Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Toxicol Mech Methods. 2021 Mar 30;31(5):393–399. doi: 10.1080/15376516.2021.1904072

A modified parachute assay for assessment of gap junction intercellular communication in placental trophoblast cells

Jeremy Gingrich 1, Yong Pu 2, Almudena Veiga-Lopez 2,3
PMCID: PMC8167828  NIHMSID: NIHMS1702573  PMID: 33784946

Abstract

Gap junction intercellular communication (GJIC) is a necessary process for placental development. GJIC can be assessed with a parachute assay, where fluorescent dye-loaded donor cells are ‘parachuted’ onto acceptor cells and dye diffuses to adjacent cells with active GJIC. During co-culture, donor cells can attach, but the assay does not allow their distinction from acceptor cells, which presents as a major limitation. We have developed a modified parachute assay that permits distinction between donor and acceptor cells, using the extravillous trophoblast cell line HTR-8/SVneo and a lentiviral transduction technique. Using PKA activator CW008 as a positive control and 12-o-tetradecanoylphorbol-13-acetate as a negative control, this modified parachute assay reliably detects both enhanced and attenuated GJIC. Importantly, the ease and accuracy of quantification over currently available methods makes this modified assay optimal for automation and represents a useful tool for in vitro placental toxicological testing.

Keywords: parachute assay, gap junction, intercellular communication, trophoblast, placenta

1. Introduction

Gap junction intercellular communication (GJIC) plays a significant role in intracellular signal transduction by allowing the transfer of signalling molecules like cAMP, cGMP and ATP between adjacent cells (Mese et al. 2007; Trosko 2011; Zong et al. 2016). Gap junction channels are formed when gap junction plaques, comprised of hexameric transmembrane proteins known as connexins, dock on appositional cells. This allows a complex cascade of phosphorylation events, through master regulators like protein kinase A, to regulate opening and closing of gap junction channels (Solan and Lampe 2016).

GJIC has been classically assessed by two assays, the scrape loading/dye transfer (SL/DT) and the parachute assay (Ziambaras et al. 1998; Babica et al. 2016b). Originally developed in 1998, the parachute assay utilizes two cellular populations: donors and acceptors (Ziambaras et al. 1998). Donor cells are loaded with a membrane permeable dye, such as calcein AM, which cannot passively diffuse out of the cell. Donor cells are then ‘parachuted’ onto a population of acceptor cells, where they are left to incubate in co-culture, allowing gap junction channel formation. The calcein dye is then passed from donor into acceptor cells via GJIC. Calcein-positive cell populations are then quantified and used as a functional assessment of GJIC. In the SL/DT assay, a mechanical scrape is performed onto a confluent monolayer of cells and a low-molecular weight gap junction permissible dye, like lucifer yellow, is added to the media (Babica et al. 2016a). Through active gap junctions, the dye gets internalized into the cells. The area in which the dye travels is then measured as a functional endpoint of GJIC. The parachute assay allows the precise quantification of dye transfer from single cells over a large area, while the SL/DT has a limited quantifiable area and is not ideal for assessing concentration-dependent effects (McKarns and Doolittle 1992). However, the method of GJIC quantification in the parachute assay is not standardized across publications and does not permit discrimination between donor and acceptor cell populations (Wang R et al. 2015; Yin et al. 2018; Zhang et al. 2018), which can lead to an overestimation of GJIC if donor cells attach during the incubation period.

GJIC is essential in the placenta, having been reported to be a key regulator of placental growth and function (Winterhager and Kidder 2015). Bipotent stem-like cells of the placenta, known as cytotrophoblasts, differentiate into two distinct lineages: 1) syncytiotrophoblasts, which are fused cytotrophoblasts or 2) extravillous cytotrophoblasts (EVTs), which invade into the uterus and remodel the maternal vasculature to provide nutrients to the developing fetus. GJIC is required for both the syncytialization process (Pidoux et al. 2010; Dunk et al. 2012) and EVT invasion during uterine spiral artery remodelling (Winterhager and Kidder 2015). Importantly, aberrant GJIC in EVTs has potential implications in the development of pregnancy complications like placenta accreta (Nishimura et al. 2004) and preeclampsia (Otto et al. 2015).

