Journal:

Article Title: Functional characterization of recombinant human ClC-4 chloride channels in cultured mammalian cells

doi: 10.1113/jphysiol.2001.013115

Figure Lengend Snippet: The properties of the whole-cell current were studied by stepping from the holding potential of −30 mV to potentials from −100 to +100 mV, in 20 mV steps for 350 ms. Whole-cell currents were measured at 345 ms after the start of the voltage pulse. Representative traces of ICl (normalized for membrane capacitance) measured at pHo 7.4 in non-transfected cells (A) and hClC-4-expressing cells (B). Continuous lines next to current traces indicate zero current level. The expression of hClC-4 (C) generates an outward-rectifying Cl− current (○, n = 9 for tsA201 cells and n = 4 for both CHO-K1 and MDCK II cells) that was not observed in cells transfected with the CD8 antigen alone (•, n = 6 for tsA201 cells, and n≥4 for both CHO-K1 and MDCK II cells). Data are means ± s.e.m. of currents (I) normalized by estimated membrane capacitance as a function of membrane potential (V).

Article Snippet: Cell culture We used the human cell line tsA201 (HEK 293 cells stably transfected with SV40 large T antigen), Chinese hamster ovary cells (CHO-K1, CRL 9618, American Type Culture Collection, Rockville, MD, USA), and the canine epithelial cell line MDCK II (a gift from Dr L. Limbird).

Techniques: Membrane, Transfection, Expressing

Journal:

Article Title: Functional characterization of recombinant human ClC-4 chloride channels in cultured mammalian cells

doi: 10.1113/jphysiol.2001.013115

Figure Lengend Snippet: A and B, typical whole-cell current (normalized by estimated membrane capacitance) recorded from tsA201, CHO-K1 and MDCK II cells expressing hClC-4 at pHo 7.4 (A) and after reducing the bath pH to 5.5 (B). For all cell lines, block was rapid and reversible. Continuous lines next to current traces indicate zero current level. C, current-voltage relationships for ICl measured at pHo 7.4 (▵), after reducing the bath pH to 5.5 (○), and after subsequent return to pHo 7.4 (▿). Data are means ± s.e.m. of five different experiments for tsA201 and four different experiments for both CHO-K1 and MDCK II cells. Voltage protocol was as described in legend of Fig. 1.

Article Snippet: Cell culture We used the human cell line tsA201 (HEK 293 cells stably transfected with SV40 large T antigen), Chinese hamster ovary cells (CHO-K1, CRL 9618, American Type Culture Collection, Rockville, MD, USA), and the canine epithelial cell line MDCK II (a gift from Dr L. Limbird).

Techniques: Membrane, Expressing, Blocking Assay

Journal:

Article Title: Functional characterization of recombinant human ClC-4 chloride channels in cultured mammalian cells

doi: 10.1113/jphysiol.2001.013115

Figure Lengend Snippet: Current-voltage relationship of ICl in hClC-4-expressing cells in the presence of several different anions. NaCl (○) was replaced with equimolar NaBr (⋄), NaI (□), NaNO3 (▿), NaSCN (▵) and Na-aspartate (•). Data are means ± s.e.m. (n ≥ 4 for tsA201 and CHO-K1 cells, and n = 2 for MDCK II cells) of currents (I) normalized by estimated membrane capacitance and plotted as a function of membrane potential (V) corrected for liquid junction potentials. Voltage protocol was as described in legend of Fig. 1.

Article Snippet: Cell culture We used the human cell line tsA201 (HEK 293 cells stably transfected with SV40 large T antigen), Chinese hamster ovary cells (CHO-K1, CRL 9618, American Type Culture Collection, Rockville, MD, USA), and the canine epithelial cell line MDCK II (a gift from Dr L. Limbird).

Techniques: Expressing, Membrane

Journal:

Article Title: Functional characterization of recombinant human ClC-4 chloride channels in cultured mammalian cells

doi: 10.1113/jphysiol.2001.013115

Figure Lengend Snippet: A. single-channel traces from a hClC-4-expressing tsA201 cell. Channel openings are in the upward direction, and filled bars indicate zero current level. Holding voltage is shown as cell interior relative to the outside. Amplitude histograms for the single-channel currents at the indicated holding potentials are shown to the right. B, single-channel traces from two hClC-4-expressing cells. Left, bath solution pH = 7.4; right, bath solution pH = 6.0. Channel openings are in the upward direction, and the filled bar indicates zero current level. The holding voltage is +60 mV (cell interior relative to the outside) for both traces. All channel traces were obtained ≤ 4 min after excision to minimize channel rundown.

