cell culture rt4  (Korean Cell Line Bank)

Journal: Biomaterials

Article Title: The Critical Chemical and Mechanical Regulation of Folic Acid on Neural Engineering

doi: 10.1016/j.biomaterials.2018.03.059

Figure Lengend Snippet: A) Overview of the folic acid niche that provides chemical and mechanical cues to enhance functional recovery in a peripheral injury model. B) Cartoon illustrating the detailed mechanism of the multifunctional and dynamic neuroregenerative folic acid niche and how it allows peripheral nerve regeneration by modulating the proliferation and migration of Schwann cells (I, II), stimulating the Schwann cells to release more neurotrophins (III), and increasing mechanical forces in the neurons that in turn boost the axonal regeneration of the neurons (IV and V) and peripheral nerve regeneration (VI).

Article Snippet: 2.2 Cell culture Rat Schwann cells (ATCC, Manassas, VA, USA; catalog #: CRL-2768TM) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% antibiotic antimycotic 100× solution.

Techniques: Functional Assay, Migration

Journal: Biomaterials

Article Title: The Critical Chemical and Mechanical Regulation of Folic Acid on Neural Engineering

doi: 10.1016/j.biomaterials.2018.03.059

Figure Lengend Snippet: Effects of folic acid on traction force and global DNA methylation levels of PC-12 cells. A) Representative phase contrast microscopic images of PC-12 cells stimulated with low (1.3 mg/L) and high (50 mg/L) concentrations of folic acid in the presence/absence of 50 ng/mL NGF after four days. B, C) Average and maximum traction forces on PC-12 cells stimulated with low (1.3 mg/L) and high (50 mg/L) concentrations of folic acid without (B) and with (C) 50 ng/mL NGF after four days. D) Phase contrast images and traction force maps of PC-12 cells stimulated with 50 mg/L of folic acid at different stages of differentiation. (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05) (Scale bar: 100 µm). E) Global DNA methylation levels in PC-12 cells treated with different concentrations of folic acid in the presence of 50 ng/mL NGF. F) Global DNA methylation levels in Schwann cells treated with different concentrations of folic acid. (****P<0.0001, **P<0.01, *P<0.05).

Article Snippet: 2.2 Cell culture Rat Schwann cells (ATCC, Manassas, VA, USA; catalog #: CRL-2768TM) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% antibiotic antimycotic 100× solution.

Techniques: DNA Methylation Assay

Journal: Biomaterials

Article Title: The Critical Chemical and Mechanical Regulation of Folic Acid on Neural Engineering

doi: 10.1016/j.biomaterials.2018.03.059

Figure Lengend Snippet: Proliferative and chemotactic effects of folic acid. A, B) Cytocompatibility and proliferative effect of different concentrations of folic acid (mg/L) on Schwann cells (A) and on PC-12 cells (B) at various time points. Error bars indicate mean ± S.E.M. (n=8). C, E) Relative cell migration of Schwann cells (C) and human astrocytes (E) determined by the number of the migrated cells in each sample normalized to the total number of the migrated cells in the control sample where the folic acid concentration in the basolateral chamber was 4 mg/L (labeled as ctrl). Each folic acid concentration on x-axes indicates the folic acid concentration in the basolateral chamber when the folic acid concentration was consistent at 4 mg/L. Data are shown as mean ± S.E.M., n=5. (****P<0.0001; ***P<0.001). D, F) Bright field images of the migrated Schwann cells (D) and human astrocytes (F) stained with crystal violet in each sample. The number on top of each picture indicates the folic acid concentration in the basolateral chamber.

Article Snippet: 2.2 Cell culture Rat Schwann cells (ATCC, Manassas, VA, USA; catalog #: CRL-2768TM) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% antibiotic antimycotic 100× solution.

Techniques: Migration, Control, Concentration Assay, Labeling, Staining

Journal: Biomaterials

Article Title: The Critical Chemical and Mechanical Regulation of Folic Acid on Neural Engineering

doi: 10.1016/j.biomaterials.2018.03.059

Figure Lengend Snippet: Effects of folic acid supplementation on neurotrophin release from Schwann cells. A) Representative microscopic images of PC-12 cells cultured for one day and three days with 1) control medium with 50 mg/L of folic acid, 2) cell culture supernatant from the Schwann cells primed with 4 mg/L of folic acid, and 3) cell culture supernatant from the Schwann cells primed with 50 mg/L of folic acid. B) Mean NT3 and NT4/5 levels measured in Schwann cultures incubated with control (4 mg/L) and high-folic acid (50 mg/L) cell media (*P<0.05).

Article Snippet: 2.2 Cell culture Rat Schwann cells (ATCC, Manassas, VA, USA; catalog #: CRL-2768TM) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% antibiotic antimycotic 100× solution.