To our knowledge, there is no established method for assessing GJIC in the placenta. Therefore, the aim of this work was to develop a functional parachute assay for placental GJIC that is capable of distinction between donor and acceptor cell populations. For this method, we have utilized the cell line HTR-8/SVneo as EVTs and a lentiviral transduction technique. We hypothesize that this improved parachute assay will enable the distinction of donor cell populations and more accurately assess GJIC than previous quantification methods.

2. Materials and Methods

For all tissue culture procedures and chemical handling, aseptic techniques were used to avoid contamination. Culture conditions for all cells were set at 5% CO2 and 37 °C. Molecular biology grade chemicals were used as available, and all dilutions or resuspensions occurred using ultrapure water unless otherwise stated.

Materials

  • Calcein AM (Corning, Corning, NY, US; Cat. #: 354217)

  • CW008 (Tocris Bioscience, Bristol, UK; Cat. #: 5495)

  • DAPI (Biotium, Hayward, CA, US; Cat. #: 40011)

  • Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, US; Cat. #: 472301)

  • DMEM/F12 cell culture media (Gibco, Waltham, MA, US; Cat. #: 12500062)

  • Dulbecco’s phosphate buffered saline, no calcium, no magnesium (DPBS) (Gibco, Waltham, MA, US; Cat. #: 14190144)

  • Fetal bovine serum (FBS) (Corning, Corning, NY, US; Cat. #: 35-010-CV)

  • Fugene 6 (Promega, Madison, WI, US; Cat. #: E2692)

  • HEK293T cell line (ATCC, Manassas, VA, US; Cat. #: CRL-3216)

  • HEPES (Sigma-Aldrich, St. Louis, MO, US; Cat. #: H3375)

  • HTR-8/SVneo cell line (ATCC, Manassas, VA, US; Cat. #: CRL-3271)

  • Lenti-X concentrator (Takara Bio, Shiga, JP; Cat. #: 631231)

  • Neutral-buffered formalin (NBF) (Sigma-Aldrich, St. Louis, MO, US; Cat. #: HT501128)

  • pLVX-mCherry-C1 vector (Takara Bio, Shiga, JP; Cat. #: 632561)

  • PMD2G plasmid (Addgene, Watertown, MA, US; Cat. #: 12259)

  • psPAX2 plasmid (Addgene, Watertown, MA, US; Cat. #: 12260)

  • Puromycin (Sigma-Aldrich, St. Louis, MO, US; Cat. #: P9620)

  • 12-o-tetradecanoylphorbol-13-acetate (TPA) (Tocris Bioscience, Bristol, UK; Cat. #: 1201)

Equipment

  • CoolSNAP-Pro CF camera (Media Cybernetics, Rockville, MD, US)

  • Eclipse TE2000-U inverted epifluorescent microscope (Nikon, Tokyo, JP)

  • Fluoview FV1000 confocal laser scanning microscope configured on an IX81 inverted microscope (Olympus, Tokyo, JP)

  • 5810R centrifuge (Eppendorf, Hamburg, DE)

Generation of donor and acceptor cells

Before the parachute assay was performed, two cell types were generated: 1) donor cells that will be pre-loaded with Calcein AM and ‘parachuted’ onto acceptor cells and 2) acceptor cells that will uptake the Calcein dye from the donor cells if gap junction channels between donor and acceptor cells become active.

Donor cells:

HTR-8/SVneomCherry cells (see lentiviral transduction method below) were seeded into a 100 mm tissue culture dish with growth medium (DMEM/F12 culture media supplemented with 1% FBS). Media was replaced after 24 h and cells were grown to 80–90% confluency. Cells were trypsin digested and seeded into 60 mm tissue culture dishes at a density of 5 × 105 cells/dish. After 18–24 h, donor populations were fully confluent, and the parachute assay was then ready to begin.

