted by antibodies against p-CREB and CREB. (D) Quantitation of p-CREB to CREB ( n = 3). (E) Differentiated H9c2 cells were treated with wogonin (10 μM) and PDBU (5 μM) for 24 h. The mRNA levels of ANP and BNP were determined by RT-qPCR ( n = 5). Data are given as mean ± SEM; ∗ p
Figure Legend Snippet: Wogonin inhibits the Akt signaling pathway initiated by isoprenaline treatment. H9c2 cells were cultured in DMEM containing 1% FBS and 1 μM RA for 5-day differentiation. (A) Differentiated H9c2 cells were treated with isoprenaline (10 μM) and/or wogonin (10 μM) with indicated dose for 24 h. Cell extracts were blotted by antibodies against pAkt, Akt, pCREB, CREB, pJNK, JNK, pERK1/2, ERK1/2, pP38 and P38. (B) Quantitation of phosphoprotein to total protein ( n = 3). (C) Differentiated H9c2 cells, pretreated with wogonin (10 μM) for 15 min, then were incubated with DBcAMP (10 μM) for another 15 min. Cell extracts were blotted by antibodies against p-CREB and CREB. (D) Quantitation of p-CREB to CREB ( n = 3). (E) Differentiated H9c2 cells were treated with wogonin (10 μM) and PDBU (5 μM) for 24 h. The mRNA levels of ANP and BNP were determined by RT-qPCR ( n = 5). Data are given as mean ± SEM; ∗ p

Techniques Used: Cell Culture, Quantitation Assay, Incubation, Aqueous Normal-phase Chromatography, Quantitative RT-PCR

25) Product Images from "Improved Establishment of Embryonic Stem (ES) Cell Lines from the Chinese Kunming Mice by Hybridization with 129 Mice"

Article Title: Improved Establishment of Embryonic Stem (ES) Cell Lines from the Chinese Kunming Mice by Hybridization with 129 Mice

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms15033389

ES cell clone shapes cultured for 7 days in the media containing 15% KSR ( A ); 14% KSR + 1% FBS ( B ); 15% FBS ( C ) when single ES cells were plated. Scale bar = 150 μm.
Figure Legend Snippet: ES cell clone shapes cultured for 7 days in the media containing 15% KSR ( A ); 14% KSR + 1% FBS ( B ); 15% FBS ( C ) when single ES cells were plated. Scale bar = 150 μm.

Techniques Used: Cell Culture

26) Product Images from "Mast Cell-derived Prostaglandin D2 Controls Hyaluronan Synthesis in Human Orbital Fibroblasts via DP1 Activation"

Article Title: Mast Cell-derived Prostaglandin D2 Controls Hyaluronan Synthesis in Human Orbital Fibroblasts via DP1 Activation

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.074534

PGD 2 and PGJ 2 induce HA synthesis in human orbital fibroblasts. A , confluent strains of human orbital fibroblasts (OF1 and OF2) were cultured in RPMI 1640 with 0.5% FBS for 3 days prior to treatment with PGJ 2 , PGD 2 , or vehicle (DMSO) for 18 h. The cell
Figure Legend Snippet: PGD 2 and PGJ 2 induce HA synthesis in human orbital fibroblasts. A , confluent strains of human orbital fibroblasts (OF1 and OF2) were cultured in RPMI 1640 with 0.5% FBS for 3 days prior to treatment with PGJ 2 , PGD 2 , or vehicle (DMSO) for 18 h. The cell

Techniques Used: Cell Culture

27) Product Images from "The inhibitory effects of camptothecin, a topoisomerase I inhibitor, on collagen synthesis in fibroblasts from patients with systemic sclerosis"

Article Title: The inhibitory effects of camptothecin, a topoisomerase I inhibitor, on collagen synthesis in fibroblasts from patients with systemic sclerosis

Journal: Arthritis Research

doi:

The effects of CPT on deposition of collagen types I (a) , III (b) , and VI (c) and elastin (d) in SSc and healthy control fibroblasts. Fibroblasts were grown to confluence in 96-well plates and then incubated for 24 h with CPT in DMEM/1% FBS. In control wells ('None'), the CPT was omitted. ELISA was performed on the cell and matrix layers with appropriate antibodies as described in the Patients and methods section. Bars show the relative amounts of collagen types I, III, and VI (a–c) from 11 pairs of SSc and healthy fibroblast cell lines and of elastin (d) from from four pairs of SSc and healthy fibroblast cell lines. All experiments were done in triplicate. CPT = camptothecin; FBS = fetal bovine serum; SSc = systemic sclerosis. * P
Figure Legend Snippet: The effects of CPT on deposition of collagen types I (a) , III (b) , and VI (c) and elastin (d) in SSc and healthy control fibroblasts. Fibroblasts were grown to confluence in 96-well plates and then incubated for 24 h with CPT in DMEM/1% FBS. In control wells ('None'), the CPT was omitted. ELISA was performed on the cell and matrix layers with appropriate antibodies as described in the Patients and methods section. Bars show the relative amounts of collagen types I, III, and VI (a–c) from 11 pairs of SSc and healthy fibroblast cell lines and of elastin (d) from from four pairs of SSc and healthy fibroblast cell lines. All experiments were done in triplicate. CPT = camptothecin; FBS = fetal bovine serum; SSc = systemic sclerosis. * P

Techniques Used: Cycling Probe Technology, Incubation, Enzyme-linked Immunosorbent Assay

28) Product Images from "Soluble LRIG2 Ectodomain Is Released from Glioblastoma Cells and Promotes the Proliferation and Inhibits the Apoptosis of Glioblastoma Cells In Vitro and In Vivo in a Similar Manner to the Full-Length LRIG2"

Article Title: Soluble LRIG2 Ectodomain Is Released from Glioblastoma Cells and Promotes the Proliferation and Inhibits the Apoptosis of Glioblastoma Cells In Vitro and In Vivo in a Similar Manner to the Full-Length LRIG2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0111419

LRIG2 or LRIG2ecto overexpression inhibits the spontaneous apoptosis of glioblastoma cells in vitro . ( A ) Cells were cultured in DMEM with 10% FBS for 48 h, stained by Annexin V/PI and subjected to flow cytometry analysis. Early apoptotic populations were in the lower-right quadrant and late apoptotic cells were in the upper-right quadrant. Representative images were shown in the left panel and percentages of early and late apoptotic cells were expressed as the mean±SD of three independent experiments, shown in the right panel (* P
Figure Legend Snippet: LRIG2 or LRIG2ecto overexpression inhibits the spontaneous apoptosis of glioblastoma cells in vitro . ( A ) Cells were cultured in DMEM with 10% FBS for 48 h, stained by Annexin V/PI and subjected to flow cytometry analysis. Early apoptotic populations were in the lower-right quadrant and late apoptotic cells were in the upper-right quadrant. Representative images were shown in the left panel and percentages of early and late apoptotic cells were expressed as the mean±SD of three independent experiments, shown in the right panel (* P

Techniques Used: Over Expression, In Vitro, Cell Culture, Staining, Flow Cytometry, Cytometry

LRIG2 or LRIG2ecto overexpression enhances EGFR signaling and regulates the cell cycle and apoptosis related proteins. ( A ) After synchronization for 24 h, cells were cultured in DMEM with 10% FBS for 48 h and cell lysates were subjected to western blotting for the levels of EGFR signaling related proteins. Analysis of quantification of the bands intensity was shown in the lower panel (* P
Figure Legend Snippet: LRIG2 or LRIG2ecto overexpression enhances EGFR signaling and regulates the cell cycle and apoptosis related proteins. ( A ) After synchronization for 24 h, cells were cultured in DMEM with 10% FBS for 48 h and cell lysates were subjected to western blotting for the levels of EGFR signaling related proteins. Analysis of quantification of the bands intensity was shown in the lower panel (* P