Techniques: Cell Culture, Control, Incubation

Journal: Journal of proteomics

Article Title: The Impact of Cruciferous Vegetable Isothiocyanates on Histone Acetylation and Histone Phosphorylation in Bladder Cancer

doi: 10.1016/j.jprot.2017.01.013

Figure Lengend Snippet: A. Human bladder cancer cells ranging from superficial (RT4) to invasive (J82 and UMUC3) were treated with DMSO vehicle control or 5, 10 and 20 μM sulforaphane (SFN) and erucin (ECN) for 48h. Protein lysates were obtained under non-denaturing conditions followed by concentration determination and normalization via the BCA Assay. HDAC activity was assessed by Fluor de Lys fluorometric HDAC activity assay. The data was collected, normalized to the DMSO “0” control and is expressed as the mean Relative HDAC Activity ± standard deviation (SD) and represents three independent experiments. Statistical significance was set at *, p<0.05 relative to DMSO controls. B. HDAC activity was also assessed using tissue from a mouse xenograft study where athymic nude mice were subcutaneously injected with 0.05 × 106 UMUC3 cells and gavaged daily with either vehicle control (soybean oil), 295 μmol/kg body weight SFN or 295 μmol/kg body weight ECN (n = 12 mice/group) for 2 weeks until the tumors reached approximately 1.2 cm in diameter and were sacrificed. Protein extraction was performed from normal bladder tissue (Bladder) and UMUC3 tumor xenografts (Tumor) from each treatment group; the amount of protein was quantified/normalized and subjected to the HDAC Activity Assay. HDAC activity assay data was collected and normalized to DMSO treated mice and is expressed as the mean Relative HDAC Activity ± SD and represents data from four bladder tissue samples (normal bladder tissue, labeled “Bladder”) and four UMUC3 tumor samples (UMUC3 tumor xenograft, labeled “Tumor”) from each treatment group. Statistical significance is set at *, p<0.05 relative to the DMSO Control.

Article Snippet: Cell Culture RT4, J82 and UMUC3 human bladder cancer cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in Gibco RPMI-1640 medium (Invitrogen Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, L-glutamine, penicillin and streptomycin.

Techniques: Control, Concentration Assay, BIA-KA, Activity Assay, HDAC Activity Assay, Standard Deviation, Injection, Protein Extraction, Labeling

Journal: Journal of proteomics

Article Title: The Impact of Cruciferous Vegetable Isothiocyanates on Histone Acetylation and Histone Phosphorylation in Bladder Cancer

doi: 10.1016/j.jprot.2017.01.013

Figure Lengend Snippet: A. RT4 and UMUC3 cells were treated with 5, 10 and 20 μM SFN and ECN for 48h, protein lysates were obtained and analyzed by western blot analysis utilizing a site-specific (K9 and K13) acetylated histone H3 (AcH3) antibody. The antibody was raised against amino acids 1–20 of histone H3 (ARTKQTAR[K*]STGG[K*]APRKQLC, where K* is acetylated). P21 and thymidylate synthase (TS) protein expression was also analyzed and protein expression was quantified by densitometry relative to GAPDH. The data was normalized to the DMSO control “C” and is expressed as the mean (AcH3, p21, and Thymidylate Synthase Protein) Relative Densitometry (ratio to control) ± SD and represents three independent experiments. Statistical significance was set at *, p<0.05 relative to the “C” control. B. UMUC3 cells were treated with 20 μM SFN or ECN for 48h, protein lysates were obtained and analyzed by western blot analysis utilizing acetylated tubulin antibody and quantified by densitometry relative to GAPDH. The data was normalized to the DMSO treated control and is expressed as the mean Ac-Tubulin Protein (Fold Change) ± standard error of the mean (SEM) and represents 3 independent experiments. Statistical significance was set as *, p<0.05 relative to the DMSO control. Representative gels in (A–B) are shown from three independent experiments.

Article Snippet: Cell Culture RT4, J82 and UMUC3 human bladder cancer cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in Gibco RPMI-1640 medium (Invitrogen Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, L-glutamine, penicillin and streptomycin.

Techniques: Western Blot, Expressing, Control

Journal: Journal of proteomics

Article Title: The Impact of Cruciferous Vegetable Isothiocyanates on Histone Acetylation and Histone Phosphorylation in Bladder Cancer

doi: 10.1016/j.jprot.2017.01.013

Figure Lengend Snippet: RT4 and UMUC3 cells were treated with DMSO vehicle control or 20 μM SFN or ECN for 3h, cells were flash frozen, histones extracted and analyzed by LC-MS. The deconvoluted mass spectra of all four variants of histone H1 (H1.2, H1.3, H1.4 and H1.5) of RT4 and UMUC3 is shown. An 80 Da shift to the right indicates an increase in phosphorylation of histone H1. A representative spectra is shown from three independent experiments.

Article Snippet: Cell Culture RT4, J82 and UMUC3 human bladder cancer cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in Gibco RPMI-1640 medium (Invitrogen Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, L-glutamine, penicillin and streptomycin.

Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics

Journal: Journal of proteomics

Article Title: The Impact of Cruciferous Vegetable Isothiocyanates on Histone Acetylation and Histone Phosphorylation in Bladder Cancer

doi: 10.1016/j.jprot.2017.01.013

Figure Lengend Snippet: Data from the treatment of RT4 and UMUC3 cells with DMSO, SFN or ECN treatment (20 μM, 3h) is shown. A. The ratio of the summed peak heights from the LC-MS data for the phosphorylated isoforms to the unphosphorylated isoform for all four variants of histone H1 (H1.2, H1.3, H1.4 and H1.5) is shown. Data represents four independent experiments, error bars represent ± standard error and statistically significant differences are indicated (*, p<0.05; **, p<0.01; ***, p<0.001). B. Histone H1 phosphorylation changes were also analyzed by western blot analysis where representative gels are shown from at least two independent experiments. C. Densitometry from the western blot analysis from B. is shown. The data was normalized to the DMSO control and is expressed as the mean Relative Densitometry (ratio to control) ± SEM and represents two independent experiments. Statistical significance was set at *, p<0.05 and **, p<0.01 relative to the DMSO control.

Article Snippet: Cell Culture RT4, J82 and UMUC3 human bladder cancer cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in Gibco RPMI-1640 medium (Invitrogen Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, L-glutamine, penicillin and streptomycin.

Techniques: Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics, Western Blot, Control