Acceptor cells:

HTR-8/SVneo cells were seeded into a 100 mm tissue culture dish with growth medium. Media was replaced after 24 h and cells were grown to 80–90% confluency. Cells were then trypsin digested and seeded at a volume of 1 ml/well (1 × 104 cells/ml) in a 12-well plate. After 18–24 h, when acceptor populations were at an optimal 50% confluency, the parachute assay was then ready to begin.

Parachute Assay

The parachute assay consists of the co-incubation of donor cells that have been ‘parachuted’ on to acceptor cells (see details of cell types above). The parachute assay scheme is shown in Figure 1.

Figure 1: Modified parachute assay methodology and experimental scheme.

Figure 1:

Open in a new tab

Step 1: Donor (1a; HTR8-SVneomCherry) and acceptor (1b; HTR8-SVneo) cells treated with exposure media (see Exposure Step for treatment groups details) for 24 h. Step 2: Under continued exposure conditions, both donor (2a) and acceptor (2b) cells are subjected to a 2 h serum starving period. Step 3: In the last 30 min of serum starvation, donor cells are exposed to the fluorescent dye, Calcein AM. Step 4: A cell suspension is made from the donor cell cultures. Step 5: TPA is added to the donor cell resuspension media (5a) and the acceptor cell media (5b) prior to parachuting. Note that this step is only needed in groups that require TPA. Step 6: Calcein-loaded donor cells are “parachuted” onto a confluent monolayer of acceptor cells. Step 7: The cell co-culture is incubated for 3 h. This allows the transfer of calcein through active gap junctions between donor and acceptor cells. Step 8: Each well is then rinsed, fixed with 4% NBF and counterstained with DAPI. After rinsing, any remaining donor cells can be distinguished from the acceptor cells as they have a green cytoplasm and red/blue (mCherry and DAPI) nucleus. Step 9: Cells are then imaged and quantified for donor and acceptor cell populations. Acceptor cells without active GJIC have a blue (DAPI) nucleus and unstained cytoplasm. Acceptor cells with active GJIC have a blue (DAPI) nucleus and a green (calcein) cytoplasm.

Parachute Assay: Exposure Step

Donor and acceptor cells:

During the Exposure Step (24 h; Figure 1, steps 1a and 1b), both donor and acceptor cells were allocated into one of four treatment groups: 1) DMSO vehicle control, 2) CW008 positive control, 3) TPA negative control, or 4) CW008+TPA rescue. Exposure media consisted in growth media supplemented with 0.1% DMSO or 0.25 μM CW008 dissolved in DMSO to a final concentration of 0.1%. In the vehicle control group, cells were exposed to 0.1% DMSO, which allowed the assessment of the baseline GJIC. The positive control group was exposed to CW008 dissolved in DMSO and used at a final concentration in media at 0.1% DMSO. CW008 is a PKA activator that allows the opening of gap junction channels. The TPA negative control group and the CW008+TPA rescue groups were exposed to 0.1% DMSO and CW008, respectively. Each group was run in triplicate and each treatment group was cultured in a separate cell culture dish.

Parachute Assay: Serum Starving & Calcein AM Loading Steps

Donor cells:

After the Exposure Step, and to induce GJIC, the exposure media from HTR-8/SVneomCherry donor cells was replaced with serum free exposure media (Figure 1, step 2a) and kept for 2 h. After 1.5 h in serum starving conditions, Calcein AM (15 μM), a fluorescent membrane permeable dye, was added to the donor cell media (Figure 1, step 3a). Calcein AM-exposed donor cells uptake the Calcein AM and rapidly convert it to calcein, which is not membrane permeable, but can be transferred between cells through active gap junctions. After 2 h of serum starvation, donor cell media was removed, and plates rinsed once with pre-warmed DPBS. Caution should be exerted in this step to avoid direct contact of donor cells to the light to prevent photobleaching.

Acceptor cells:

After the Exposure Step, the exposure media from HTR-8/SVneo acceptor cells, was replaced with serum-free exposure media (Figure 1, step 2b). After 2 h of serum starvation, the cells were ready for the donor cells to be ‘parachuted’ onto them. At the time of parachuting, acceptor cells were in a confluent monolayer in a 12-well plate.