Article Snippet: Cell culture We used the human cell line tsA201 (HEK 293 cells stably transfected with SV40 large T antigen), Chinese hamster ovary cells (CHO-K1, CRL 9618, American Type Culture Collection, Rockville, MD, USA), and the canine epithelial cell line MDCK II (a gift from Dr L. Limbird).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Induction of t(11;14) IgH enhancer/promoter- cyclin D1 gene translocation using CRISPR/Cas9

doi: 10.3892/ol.2019.10303

Figure Lengend Snippet: Confirmation of DNA cutting activity of the gRNA candidates. (A) 293T cells were introduced with target DNA ( IgH, CCND1 or empty)-containing EGxxFP and gRNA ( IgH and CCND1 or empty) expressing lentiCRISPRv2 vectors. Cells with high fluorescence indicate efficient gRNA activity to cut target DNA region. Scale bars, 50 µm. (B) gRNA activity are compared among IgH and CCND1 gRNA candidates-coding lentiCRISPRv2. Bars A and B indicate the negative control (empty lentiCRISPRv2) and positive control (pX330 Cetn1 ), respectively. Every gRNA vector was co-transfected with the gRNA target DNA-containing EGxxFP vector, and the intensity of cell fluorescence was analyzed by flow cytometry. Relative fluorescence compared to positive control is indicated (n=3). IgH -B and CCND1 -B exhibited stronger fluorescence compared to IgH -A and CCND1 -A, respectively. *P<0.001, as indicated. (C) Activity of single ( IgH -B or CCND1 -B) gRNA and dual ( IgH -B and CCND1 -B) gRNA expressing vectors were compared by co-transfecting with the gRNA target DNA-containing EGxxFP vectors (indicates as EGxxFP:). Negative and positive controls are the same as those in 2B. IgH, immunoglobulin heavy chain; CCND1, cyclin D1; ph, phase contrast micrograph; GFP, green fluorescent protein; nega, negative; posi, positive.

Article Snippet: 293 and 293T (SV40 T antigen introduced 293, 293T) cells originated from Japanese Collection of Research Bioresources Cell Bank were cultured in Dulbecco's modified Eagle medium containing 10% fetal calf serum and incubated in a humidified chamber at 37°C with 95% air and 5% CO 2 .

Techniques: Activity Assay, Expressing, Fluorescence, Negative Control, Positive Control, Plasmid Preparation, Transfection, Flow Cytometry

Journal: Oncology Letters

Article Title: Induction of t(11;14) IgH enhancer/promoter- cyclin D1 gene translocation using CRISPR/Cas9

doi: 10.3892/ol.2019.10303

Figure Lengend Snippet: Confirmation of genome editing activity in IgH and CCND1 loci and induced translocation. (A) IgH and CCND1 target regions were PCR-amplified from infected 293T cells and forced using a mismatch cleavage assay. Fragments cleaved by Guide-IT nuclease are indicated by arrows with dotted lines (IgH), and arrowheads (CCND1). (B) t(11;14) translocation-specific PCR products from infected cells were cloned into the TA cloning vector, followed by DNA sequencing. Sequences of 6 clones are aligned against the simulated IgH / CCND1 sequence. The junction between IgH and CCND1 is indicated by a dotted line. Sequence differences relative to the simulated sequence are highlighted in gray boxes. IgH, immunoglobulin heavy chain; CCND1, cyclin D1; empty, empty lentiCRISPRv2-introduced; IgH-CCND1, IgH-CCND1 lentiCRISPRv2-introduced; IgH and CCND1 groups, PCR-amplified regions; - and +, Guide-IT nuclease addition; M, DNA marker.

Article Snippet: 293 and 293T (SV40 T antigen introduced 293, 293T) cells originated from Japanese Collection of Research Bioresources Cell Bank were cultured in Dulbecco's modified Eagle medium containing 10% fetal calf serum and incubated in a humidified chamber at 37°C with 95% air and 5% CO 2 .

Techniques: Activity Assay, Translocation Assay, Amplification, Infection, Cleavage Assay, Clone Assay, TA Cloning, Plasmid Preparation, DNA Sequencing, Sequencing, Marker

Journal: Oncology Letters

Article Title: Induction of t(11;14) IgH enhancer/promoter- cyclin D1 gene translocation using CRISPR/Cas9

doi: 10.3892/ol.2019.10303

Figure Lengend Snippet: Characteristics of 293T cells harboring t(11;14). (A) Growth curves of t(11;14) 293T cells and parental cells. Viable cell numbers are indicated on days 0, 2, and 4 (n=3). Error bars indicate the standard deviation. *P<0.05 vs. 293T. (B) Reverse transcription-quantitative PCR analysis of CCND1 . Relative copy numbers were calculated using GAPDH as a reference (n=3). CCND1, cyclin D1.

Article Snippet: 293 and 293T (SV40 T antigen introduced 293, 293T) cells originated from Japanese Collection of Research Bioresources Cell Bank were cultured in Dulbecco's modified Eagle medium containing 10% fetal calf serum and incubated in a humidified chamber at 37°C with 95% air and 5% CO 2 .

Techniques: Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction