Scrape-loading and dye transfer

El‐Fouly, Mohamed H.; Trosko, James E.; Chang, Chia‐Cheng

doi:10.1016/0014-4827(87)90014-0

Cited by 625 publications

(135 citation statements)

References 27 publications

Supporting

Mentioning

131

Contrasting

Order By: Relevance

“…It does not diffuse through intact plasma membranes, and its low molecular weight (M r 457.2) permits its transmission from one cell to another presumably through patent gap junctions. Rhodamine dextran is a high molecular weight polymer (M r 10,000), and it cannot diffuse through intact plasma membranes nor cross the gap junctions; therefore, it is used as a control to differentiate dye transfer from diffusion because of cell damage (30). Cells were then fixed with 2.5% (w/v) paraformaldehyde, and the numbers of cells transferring dye are counted using a standard fluorescent or laser-scanning confocal microscope.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

Nguyen¹,

Takemoto

Takemoto³

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Gap junction activity in lens epithelial cells is regulated by PKC␥-mediated phosphorylation of Cx43. PKC␥ activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKC␥-C1B domain that are responsible for its interaction with 14-3-3⑀. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKC␥. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3⑀, resulting in the release of endogenous cellular PKC␥ from 14-3-3⑀, in vivo or in vitro, in activation of PKC␥ enzyme activity, phosphorylation of PKC␥, in the subsequent translocation of PKC␥ to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 M of C1B1 or C1B5 peptides also caused a 10-or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKC␥ are regulated by its binding to 14-3-3⑀ at the C1B domain of PKC␥. Synthetic peptides corresponding to these regions of PKC␥ successfully competed for the binding of 14-3-3⑀ to endogenous PKC␥, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.

show abstract

Section: Methodsmentioning

confidence: 99%

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

Nguyen¹,

Takemoto

Takemoto³

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The scrape-loading dye transfer assay was performed based on the modified procedure described by El-Fouly et al (49). In this method, cells were scratched in the presence of two types of fluorescence dyes: Lucifer Yellow (LY) (457 Da), which can penetrate through gap junction channels, and rhodamine dextran (RD) (10 kDa), which is too large to pass through the channels, thus serving as a tracer dye for the cells originally receiving the dye.…”

Section: Sds-page and Westernmentioning

confidence: 99%

Effects of Mechanical Strain on the Function of Gap Junctions in Osteocytes Are Mediated through the Prostaglandin EP2 Receptor

Cherian

Cheng

et al. 2003

Journal of Biological Chemistry

190

146

View full text Add to dashboard Cite

Fluid flow conditioned medium and PGE 2 stimulated cAMP production and PKA activity suggesting that PGE 2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE 2 on gap junctions. These studies suggest that the EP 2 receptor mediates the effects of autocrine PGE 2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP 2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.

show abstract

“…GJIC was assessed in WB cells by scrape-loading/dye transfer (SL/DT) [11] [12]. Cells were cultured to confluence in six well dishes then fluorescent Lucifer Yellow CH was added from stock solution (5% w/v in PBS) to a final concentration of 0.05%.…”

Section: Assessment Of Gjicmentioning

confidence: 99%

Gap Junctional Intercellular Communication Increases Cytotoxicity and Reduces Resistance to Hydroxyurea

Ruch¹,

Boucher²,

Gentry³

et al. 2014

JCT

View full text Add to dashboard Cite

Background: Gap junctions enable small molecules to diffuse between adjacent cells and have been associated with greater cytotoxicity of radiation and anti-cancer drugs. We investigated whether this gap junctional intercellular communication (GJIC) affected the cytotoxicity of the classic ribonucleotide reductase (RR) inhibitor and anti-cancer agent, hydroxyurea (HU). Materials and Methods: We used GJIC-proficient and deficient, connexin 43-expressing WB rat liver epithelial cell lines. We compared HU toxicity by crystal violet assay, effects of the drug on deoxynucleotide pools by HPLC, and ability of GJIC to increase toxicity of HU-resistant cells through a bystander effect in co-culture experiments. Results: GJIC-proficient cells were three-to five-fold more sensitive (IC50 0.1 mM) to HU than GJIC-deficient derivatives (IC 50 0.3 -0.5 mM). This sensitivity depended upon GJIC because treatment of GJIC-proficient cells with the GJIC blocker oleamide decreased HU toxicity by approximately 60% -80% and restoration of GJIC in GJIC-deficient cells by stable transduction of connexin 32-encoding Gjb1 increased HU toxicity (IC 50 0.1 mM). The effects were not due to connexin expression per se or its localization since all cell lines expressed comparable quantities of connexin 43 that was localized to the plasma membrane. Also HU sensitivity was not related to differential effects on nucleotide metabolism in the cells. Thymidine triphosphate levels increased and deoxyadenosine triphosphate levels decreased similarly (15% -20%) in GJIC-proficient and deficient cells over 24 h of HU treatment. More importantly, when HU-resistant cells were co-cultured with sensitive cells, the resistant cells were killed only when GJIC was present. Conclusion: The data suggest that GJIC enhances cytotoxicity and decreases resistance to HU. These results may be important clinically if GJIC can be enhanced in drug-resistant cells.

show abstract

Scrape-loading and dye transfer

Cited by 625 publications

References 27 publications

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

Effects of Mechanical Strain on the Function of Gap Junctions in Osteocytes Are Mediated through the Prostaglandin EP2 Receptor

Gap Junctional Intercellular Communication Increases Cytotoxicity and Reduces Resistance to Hydroxyurea

Contact Info

Product

Resources

About