Parachute Assay: Cell suspension, Parachute, and Incubation Steps

Calcein AM-loaded donor cells were trypsin digested, resuspended in growth media to count cell number, and centrifuged at 300 × g for 5 min (Figure 1, step 4a). Then, media was decanted, cell pellet resuspended with 5 ml pre-warmed DPBS, and centrifuged at 300 × g for 5 min. DPBS was then decanted, and cell pellet resuspended in growth media to a concentration of 1 × 105 cells/ml. In both the TPA negative control group and CW008+TPA rescue group, a short-acting GJIC inhibitor (10 nM TPA) was added into both and acceptor cell media (Figure 1, step 5a) and donor cell resuspension media (Figure 1, step 5b) prior to parachuting.

The donor cell suspension (100 μl; 10,000 donor cells/well) was then ‘parachuted’ onto the corresponding acceptor cell group (DMSO, CW008, TPA, or CW008+TPA) (Figure 1, step 6). Treatment groups were run in triplicate. The 12-well tissue culture dish with donor and acceptor cells was then returned to the incubator (5% CO2, 37 °C) for 3 h (Figure 1, step 7).

Parachute Assay: Cell Fixation & Staining Steps

Media was gently removed from all wells, rinsed three times with pre-warmed DBPS, and fixed with 4% NBF. Caution should be exercised in the remaining steps to protect cells from exposure to direct light to avoid calcein photobleaching. Cells were fixed overnight at 4 °C. NBF was then removed, and each well gently rinsed with room temperature DPBS. Freshly prepared DAPI solution (1 ml of 2.5 μM DAPI diluted in DPBS) was then added to each well and incubated at room temperature for 5 min (Figure 1, step 8). Each well was then gently rinsed with DPBS twice, leaving 1 ml DPBS per well. Plates may be stored at 4 °C and protected from light, for later imaging (Figure 1, step 9).

Lentiviral transduction

The fluorescent protein, mCherry, was stably expressed in HTR-8/SVneo cells (HTR-8/SVneomCherry) through a lentiviral transduction method as follows. Lentivirus particles were generated using HEK293T cells via the pLVX-mCherry-C1 vector, a second-generation packaging system. At 60% confluency, HEK293T cells cultured in a 100 mm dish were transfected with a plasmid cocktail of pLVX-mCherry-C1 (2.5 μg), PMD2G (0.875 μg), and psPAX2 (1.62 μg) via a balanced Fugene 6 working solution (45 μl Fugene with 435 μl Opti-MEM) in a 500 μl transduction volume. After 24 h, media was replaced with fresh growth medium (DMEM/F12, 10% FBS, 10 mM HEPES). After 72 h, supernatant was collected and mixed with Lenti-X concentrator (3:1 v/v). After 45 min of centrifugation at 4 °C, supernatant was trashed, and the lentiviral particles were resuspended with 1 ml of growth medium. HTR-8/SVneo cells were then transduced with the mCherry lentiviral particles, which additionally confer puromycin resistance. Cells were then selected through exposure to growth medium supplemented with 1 μg/ml puromycin. Following one week of antibiotic selection, mCherry-positive cell colonies were harvested with a cloning cylinder and frozen (LN2, −196 °C) until further use.

Imaging

Each well can be directly imaged with an epifluorescent microscope for DAPI (461 nm), calcein (515 nm), and mCherry (590 nm). Confocal microscopy was used to confirm dye transfer between cells under control exposure conditions. For quantification, a minimum of 5 random images per exposure group (20X magnification) were taken, avoiding areas too close to the perimeter. Exposure groups were run in triplicate. GJIC was expressed as either the average sum of all calcein-positive cells (Σcalcein) like previous methods, or the summation of all calcein-positive cells minus all mCherry-positive cells divided by all DAPI-positive cells (% transfer events) (see Figure 1, step 9 for details on the distinction between donor and acceptor cells). Each treatment was normalized to its respective vehicle group.