Techniques Used: Over Expression, Cell Culture, Western Blot

29) Product Images from "Development of PLGA-lipid nanoparticles with covalently conjugated indocyanine green as a versatile nanoplatform for tumor-targeted imaging and drug delivery"

Article Title: Development of PLGA-lipid nanoparticles with covalently conjugated indocyanine green as a versatile nanoplatform for tumor-targeted imaging and drug delivery

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S119999

Fluorescence stability and stability in water of FA-RIPNPs. Notes: ICG fluorescence stability of free ICG and FA-RIPNPs in aqueous solution at ( A ) 4°C and ( B ) 37°C over 4 wk. Insets show photos of free ICG and FA-RIPNPs in aqueous solution after 4 wk of storage at 4°C and 37°C, respectively. ( C ) Colloidal stability test of FA-RIPNPs in different media, including water, MEM, PBS and FBS. ( D ) Change in ratio of absorbances at 304 nm and 280 nm (A 304 nm /A 280 nm ) of the FA-RIPNPs over 4 wk at 37°C. Inset shows the absorbance spectrum of FA-RIPNPs. Abbreviations: RSV, resveratrol; ICG, indocyanine green; FA, folic acid; PLGA, poly( d , l -lactide- co -glycolide); NPs, nanoparticles; FA-RIPNPs, FA-RSV/ICG-PLGA-lipid NPs; MEM, minimum essential medium; PBS, phosphate-buffered saline; FBS, fetal bovine serum.
Figure Legend Snippet: Fluorescence stability and stability in water of FA-RIPNPs. Notes: ICG fluorescence stability of free ICG and FA-RIPNPs in aqueous solution at ( A ) 4°C and ( B ) 37°C over 4 wk. Insets show photos of free ICG and FA-RIPNPs in aqueous solution after 4 wk of storage at 4°C and 37°C, respectively. ( C ) Colloidal stability test of FA-RIPNPs in different media, including water, MEM, PBS and FBS. ( D ) Change in ratio of absorbances at 304 nm and 280 nm (A 304 nm /A 280 nm ) of the FA-RIPNPs over 4 wk at 37°C. Inset shows the absorbance spectrum of FA-RIPNPs. Abbreviations: RSV, resveratrol; ICG, indocyanine green; FA, folic acid; PLGA, poly( d , l -lactide- co -glycolide); NPs, nanoparticles; FA-RIPNPs, FA-RSV/ICG-PLGA-lipid NPs; MEM, minimum essential medium; PBS, phosphate-buffered saline; FBS, fetal bovine serum.

Techniques Used: Fluorescence

30) Product Images from "Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity"

Article Title: Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity

Journal: BMC Medical Genetics

doi: 10.1186/1471-2350-13-26

Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of Wnt3a-CM (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p
Figure Legend Snippet: Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of Wnt3a-CM (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p

Techniques Used: Luciferase, Activity Assay, Transfection, Mutagenesis

31) Product Images from "Thy-1 Expression Regulates the Ability of Rat Lung Fibroblasts to Activate Transforming Growth Factor-? in Response to Fibrogenic Stimuli"

Article Title: Thy-1 Expression Regulates the Ability of Rat Lung Fibroblasts to Activate Transforming Growth Factor-? in Response to Fibrogenic Stimuli

Journal: The American Journal of Pathology

doi:

Thy-1 is mechanistically important for differential regulation of TGF-β activation. A: Thy-1-transfected RFL-6 CD90 cells express Thy-1 on the cell surface, whereas empty vector-transfected RFL-6 EV cells do not. RFL-6 EV ( top right ) and RFL-6 CD90 cells ( bottom left ) were harvested and incubated with fluorescein isothiocyanate-conjugated anti-CD90.2 antibody. Expression of Thy-1 on the cell surface was determined by flow cytometry. RFL-6 EV cells treated with fluorescein isothiocyanate-conjugated mIgG1κ ( top left ) were used as a control. B: RFL-6 EV and RFL-6 CD90 cells were cultured in six-well plates until 70 to 80% confluent. Cells were made quiescent in F12K media with 0.1% FBS for 24 hours and in serum-free media for 2 hours. Cells were treated with increasing concentrations of IL-4, PDGF-BB, IL-1β, or BLM for 24 hours. Conditioned media were harvested and assayed for active TGF-β activity and total TGF-β activity. Cell number in each well was determined by cell counting. Values of TGF-β activity were normalized for cell number. Results are shown as mean ± SD of triplicate wells. *, P
Figure Legend Snippet: Thy-1 is mechanistically important for differential regulation of TGF-β activation. A: Thy-1-transfected RFL-6 CD90 cells express Thy-1 on the cell surface, whereas empty vector-transfected RFL-6 EV cells do not. RFL-6 EV ( top right ) and RFL-6 CD90 cells ( bottom left ) were harvested and incubated with fluorescein isothiocyanate-conjugated anti-CD90.2 antibody. Expression of Thy-1 on the cell surface was determined by flow cytometry. RFL-6 EV cells treated with fluorescein isothiocyanate-conjugated mIgG1κ ( top left ) were used as a control. B: RFL-6 EV and RFL-6 CD90 cells were cultured in six-well plates until 70 to 80% confluent. Cells were made quiescent in F12K media with 0.1% FBS for 24 hours and in serum-free media for 2 hours. Cells were treated with increasing concentrations of IL-4, PDGF-BB, IL-1β, or BLM for 24 hours. Conditioned media were harvested and assayed for active TGF-β activity and total TGF-β activity. Cell number in each well was determined by cell counting. Values of TGF-β activity were normalized for cell number. Results are shown as mean ± SD of triplicate wells. *, P

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Incubation, Expressing, Flow Cytometry, Cytometry, Cell Culture, Activity Assay, Cell Counting

Thy-1 (−) cells express increased α-SMA and increase expression in response to fibrogenic stimuli. A: Thy-1 (+) and (−) fibroblasts were made quiescent in DMEM with 0.1% FBS for 48 hours and in serum-free DMEM for 2 hours. Cell lysates were collected and subjected to electrophoresis on 10% SDS-PAGE under reducing conditions. Levels of α-SMA were detected by immunoblotting. To verify equal loading, the same blot was stripped out and reprobed with anti-β-tubulin antibody. B: Quiescent cells were treated with 8 pmol/L TGF-β1, 10 ng/ml of IL-4, 20 ng/ml of PDGF-BB, 5 ng/ml of IL-1β, or 1 μg/ml of BLM for 24 hours. Cell lysates were collected and subjected to immunoblotting. Relative α-SMA protein levels were determined by scanning densitometry of the blots and were normalized to β-tubulin. The level of α-SMA expressed in Thy-1 (+) cells was set to 1. Results are the means of three independent experiments ± SD. *, P
Figure Legend Snippet: Thy-1 (−) cells express increased α-SMA and increase expression in response to fibrogenic stimuli. A: Thy-1 (+) and (−) fibroblasts were made quiescent in DMEM with 0.1% FBS for 48 hours and in serum-free DMEM for 2 hours. Cell lysates were collected and subjected to electrophoresis on 10% SDS-PAGE under reducing conditions. Levels of α-SMA were detected by immunoblotting. To verify equal loading, the same blot was stripped out and reprobed with anti-β-tubulin antibody. B: Quiescent cells were treated with 8 pmol/L TGF-β1, 10 ng/ml of IL-4, 20 ng/ml of PDGF-BB, 5 ng/ml of IL-1β, or 1 μg/ml of BLM for 24 hours. Cell lysates were collected and subjected to immunoblotting. Relative α-SMA protein levels were determined by scanning densitometry of the blots and were normalized to β-tubulin. The level of α-SMA expressed in Thy-1 (+) cells was set to 1. Results are the means of three independent experiments ± SD. *, P

Techniques Used: Expressing, Electrophoresis, SDS Page

32) Product Images from "Effects of metformin on colorectal cancer stem cells depend on alterations in glutamine metabolism"

Article Title: Effects of metformin on colorectal cancer stem cells depend on alterations in glutamine metabolism

Journal: Scientific Reports

doi: 10.1038/s41598-017-18762-4

The different effects of low-glucose or glutamine-free conditions on CSCs of metformin-treated SW620 and HT29 cells. The control medium (Dulbecco’s modified Eagle’s medium) contained 4 mM L-glutamine and 4,500 mg/L glucose. ( a and b ) The low-glucose medium had the same composition except for 1,000 mg/L glucose. The proportion of CD133 + CD44 + cells (CSCs) among SW620 and HT29 cells was analyzed by flow cytometry after 48 h treatment with control vehicle, 10 mM metformin, or 20 mM metformin. ( c and d ) The glutamine-free medium included DMEM with 10% FBS, 1% P/S, and 4,500 mg/L glucose. The proportion of CSCs among the SW620 cells and HT29 cells was analyzed by flow cytometry after 48 h treatment with control vehicle or 10 mM metformin. ( e and f ) In tumor-sphere culture, the number of colospheres was counted at day 14 in each concentration of glutamine. ( g ) In Western-blot analysis, the expression of p-S6/S6 and p-AMPK/AMPK in SW620 cells and HT29 cells was analyzed after 48 h treatment with control vehicle, L-glutamine, or metformin. Data are expressed as the mean ± standard error of three independent experiments; * P
Figure Legend Snippet: The different effects of low-glucose or glutamine-free conditions on CSCs of metformin-treated SW620 and HT29 cells. The control medium (Dulbecco’s modified Eagle’s medium) contained 4 mM L-glutamine and 4,500 mg/L glucose. ( a and b ) The low-glucose medium had the same composition except for 1,000 mg/L glucose. The proportion of CD133 + CD44 + cells (CSCs) among SW620 and HT29 cells was analyzed by flow cytometry after 48 h treatment with control vehicle, 10 mM metformin, or 20 mM metformin. ( c and d ) The glutamine-free medium included DMEM with 10% FBS, 1% P/S, and 4,500 mg/L glucose. The proportion of CSCs among the SW620 cells and HT29 cells was analyzed by flow cytometry after 48 h treatment with control vehicle or 10 mM metformin. ( e and f ) In tumor-sphere culture, the number of colospheres was counted at day 14 in each concentration of glutamine. ( g ) In Western-blot analysis, the expression of p-S6/S6 and p-AMPK/AMPK in SW620 cells and HT29 cells was analyzed after 48 h treatment with control vehicle, L-glutamine, or metformin. Data are expressed as the mean ± standard error of three independent experiments; * P

Techniques Used: Modification, Flow Cytometry, Cytometry, Concentration Assay, Western Blot, Expressing

33) Product Images from "Anticancer Effect of Fucoidan on DU-145 Prostate Cancer Cells through Inhibition of PI3K/Akt and MAPK Pathway Expression"

Article Title: Anticancer Effect of Fucoidan on DU-145 Prostate Cancer Cells through Inhibition of PI3K/Akt and MAPK Pathway Expression

Journal: Marine Drugs

doi: 10.3390/md14070126

Effect of fucoidan on the cell viability of DU-145 cells. DU-145 cells (2 × 10 4 cells/mL) were treated with 0, 250, 500, 750, 1000 μg/mL fucoidan in RPMI-1640 medium containing 5% FBS for 24 h. The growth inhibition was measured by the MTT assay. Data are mean standard deviation (SD) for three samples. The significance was determined by Student’s t -test (* p
Figure Legend Snippet: Effect of fucoidan on the cell viability of DU-145 cells. DU-145 cells (2 × 10 4 cells/mL) were treated with 0, 250, 500, 750, 1000 μg/mL fucoidan in RPMI-1640 medium containing 5% FBS for 24 h. The growth inhibition was measured by the MTT assay. Data are mean standard deviation (SD) for three samples. The significance was determined by Student’s t -test (* p

Techniques Used: Inhibition, MTT Assay, Standard Deviation

Effect of fucoidan on the chromatin condensation in DU-145 cells. ( A ) DU-145 cells were treated with 0, 500, 1000 μg/mL fucoidan or vehicle in RPMI-1640 medium containing 5% FBS for 24 h, and cell were stained with DAPI. The arrows indicate chromatin condensation in the cancer cell. ( B ) DU-145 cells were treated with fucoidan (0, 500, 1000 μg/mL) for 24 h. Apoptosis cells were counted under a light microscope and expressed as the average of five fields. Each bar represents the mean ± SD calculated from independent experiments. Significance was determined by Dunnett’s t -test with * p
Figure Legend Snippet: Effect of fucoidan on the chromatin condensation in DU-145 cells. ( A ) DU-145 cells were treated with 0, 500, 1000 μg/mL fucoidan or vehicle in RPMI-1640 medium containing 5% FBS for 24 h, and cell were stained with DAPI. The arrows indicate chromatin condensation in the cancer cell. ( B ) DU-145 cells were treated with fucoidan (0, 500, 1000 μg/mL) for 24 h. Apoptosis cells were counted under a light microscope and expressed as the average of five fields. Each bar represents the mean ± SD calculated from independent experiments. Significance was determined by Dunnett’s t -test with * p

Techniques Used: Staining, Light Microscopy

34) Product Images from "Combined tests of prostate specific antigen and testosterone will improve diagnosis and monitoring the progression of prostate cancer"

Article Title: Combined tests of prostate specific antigen and testosterone will improve diagnosis and monitoring the progression of prostate cancer

Journal: Asian Journal of Andrology

doi: 10.4103/1008-682X.148721

LNCaP cell were seeded in 24 well poly-lysine coated culture plates in RPMI-1640 medium supplemented with 10% FBS for 48 h. Medium was changed to fresh serum free medium (day 0), serum free medium was changed and collected for PSA test every day till day 4. Different concentrations of T were added to different wells on day 4 and PSA level in each well was tested on day 5. *** P
Figure Legend Snippet: LNCaP cell were seeded in 24 well poly-lysine coated culture plates in RPMI-1640 medium supplemented with 10% FBS for 48 h. Medium was changed to fresh serum free medium (day 0), serum free medium was changed and collected for PSA test every day till day 4. Different concentrations of T were added to different wells on day 4 and PSA level in each well was tested on day 5. *** P

Techniques Used:

35) Product Images from "Extracellular Interactions between Hepatitis C Virus and Secreted Apolipoprotein E"

Article Title: Extracellular Interactions between Hepatitis C Virus and Secreted Apolipoprotein E