Statistics

For experiment output, all assays were run in triplicate, each using triplicate exposure groups. Quantitative data between exposure groups for percent transfer events and Σcalcein were compared against vehicle groups by using the MIXED procedure ANOVA in SAS (version 9.4). Treatment groups and run (n = 3) were considered fixed effects. Data were tested for normality by the Shapiro-Wilk normality test. Significance was set at P < 0.05.

3. Results

In this modified parachute assay for assessment of GJIC in placental EVTs, donor cells, which were calcein-negative and mCherry-positive, were easily distinguished from acceptor cells, which present as only calcein-positive for those involved in a gap junction transfer event (Figure 2). Additionally, confocal microscopy was able to capture gap junction intercellular dye transfer between cells under control conditions (Figure 3).

Figure 2: Lentiviral transfection allows for the distinction between donor and acceptor cells.

Figure 2:

Open in a new tab

Calcein-positive HTR8-SVneo acceptor cells (left, green asterisk) can be easily distinguished from HTR8-SVneomCherry donor cells, which are both calcein-positive and express the lentiviral vector for mCherry (right, red asterisk).

Figure 3: Confocal microscopy demonstrates intercellular dye transfer.

Figure 3:

Open in a new tab

Lentiviral transfected HTR8-SvneomCherry donors (red) transfers calcein (green) through pseudopodial contact with acceptor cell (arrow; top) under control conditions. This is also apparent through a 0.5 μm z-stack (arrow; bottom images).

When quantifying the calcein gap junction intercellular transfer events using this modified parachute assay that permits distinction of donor cells that have attached to the acceptor plate, exposure of EVTs to the positive control, CW008, achieved significantly higher transfer events compared to the vehicle, TPA, or CW008+TPA treatment groups (Figure 4A). The enhanced transfer events upon CW008 exposure were fully attenuated when co-incubated with the negative control, TPA. By only accounting calcein-positive cells, an accepted quantification method for parachute assays, we were unable to detect a statistical difference among any of our treatment groups (Figure 4B).

Figure 4: Modified parachute assay improves assay quantification.

Figure 4:

Open in a new tab

(A) Image analysis, expressed as the sum of calcein-positive cells per group (n = 3) normalized to the vehicle group after exposure to CW008 (black, positive control), TPA (grey, negative control), or co-treatment of CW008 and TPA (striped). This method does not account for donor cell populations. (B) Image analysis quantification of the same data accounting for donor cells and total cell number, expressed as the percent of transfer events (calcein-positive cells minus mCherry-positive cells divided by DAPI-positive cells) normalized to the vehicle group. Different letters denote a significance of P < 0.05.

4. Discussion

Through results generated in this manuscript, we have demonstrated the successful development of a modified parachute assay representative of homocellular GJIC in the placenta among EVTs, and differs from previous methods in accuracy, the use of lentiviral transduction, and the fluorescent dye used.

The addition of fluorescently tagged donor cells improves quantification from previously reported methods (Wang R et al. 2015; Yin et al. 2018; Zhang et al. 2018) as it can accurately distinguish donor from acceptor cell populations. This distinction has demonstrated that the classic parachute assay results in an overestimation of gap junction intercellular transfer events. We have additionally demonstrated that this assay can detect chemically enhanced GJIC, as well as attenuation of that enhancement by TPA, a signal pathway inhibitor.

Previous modifications to the parachute assay in other cell types have used the fluorescent dye, DiI, to discriminate between donor and acceptor populations (Wang SQ et al. 2017; Luo et al. 2019; Shi et al. 2019). DiI is marketed as a non-gap junction permissible dye with no observed off-target effects in vitro (De Clerck et al. 1994). This non-transmittable dye is also used in a parachute assay which investigates heterocellular GJIC between carcinogenic and non-carcinogenic placental EVT cell lines (Khoo et al. 1998). However, DiI has been reported to leak out of cells over time (Lassailly et al. 2010). DiI leakage can contaminate the cell media and internalize into acceptor cells independent of active GJIC between cells, resulting in an over representation of transfer events. Our method of donor distinction, which uses a lentiviral transduction technique to provide stable expression of the mCherry fluorescent protein prevents such overrepresentation of acceptor cells.