Journal: Journal of Virology

doi: 10.1128/JVI.02227-16

Infectivity of reporter HCV is influenced by sApoE in a dose-dependent manner. (A) Genomic organization of the JFH1-derived luciferase reporter construct (HCV-Rluc). (B) Schematic representation of full-length (FL; 1, 3, and 4A) and N- or C-terminal domain deletion mutants of sApoE. (C to F) Equivalent amounts of reporter HCV were incubated with increasing concentrations of sApoE1 (C), sApoE3 (D), sApoE4A (E), or sApoE3 deletion mutants (F) at room temperature for 30 min and subsequently used to infect Huh7.5 cells in 24-well culture plates. At 5 h h.p.i., the mixture of HCV-Rluc and sApoE was removed, and the cells were washed twice with PBS and then incubated with DMEM containing 10% FBS. After 24 h, the cells were lysed for luciferase assays. The quantitative results derived from the data were converted to percentages of the control in the bar graph. The data are shown as the mean ± SDs (error bars) pooled from three independent experiments. *, P
Figure Legend Snippet: Infectivity of reporter HCV is influenced by sApoE in a dose-dependent manner. (A) Genomic organization of the JFH1-derived luciferase reporter construct (HCV-Rluc). (B) Schematic representation of full-length (FL; 1, 3, and 4A) and N- or C-terminal domain deletion mutants of sApoE. (C to F) Equivalent amounts of reporter HCV were incubated with increasing concentrations of sApoE1 (C), sApoE3 (D), sApoE4A (E), or sApoE3 deletion mutants (F) at room temperature for 30 min and subsequently used to infect Huh7.5 cells in 24-well culture plates. At 5 h h.p.i., the mixture of HCV-Rluc and sApoE was removed, and the cells were washed twice with PBS and then incubated with DMEM containing 10% FBS. After 24 h, the cells were lysed for luciferase assays. The quantitative results derived from the data were converted to percentages of the control in the bar graph. The data are shown as the mean ± SDs (error bars) pooled from three independent experiments. *, P

Techniques Used: Infection, Derivative Assay, Luciferase, Construct, Incubation

36) Product Images from "Angiocidin Inhibits Breast Cancer Proliferation Through Activation of Epidermal Growth Factor Receptor and Nuclear Factor ?B (Nf-?B)"

Article Title: Angiocidin Inhibits Breast Cancer Proliferation Through Activation of Epidermal Growth Factor Receptor and Nuclear Factor ?B (Nf-?B)

Journal: Experimental and molecular pathology

doi: 10.1016/j.yexmp.2011.01.002

Angiocidin promotes the activation of NFκB and phosphorylation of IκBα. Panel A-NFκB p65 Western Blot of Nuclear and cytoplasmic extracts of MB-231 cells treated with 10 μg/ml of angiocidin in 2% FBS DMEM for a period of time ranging from 5 minutes to 90 minutes. Panel B-Phospho-IKBα Western Blot of MB-231 cells treated for twenty-four hours with 0 μg/ml, 10 μg/ml, or 20 μg/ml of angiocidin in DMEM containing 0.1% BSA. Experiments were repeated three times and the results of a representative experiment are shown in the figure.
Figure Legend Snippet: Angiocidin promotes the activation of NFκB and phosphorylation of IκBα. Panel A-NFκB p65 Western Blot of Nuclear and cytoplasmic extracts of MB-231 cells treated with 10 μg/ml of angiocidin in 2% FBS DMEM for a period of time ranging from 5 minutes to 90 minutes. Panel B-Phospho-IKBα Western Blot of MB-231 cells treated for twenty-four hours with 0 μg/ml, 10 μg/ml, or 20 μg/ml of angiocidin in DMEM containing 0.1% BSA. Experiments were repeated three times and the results of a representative experiment are shown in the figure.

Techniques Used: Activation Assay, Western Blot

37) Product Images from "Self-assembled supramolecular hetero-bimetallacycles for anticancer potency via intracellular release"

Article Title: Self-assembled supramolecular hetero-bimetallacycles for anticancer potency via intracellular release

Journal: Chemistry (Weinheim an der Bergstrasse, Germany)

doi: 10.1002/chem.201403372

Inhibitory effect of complex 4 and 5 on T98G solid brain tumor cells. Percentage of viable T98G cells counts after treatment by (A) complexes 4 , and (B) complex 5 at 24, 48 and 72 h. Data obtained by growth kinetics assay (A and B) showed that complexes 4 and 5 have an inhibitory effect on T98G cells that is dependent upon incubation time. The cultured cells were treated with 0.5 to 16 μM concentrations of the complexes. (C) Clonogenic capacity of T98G cells treated with complex 4 , and 5 . (D) Loss of growth-inhibiting activity of the complexes pre-incubated in culture medium. Complexes 4 and 5 , at a concentration of 6 μM, were pre-incubated in DMSO and cell culture medium (DMEM) supplemented with 10% FBS at 37°C for the indicated times before being added to cultures of T98G cells.
Figure Legend Snippet: Inhibitory effect of complex 4 and 5 on T98G solid brain tumor cells. Percentage of viable T98G cells counts after treatment by (A) complexes 4 , and (B) complex 5 at 24, 48 and 72 h. Data obtained by growth kinetics assay (A and B) showed that complexes 4 and 5 have an inhibitory effect on T98G cells that is dependent upon incubation time. The cultured cells were treated with 0.5 to 16 μM concentrations of the complexes. (C) Clonogenic capacity of T98G cells treated with complex 4 , and 5 . (D) Loss of growth-inhibiting activity of the complexes pre-incubated in culture medium. Complexes 4 and 5 , at a concentration of 6 μM, were pre-incubated in DMSO and cell culture medium (DMEM) supplemented with 10% FBS at 37°C for the indicated times before being added to cultures of T98G cells.

Techniques Used: Incubation, Cell Culture, Activity Assay, Concentration Assay

38) Product Images from "Mechanism Underlying Defective Interferon Gamma-Induced IDO Expression in Non-obese Diabetic Mouse Fibroblasts"

Article Title: Mechanism Underlying Defective Interferon Gamma-Induced IDO Expression in Non-obese Diabetic Mouse Fibroblasts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0037747

IFN-γ-induced-STAT1 phosphorylation in C57BL/6 and NOD dermal fibroblasts. Following starvation for 18 hours, dermal fibroblasts from NOD (open bars) and C57BL/6 (solid bars) mice were remained untreated or treated with 1000 U IFN-γ per ml of DMEM plus 2% FBS for 15, 30 or 60 minutes. Cell lysates were collected for western blot analysis. A : STAT 1 phosphorylation shown by western blotting. B : the Mean±SEM ratio of phospho-STAT1 (P-STAT1), to the ratio of β-actin to total STAT1. Total STAT1 and β-actin expressions were used as loading controls. *denotes significant difference between related bars (p
Figure Legend Snippet: IFN-γ-induced-STAT1 phosphorylation in C57BL/6 and NOD dermal fibroblasts. Following starvation for 18 hours, dermal fibroblasts from NOD (open bars) and C57BL/6 (solid bars) mice were remained untreated or treated with 1000 U IFN-γ per ml of DMEM plus 2% FBS for 15, 30 or 60 minutes. Cell lysates were collected for western blot analysis. A : STAT 1 phosphorylation shown by western blotting. B : the Mean±SEM ratio of phospho-STAT1 (P-STAT1), to the ratio of β-actin to total STAT1. Total STAT1 and β-actin expressions were used as loading controls. *denotes significant difference between related bars (p

Techniques Used: Mouse Assay, Western Blot

LPS-induced IDO expression in C57BL/6 and NOD dermal fibroblasts. In panels A and B, C57BL/6 (solid bars) and NOD (open bars) were treated with 0 or 1 µg of LPS from Pseudomonas aeruginosa per ml of DMEM plus 2% FBS, for 24 hours. A : RT-PCR analysis of IDO mRNA expression. B : the Mean±SEM ratio of densities of IDO to β-actin. GAPDH was used as a loading control. In panels C, D and E, cells were treated with 1 g/ml of LPS in the presence or absence of SP600125 (10 M), a JNK inhibitor, for 48 hours. C: western blot analysis of IDO expression, D: the Mean±SEM ratio of densities of IDO to β-actin. E : The Kyn levels indicate that LPS induced the expression of active IDO enzyme in NOD and C57BL/6 fibroblasts, which was mediated through JNK pathway. *denotes significant difference between cells from the same strain treated with LPS with or without SP600125 (p
Figure Legend Snippet: LPS-induced IDO expression in C57BL/6 and NOD dermal fibroblasts. In panels A and B, C57BL/6 (solid bars) and NOD (open bars) were treated with 0 or 1 µg of LPS from Pseudomonas aeruginosa per ml of DMEM plus 2% FBS, for 24 hours. A : RT-PCR analysis of IDO mRNA expression. B : the Mean±SEM ratio of densities of IDO to β-actin. GAPDH was used as a loading control. In panels C, D and E, cells were treated with 1 g/ml of LPS in the presence or absence of SP600125 (10 M), a JNK inhibitor, for 48 hours. C: western blot analysis of IDO expression, D: the Mean±SEM ratio of densities of IDO to β-actin. E : The Kyn levels indicate that LPS induced the expression of active IDO enzyme in NOD and C57BL/6 fibroblasts, which was mediated through JNK pathway. *denotes significant difference between cells from the same strain treated with LPS with or without SP600125 (p

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

39) Product Images from "Diazinon exposure activated transcriptional factors CCAAT-enhancer-binding proteins α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) and induced adipogenesis in 3T3-L1 preadipocytes"

Article Title: Diazinon exposure activated transcriptional factors CCAAT-enhancer-binding proteins α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) and induced adipogenesis in 3T3-L1 preadipocytes

Journal: Pesticide biochemistry and physiology

doi: 10.1016/j.pestbp.2018.07.003

Illustration of the treatment schedule and maturation stages of 3T3-L1 preadipocytes. 3T3-L1 cells were cultured to 100% confluence in 10% FBS DMEM (M1 medium), denoted as day 0. To differentiate the pre-adipocyte 3T3-L1 cells as a positive control (Pos), cells were incubated with 1 μM DEX in adipogenic induction medium (M2 medium: DMEM containing, 0.5 mM IBMX, 167 nM insulin and 10% FBS) for 2 days (day 2). To examine the effect of a compound on the adipogenesis, diazinon was added to the M2 medium for two days, M3 medium for two days, and M1 medium for another four days. Cells cultured in the vehicle (DMSO 0.1%) in all medium was set as the untreated control (CTL). The cellular differentiation was processed into three stages: induction (day 0–2), differentiation (day 2–4) and maturation (day 4–8).
Figure Legend Snippet: Illustration of the treatment schedule and maturation stages of 3T3-L1 preadipocytes. 3T3-L1 cells were cultured to 100% confluence in 10% FBS DMEM (M1 medium), denoted as day 0. To differentiate the pre-adipocyte 3T3-L1 cells as a positive control (Pos), cells were incubated with 1 μM DEX in adipogenic induction medium (M2 medium: DMEM containing, 0.5 mM IBMX, 167 nM insulin and 10% FBS) for 2 days (day 2). To examine the effect of a compound on the adipogenesis, diazinon was added to the M2 medium for two days, M3 medium for two days, and M1 medium for another four days. Cells cultured in the vehicle (DMSO 0.1%) in all medium was set as the untreated control (CTL). The cellular differentiation was processed into three stages: induction (day 0–2), differentiation (day 2–4) and maturation (day 4–8).

Techniques Used: Cell Culture, Positive Control, Incubation, CTL Assay, Cell Differentiation

40) Product Images from "Inhibition of nuclear factor-kappa B differentially affects thyroid cancer cell growth, apoptosis, and invasion"

Article Title: Inhibition of nuclear factor-kappa B differentially affects thyroid cancer cell growth, apoptosis, and invasion

Journal: Molecular Cancer

doi: 10.1186/1476-4598-9-117

Differential Inhibition of Thyroid Cancer Cell Line Invasiveness by Blocking NF-κB Signaling . BCPAP, SW1736, TPC1, and C643 cells were transduced with either Ad-GFP or Ad-mIκBα at an MOI of 200, 100, 50, and 100, respectively. After 24 hours, cells were serum-starved in RPMI (0.1% FBS) for 6 hours. Cells were then harvested by trypsinization and applied to Matrigel-coated transwell chambers in RPMI supplemented with 0.1% FBS. Invasion was promoted by the presence of RPMI supplemented with 10% FBS in the lower chamber. Invaded cells were then fixed, stained with DAPI, and counted, as described in Materials and Methods. Data is represented as percent cell invasion compared to Ad-GFP control (normalized to 100%), which is denoted by the dashed line. Mean ± S.E.M. of 3 independent experiments performed in duplicate is reported. [p
Figure Legend Snippet: Differential Inhibition of Thyroid Cancer Cell Line Invasiveness by Blocking NF-κB Signaling . BCPAP, SW1736, TPC1, and C643 cells were transduced with either Ad-GFP or Ad-mIκBα at an MOI of 200, 100, 50, and 100, respectively. After 24 hours, cells were serum-starved in RPMI (0.1% FBS) for 6 hours. Cells were then harvested by trypsinization and applied to Matrigel-coated transwell chambers in RPMI supplemented with 0.1% FBS. Invasion was promoted by the presence of RPMI supplemented with 10% FBS in the lower chamber. Invaded cells were then fixed, stained with DAPI, and counted, as described in Materials and Methods. Data is represented as percent cell invasion compared to Ad-GFP control (normalized to 100%), which is denoted by the dashed line. Mean ± S.E.M. of 3 independent experiments performed in duplicate is reported. [p

Techniques Used: Inhibition, Blocking Assay, Transduction, Staining

Related Articles

Protease Inhibitor:

Article Title: Sulforaphane Protects Astrocytes against Oxidative Stress and Delayed Death Caused by Oxygen and Glucose Deprivation
Article Snippet: .. DMEM/F12 50/50 culture medium was purchased from Cellgro (Manassas, VA, USA), Fetal bovine serum (FBS) from HyClone (Logan, UT, USA), penicillin-streptomycin solution from Gemini Bio-Products (West Sacramento, CA, USA), mounting medium from Vector Laboratories Inc. (Burlingame, CA, USA), phosphate buffered saline (PBS) from Cambrex BioScience (Walkersville, MD), trypsin EDTA from Cellgro (Manassas, VA, USA) protease inhibitor cocktail from Calbiochem (San Diego, CA, USA), RNA-Bee from TEL-TEST, Inc. (Friendswood, TX, USA). ..

Avidin-Biotin Assay:

Article Title: Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-?-Mediated and Non-Transforming Growth Factor-?-Mediated Transdifferentiation in Vitro
Article Snippet: .. Material used were endothelial basal medium (EBM) (CC-3121; Clonetics, San Diego, CA); fetal bovine serum (FBS) (Hyclone, Logan, UT); 100× GPS (29.2 mg/ml l -glutamine, 10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate); gentamicin sulfate and 100× PSF (10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate, 25 μg/ml amphotericin B) (Life Technologies, Inc., Grand Island, NY); collagenase A (Boehringer Mannheim, Indianapolis, IN); Immobilon-P membrane (Millipore, Bedford, MA); Hyperfilm ECL, fluorescein-streptavidin, and Texas Red-streptavidin (Amersham Life Sciences, Arlington Heights, IL); Lumiglo (KPL); human TGF-β1, recombinant human TGF-β2 and -β3, recombinant human platelet-derived growth factor (PDGF)-BB, and anti-PDFG-BB (R & D Systems, Minneapolis, MN); Vectastain Elite ABC kit, avidin/biotin blocking kit, fluorescein anti-mouse IgG, Texas red anti-rabbit IgG, peroxidase-conjugated anti-goat IgG, biotinylated horse anti-mouse IgG, avidin-peroxidase, peroxidase-conjugated anti-mouse IgG, and 3,3′5,5′-tetramethylbenzidine (Vector Laboratories, Burlingame, CA); 3-amino-9-ethyl carbazol and mouse anti-human α-SMA (clone 1A4 ) (Sigma Chemical Co., St. Louis, MO); goat anti-human CD31/PECAM-1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-human von Willebrand factor (vWF) and mouse anti-human CD31/PECAM-1 (DAKO, Carpinteria, CA); polycarbonate PVP-F membranes (Neuro Probe, Inc, Gaithersburg, MD). .. Recombinant human bFGF was kindly provided by Scios Nova Inc., Mountain View, CA; soluble recombinant TGF-β type II receptor, prepared as described, was kindly provided by Philip Gotwals, Biogen, Cambridge, MA; SM1 antibody was kindly provided by Masanori Aikawa, Brigham and Women’s Hospital, Boston; rabbit anti-bovine CD31/PECAM-1 was kindly provided by Steven Albelda, University of Pennsylvania.

Cell Culture:

Article Title: Conditioned Media from Human Adipose Tissue-Derived Mesenchymal Stem Cells and Umbilical Cord-Derived Mesenchymal Stem Cells Efficiently Induced the Apoptosis and Differentiation in Human Glioma Cell Lines In Vitro
Article Snippet: .. U251 cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) until confluency. ..

Blocking Assay:

Article Title: Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-?-Mediated and Non-Transforming Growth Factor-?-Mediated Transdifferentiation in Vitro
Article Snippet: .. Material used were endothelial basal medium (EBM) (CC-3121; Clonetics, San Diego, CA); fetal bovine serum (FBS) (Hyclone, Logan, UT); 100× GPS (29.2 mg/ml l -glutamine, 10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate); gentamicin sulfate and 100× PSF (10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate, 25 μg/ml amphotericin B) (Life Technologies, Inc., Grand Island, NY); collagenase A (Boehringer Mannheim, Indianapolis, IN); Immobilon-P membrane (Millipore, Bedford, MA); Hyperfilm ECL, fluorescein-streptavidin, and Texas Red-streptavidin (Amersham Life Sciences, Arlington Heights, IL); Lumiglo (KPL); human TGF-β1, recombinant human TGF-β2 and -β3, recombinant human platelet-derived growth factor (PDGF)-BB, and anti-PDFG-BB (R & D Systems, Minneapolis, MN); Vectastain Elite ABC kit, avidin/biotin blocking kit, fluorescein anti-mouse IgG, Texas red anti-rabbit IgG, peroxidase-conjugated anti-goat IgG, biotinylated horse anti-mouse IgG, avidin-peroxidase, peroxidase-conjugated anti-mouse IgG, and 3,3′5,5′-tetramethylbenzidine (Vector Laboratories, Burlingame, CA); 3-amino-9-ethyl carbazol and mouse anti-human α-SMA (clone 1A4 ) (Sigma Chemical Co., St. Louis, MO); goat anti-human CD31/PECAM-1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-human von Willebrand factor (vWF) and mouse anti-human CD31/PECAM-1 (DAKO, Carpinteria, CA); polycarbonate PVP-F membranes (Neuro Probe, Inc, Gaithersburg, MD). .. Recombinant human bFGF was kindly provided by Scios Nova Inc., Mountain View, CA; soluble recombinant TGF-β type II receptor, prepared as described, was kindly provided by Philip Gotwals, Biogen, Cambridge, MA; SM1 antibody was kindly provided by Masanori Aikawa, Brigham and Women’s Hospital, Boston; rabbit anti-bovine CD31/PECAM-1 was kindly provided by Steven Albelda, University of Pennsylvania.

Modification:

Article Title: Marinobufagenin inhibits glioma growth through sodium pump α1 subunit and ERK signaling‐mediated mitochondrial apoptotic pathway
Article Snippet: .. Trypsin, fetal bovine serum (FBS), and Dulbecco's modified Eagle's medium (DMEM) were obtained from HyClone Laboratories (HyClone Laboratories Inc., WEST 1800 SOUTH LOGANUT 84321). ..

Article Title: Nonproteolytic Properties of Murine Alternatively Spliced Tissue Factor: Implications for Integrin-Mediated Signaling in Murine Models
Article Snippet: .. Murine endothelial cells (ECs) (bEnd.3 from the American Type Culture Collection [ATCC], Manassas, VA, USA) and murine embryonic endothelial cells [MEECs], provided by MJ Goumans, Leiden University Medical Center [LUMC], Leiden, the Netherlands), primary human retinal endothelial cells (HRECs, Cell Systems) and murine monocytes/ macrophages (J774A.1, ATCC) were grown in filter-cap tissue culture flasks containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/ streptomycin (all from Hyclone/ Thermo Scientific, Rockford, IL, USA). ..

Article Title: A Functional Variant of IC53 Correlates with the Late Onset of Colorectal Cancer
Article Snippet: .. The colon cancer adenocarcinoma cell lines HCT-116, HT-29 and mouse embryonic fibroblast cell line NIH3T3 were obtained from the Institute of Cell Biology, Academic Sinica, and propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% penicillin/ streptomycin. ..

Recombinant:

Article Title: Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-?-Mediated and Non-Transforming Growth Factor-?-Mediated Transdifferentiation in Vitro
Article Snippet: .. Material used were endothelial basal medium (EBM) (CC-3121; Clonetics, San Diego, CA); fetal bovine serum (FBS) (Hyclone, Logan, UT); 100× GPS (29.2 mg/ml l -glutamine, 10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate); gentamicin sulfate and 100× PSF (10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate, 25 μg/ml amphotericin B) (Life Technologies, Inc., Grand Island, NY); collagenase A (Boehringer Mannheim, Indianapolis, IN); Immobilon-P membrane (Millipore, Bedford, MA); Hyperfilm ECL, fluorescein-streptavidin, and Texas Red-streptavidin (Amersham Life Sciences, Arlington Heights, IL); Lumiglo (KPL); human TGF-β1, recombinant human TGF-β2 and -β3, recombinant human platelet-derived growth factor (PDGF)-BB, and anti-PDFG-BB (R & D Systems, Minneapolis, MN); Vectastain Elite ABC kit, avidin/biotin blocking kit, fluorescein anti-mouse IgG, Texas red anti-rabbit IgG, peroxidase-conjugated anti-goat IgG, biotinylated horse anti-mouse IgG, avidin-peroxidase, peroxidase-conjugated anti-mouse IgG, and 3,3′5,5′-tetramethylbenzidine (Vector Laboratories, Burlingame, CA); 3-amino-9-ethyl carbazol and mouse anti-human α-SMA (clone 1A4 ) (Sigma Chemical Co., St. Louis, MO); goat anti-human CD31/PECAM-1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-human von Willebrand factor (vWF) and mouse anti-human CD31/PECAM-1 (DAKO, Carpinteria, CA); polycarbonate PVP-F membranes (Neuro Probe, Inc, Gaithersburg, MD). .. Recombinant human bFGF was kindly provided by Scios Nova Inc., Mountain View, CA; soluble recombinant TGF-β type II receptor, prepared as described, was kindly provided by Philip Gotwals, Biogen, Cambridge, MA; SM1 antibody was kindly provided by Masanori Aikawa, Brigham and Women’s Hospital, Boston; rabbit anti-bovine CD31/PECAM-1 was kindly provided by Steven Albelda, University of Pennsylvania.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    GE Healthcare fbs
    Light micrographs of Oil red O staining. ASCs bought from a commercial vendor (RB) were compared to ASCs derived from abdominoplasty (HAP) or burned human skin (BH) cultured in <t>DMEM</t> with 10% <t>FBS</t> and 10% hPL and differentiated under adipogenic conditions with identical supplements for 14 days, then stained for lipid accumulation by Oil red O. Representative images of undifferentiated RB (a), HAP cells (b, c), and BH cells (d, e) from control media conditions and differentiated RB (f), HAP cells (g, h), and BH (i, j) are given. White scale bar represents 100 μ m.
    Fbs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 3656 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/GE Healthcare
    Average 94 stars, based on 3656 article reviews
    Price from $9.99 to $1999.99
    fbs - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    91
    GE Healthcare mouse nih 3t3 cells
    IRF8 needs cooperative factors to induce Th9 polarisation. a Transactivation of the Il9 (left) and Il21 (right) promoters by IRF8. Il9 or Il21 promoter reporter plasmids were transfected into <t>NIH-3T3</t> cells with an IRF8-encoding vector. b Determination of IRF8-binding motifs by analysis of IRF8 ChIP-Seq peaks from WT Th9 cells. Left, letter size indicates frequency of nucleotide. Right, significance of motif occurrence. c Immunoprecipitation assay on Th9 cells differentiated for 24 h using anti-IRF8 antibody (above) or anti-PU.1 antibody (below), followed by immunoblot analysis with indicated specific antibodies. Ig is used as a control. d , e PLA in WT naive CD4 + T cells and in Th9 cells after 24 h of differentiation. DAPI staining (blue) indicates the nucleus. Green dots represent proximity between IRF8/PU.1, IRF4/PU.1 or IRF8/IRF4 ( d ) or between BATF and IRF8 or PU.1 or IRF4 ( e ) Magnifications are ×63, scale bar represents 5 µm. f Top panel represent a screenshot from the ECR (evolutionary conserved regions) Browser web site of the mouse Il9 (left) and Il21 (right) genes. Exons are in blue, introns in pink, UTRs in yellow and CNS are in red. Bottom panel is a representation of binding peaks of ChIP-Seq data of BATF, IRF4, PU.1 and IRF8 in WT Th9 cells differentiated for 24 h. The red square shows the overlap between BATF, IRF4, PU.1 and IRF8 peaks in Il9 or Il21 CNS1. g Co-location of IRF8, PU.1, IRF4 and BATF-binding peaks in Th9 cells were determined by multiple overlap analysis of the respective ChIP-Seq data. IRF8 can bind DNA alone or in combination with one, two or three factors (IRF4, BATF and PU.1). h The average density of IRF4, PU.1 and BATF in Th9 cells is shown for regions centred on the summits of the IRF8 peaks. i Transactivation of the Il9 promoters by IRF8, PU.1, IRF4 and BATF. Il9 promoter reporter plasmids were transfected into NIH-3T3 cells with vectors encoding IRF8, PU.1, IRF4 and BATF alone or in combination. ns, not significant; * P
    Mouse Nih 3t3 Cells, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse nih 3t3 cells/product/GE Healthcare
    Average 91 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    mouse nih 3t3 cells - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

    92
    GE Healthcare dmem
    Metabolic inhibition of sulfation of HS chains leads to a loss of sulfated groups of HS, Hsp90α, and Hsp90β from the cell surface ( a ) and to an inhibition of the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with sodium chlorate, after which cells were grown at 37 °C in <t>DMEM-FBS</t> without chlorate. At indicated times, cell surface HS and proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of chlorate-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of chlorate-treated cells. The basal migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p
    Dmem, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 1040 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dmem/product/GE Healthcare
    Average 92 stars, based on 1040 article reviews
    Price from $9.99 to $1999.99
    dmem - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    Image Search Results


    Light micrographs of Oil red O staining. ASCs bought from a commercial vendor (RB) were compared to ASCs derived from abdominoplasty (HAP) or burned human skin (BH) cultured in DMEM with 10% FBS and 10% hPL and differentiated under adipogenic conditions with identical supplements for 14 days, then stained for lipid accumulation by Oil red O. Representative images of undifferentiated RB (a), HAP cells (b, c), and BH cells (d, e) from control media conditions and differentiated RB (f), HAP cells (g, h), and BH (i, j) are given. White scale bar represents 100 μ m.

    Journal: Stem Cells International

    Article Title: Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

    doi: 10.1155/2017/7108458

    Figure Lengend Snippet: Light micrographs of Oil red O staining. ASCs bought from a commercial vendor (RB) were compared to ASCs derived from abdominoplasty (HAP) or burned human skin (BH) cultured in DMEM with 10% FBS and 10% hPL and differentiated under adipogenic conditions with identical supplements for 14 days, then stained for lipid accumulation by Oil red O. Representative images of undifferentiated RB (a), HAP cells (b, c), and BH cells (d, e) from control media conditions and differentiated RB (f), HAP cells (g, h), and BH (i, j) are given. White scale bar represents 100 μ m.

    Article Snippet: Immunophenotyping by FACS Three isolates of cryopreserved BH and HAP ASCs were resuspended in low-glucose Dulbecco's modified Eagle's medium (Gibco® DMEM; Thermo Fisher Scientific, Frederick, MD) supplemented with either 10% FBS (GE Life Sciences; Logan, Utah) or 10% hPL.

    Techniques: Staining, Derivative Assay, Cell Culture

    Effect of LPE on phosphorylation or expression of rate-limiting enzymes in the arachidonate cascade. RBL-2H3 cells were seeded on a 6-well plate (5 × 10 5 cells/well) in MEM- α with 10% FBS at 37°C overnight and further incubated with DNP-IgE for 24 h. IgE-sensitized RBL-2H3 cells were preincubated with LPE (0 to 350 μ g/mL) prior to antigen challenge. The above cells were washed with 1x DPBS and lysed with cell lysis buffer. The expression of p-cPLA 2 , p-5-LO, COX-2, or β -actin was determined as described in Section 2 . Similar results were obtained in three independent experiments. ∗∗ P

    Journal: Mediators of Inflammation

    Article Title: Inhibitory Effect of Loranthus parasiticus on IgE-Mediated Allergic Responses in RBL-2H3 Cells

    doi: 10.1155/2016/8742562

    Figure Lengend Snippet: Effect of LPE on phosphorylation or expression of rate-limiting enzymes in the arachidonate cascade. RBL-2H3 cells were seeded on a 6-well plate (5 × 10 5 cells/well) in MEM- α with 10% FBS at 37°C overnight and further incubated with DNP-IgE for 24 h. IgE-sensitized RBL-2H3 cells were preincubated with LPE (0 to 350 μ g/mL) prior to antigen challenge. The above cells were washed with 1x DPBS and lysed with cell lysis buffer. The expression of p-cPLA 2 , p-5-LO, COX-2, or β -actin was determined as described in Section 2 . Similar results were obtained in three independent experiments. ∗∗ P

    Article Snippet: Reagents MEM-α medium, 1x DPBS, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from GE Healthcare Life Sciences (Hyclone™ , Logan, UT, USA).

    Techniques: Expressing, Incubation, Lysis

    Effect of LPE on degranulation and cell viability in IgE-mediated RBL-2H3 cells. RBL-2H3 cells were seeded on a 24-well plate (1 × 10 5 cells/well) or a 96-well plate (1 × 10 4 cells/well) in MEM- α with 10% FBS at 37°C overnight and further incubated with DNP-IgE for 24 h. IgE-sensitized cells were preincubated with LPE (0 to 400 μ g/mL) for 1 h and then stimulated with DNP-HSA (0.1 μ g/mL) for 4 h. β -Hexosaminidase activity and cell viability were determined as described in Section 2 . Data are the mean ± SD values of triple or octuple determinations. ∗∗ P

    Journal: Mediators of Inflammation

    Article Title: Inhibitory Effect of Loranthus parasiticus on IgE-Mediated Allergic Responses in RBL-2H3 Cells

    doi: 10.1155/2016/8742562

    Figure Lengend Snippet: Effect of LPE on degranulation and cell viability in IgE-mediated RBL-2H3 cells. RBL-2H3 cells were seeded on a 24-well plate (1 × 10 5 cells/well) or a 96-well plate (1 × 10 4 cells/well) in MEM- α with 10% FBS at 37°C overnight and further incubated with DNP-IgE for 24 h. IgE-sensitized cells were preincubated with LPE (0 to 400 μ g/mL) for 1 h and then stimulated with DNP-HSA (0.1 μ g/mL) for 4 h. β -Hexosaminidase activity and cell viability were determined as described in Section 2 . Data are the mean ± SD values of triple or octuple determinations. ∗∗ P

    Article Snippet: Reagents MEM-α medium, 1x DPBS, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from GE Healthcare Life Sciences (Hyclone™ , Logan, UT, USA).

    Techniques: Incubation, Activity Assay

    IRF8 needs cooperative factors to induce Th9 polarisation. a Transactivation of the Il9 (left) and Il21 (right) promoters by IRF8. Il9 or Il21 promoter reporter plasmids were transfected into NIH-3T3 cells with an IRF8-encoding vector. b Determination of IRF8-binding motifs by analysis of IRF8 ChIP-Seq peaks from WT Th9 cells. Left, letter size indicates frequency of nucleotide. Right, significance of motif occurrence. c Immunoprecipitation assay on Th9 cells differentiated for 24 h using anti-IRF8 antibody (above) or anti-PU.1 antibody (below), followed by immunoblot analysis with indicated specific antibodies. Ig is used as a control. d , e PLA in WT naive CD4 + T cells and in Th9 cells after 24 h of differentiation. DAPI staining (blue) indicates the nucleus. Green dots represent proximity between IRF8/PU.1, IRF4/PU.1 or IRF8/IRF4 ( d ) or between BATF and IRF8 or PU.1 or IRF4 ( e ) Magnifications are ×63, scale bar represents 5 µm. f Top panel represent a screenshot from the ECR (evolutionary conserved regions) Browser web site of the mouse Il9 (left) and Il21 (right) genes. Exons are in blue, introns in pink, UTRs in yellow and CNS are in red. Bottom panel is a representation of binding peaks of ChIP-Seq data of BATF, IRF4, PU.1 and IRF8 in WT Th9 cells differentiated for 24 h. The red square shows the overlap between BATF, IRF4, PU.1 and IRF8 peaks in Il9 or Il21 CNS1. g Co-location of IRF8, PU.1, IRF4 and BATF-binding peaks in Th9 cells were determined by multiple overlap analysis of the respective ChIP-Seq data. IRF8 can bind DNA alone or in combination with one, two or three factors (IRF4, BATF and PU.1). h The average density of IRF4, PU.1 and BATF in Th9 cells is shown for regions centred on the summits of the IRF8 peaks. i Transactivation of the Il9 promoters by IRF8, PU.1, IRF4 and BATF. Il9 promoter reporter plasmids were transfected into NIH-3T3 cells with vectors encoding IRF8, PU.1, IRF4 and BATF alone or in combination. ns, not significant; * P

    Journal: Nature Communications

    Article Title: IRF8-dependent molecular complexes control the Th9 transcriptional program

    doi: 10.1038/s41467-017-01070-w

    Figure Lengend Snippet: IRF8 needs cooperative factors to induce Th9 polarisation. a Transactivation of the Il9 (left) and Il21 (right) promoters by IRF8. Il9 or Il21 promoter reporter plasmids were transfected into NIH-3T3 cells with an IRF8-encoding vector. b Determination of IRF8-binding motifs by analysis of IRF8 ChIP-Seq peaks from WT Th9 cells. Left, letter size indicates frequency of nucleotide. Right, significance of motif occurrence. c Immunoprecipitation assay on Th9 cells differentiated for 24 h using anti-IRF8 antibody (above) or anti-PU.1 antibody (below), followed by immunoblot analysis with indicated specific antibodies. Ig is used as a control. d , e PLA in WT naive CD4 + T cells and in Th9 cells after 24 h of differentiation. DAPI staining (blue) indicates the nucleus. Green dots represent proximity between IRF8/PU.1, IRF4/PU.1 or IRF8/IRF4 ( d ) or between BATF and IRF8 or PU.1 or IRF4 ( e ) Magnifications are ×63, scale bar represents 5 µm. f Top panel represent a screenshot from the ECR (evolutionary conserved regions) Browser web site of the mouse Il9 (left) and Il21 (right) genes. Exons are in blue, introns in pink, UTRs in yellow and CNS are in red. Bottom panel is a representation of binding peaks of ChIP-Seq data of BATF, IRF4, PU.1 and IRF8 in WT Th9 cells differentiated for 24 h. The red square shows the overlap between BATF, IRF4, PU.1 and IRF8 peaks in Il9 or Il21 CNS1. g Co-location of IRF8, PU.1, IRF4 and BATF-binding peaks in Th9 cells were determined by multiple overlap analysis of the respective ChIP-Seq data. IRF8 can bind DNA alone or in combination with one, two or three factors (IRF4, BATF and PU.1). h The average density of IRF4, PU.1 and BATF in Th9 cells is shown for regions centred on the summits of the IRF8 peaks. i Transactivation of the Il9 promoters by IRF8, PU.1, IRF4 and BATF. Il9 promoter reporter plasmids were transfected into NIH-3T3 cells with vectors encoding IRF8, PU.1, IRF4 and BATF alone or in combination. ns, not significant; * P

    Article Snippet: Mouse NIH-3T3 cells (cultured in DMEM 4.5 g/l + glutamine + FBS at 37 °C, 5% CO2 ) were transiently transfected for 48 h with reporter plasmid and the pCMV-SPORT6-IRF8 (GE Healthcare), pCMV-SPORT6-PU.1 (GE Healthcare), pCR4-TOPO-IRF4 (Thermo Scientific) and pcDNA3.1-mBATF (Addgene) using Lipofectamine 2000 (Invitrogen).

    Techniques: Transfection, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Proximity Ligation Assay, Staining

    Metabolic inhibition of sulfation of HS chains leads to a loss of sulfated groups of HS, Hsp90α, and Hsp90β from the cell surface ( a ) and to an inhibition of the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with sodium chlorate, after which cells were grown at 37 °C in DMEM-FBS without chlorate. At indicated times, cell surface HS and proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of chlorate-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of chlorate-treated cells. The basal migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Journal: Cell Stress & Chaperones

    Article Title: Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells

    doi: 10.1007/s12192-018-0955-5

    Figure Lengend Snippet: Metabolic inhibition of sulfation of HS chains leads to a loss of sulfated groups of HS, Hsp90α, and Hsp90β from the cell surface ( a ) and to an inhibition of the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with sodium chlorate, after which cells were grown at 37 °C in DMEM-FBS without chlorate. At indicated times, cell surface HS and proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of chlorate-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of chlorate-treated cells. The basal migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Article Snippet: FBS and DMEM were purchased from GE Healthcare.

    Techniques: Inhibition, Migration, Staining, Flow Cytometry, Cytometry, Wound Healing Assay

    Digestion of HS moieties of HSPGs with a heparinase reduces the level of HS and Hsp90s at the cell surface ( a ) and inhibits the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with a heparinase, after which cells were grown at 37 °C in DMEM-FBS. At indicated times, cell surface proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of heparinase-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of heparinase-treated cells. The migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Journal: Cell Stress & Chaperones

    Article Title: Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells

    doi: 10.1007/s12192-018-0955-5

    Figure Lengend Snippet: Digestion of HS moieties of HSPGs with a heparinase reduces the level of HS and Hsp90s at the cell surface ( a ) and inhibits the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with a heparinase, after which cells were grown at 37 °C in DMEM-FBS. At indicated times, cell surface proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of heparinase-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of heparinase-treated cells. The migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Article Snippet: FBS and DMEM were purchased from GE Healthcare.

    Techniques: Migration, Staining, Flow Cytometry, Cytometry, Wound Healing Assay