Since lentiviral transduction represents a simple way to produce long-lasting cell lines that stably express fluorescent proteins (Godecke et al. 2018), our modified parachute assay model could also be adapted to high through-put screening for placental toxicity. Similar parachute assays have been adapted to high through-put screening for onco-pharmacological screening purposes (Lee et al. 2015; Yeo and Lee 2019), but no such assay exists for the placenta. Importantly, toxicological studies have implicated exogenous chemical exposures in the development of placental pathologies (Gingrich et al. 2020), including potential modulation of GJIC (Gingrich et al. 2018).

GJIC among EVTs is important for regulating their proliferation and invasion (Winterhager and Kidder 2015), although the mechanisms regulating GJIC in EVTs have yet to be fully elucidated. During the invasion and spiral artery remodelling processes, intercellular communication among different cell types (heterocellular GJIC), such as EVTs, uterine arteriole endothelium and the endometrium is also required (Choudhury et al. 2017). Importantly, since connexin protein expression in the endometrium has been negatively associated with early pregnancy loss or stillbirth (Winterhager and Kidder 2015), this model would be a useful tool for studying the heterocellular GJIC between EVTs and uterine arteriole or endometrial cells that may be altered in the etiology of these conditions.

In summary, the modified parachute assay presented in this study allows accurate measurements of GJIC in placental EVTs. This modified parachute assay could easily be adapted to heterocellular populations, such as EVTs communicating with the maternal spiral artery endothelium, providing valuable insights into placental health and development upon stressor exposures. Additionally, future efforts could implement this technology as a high throughput chemical screening tool for toxicological risk assessment.

Acknowledgements

We thank Mr. Kwame Kannatey-Asibu for technical assistance. Figure 1 was developed using biorender.com.

Funding

Research reported in this publication was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under awards R01 ES027863 to A.V-L, and the Eunice Kennedy Shriver National Institute of Child Health & Development of the National Institutes of Health under award T32 HD087166 to J.G. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

Conflicts of Interest

All author(s) report no conflicts of interest.

References

  1. Babica P, Sovadinova I, Upham BL. 2016a. Scrape Loading/Dye Transfer Assay. Gap Junction Protocols. 1437:133–144. English. [DOI] [PubMed] [Google Scholar]
  2. Babica P, Sovadinova I, Upham BL. 2016b. Scrape Loading/Dye Transfer Assay. Methods Mol Biol. 1437:133–144. [DOI] [PubMed] [Google Scholar]
  3. Choudhury RH, Dunk CE, Lye SJ, Aplin JD, Harris LK, Jones RL. 2017. Extravillous Trophoblast and Endothelial Cell Crosstalk Mediates Leukocyte Infiltration to the Early Remodeling Decidual Spiral Arteriole Wall. J Immunol. 198(10):4115–4128. [DOI] [PubMed] [Google Scholar]
  4. De Clerck LS, Bridts CH, Mertens AM, Moens MM, Stevens WJ. 1994. Use of fluorescent dyes in the determination of adherence of human leucocytes to endothelial cells and the effect of fluorochromes on cellular function. J Immunol Methods. 172(1):115–124. [DOI] [PubMed] [Google Scholar]
  5. Dunk CE, Gellhaus A, Drewlo S, Baczyk D, Potgens AJ, Winterhager E, Kingdom JC, Lye SJ. 2012. The molecular role of connexin 43 in human trophoblast cell fusion. Biol Reprod. 86(4):115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gingrich J, Pu Y, Roberts J, Karthikraj R, Kannan K, Ehrhardt R, Veiga-Lopez A. 2018. Gestational bisphenol S impairs placental endocrine function and the fusogenic trophoblast signaling pathway. Arch Toxicol. 92(5):1861–1876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gingrich J, Ticiani E, Veiga-Lopez A. 2020. Placenta Disrupted: Endocrine Disrupting Chemicals and Pregnancy. Trends in endocrinology and metabolism: TEM. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Godecke N, Hauser H, Wirth D. 2018. Stable Expression by Lentiviral Transduction of Cells. Methods Mol Biol. 1850:43–55. [DOI] [PubMed] [Google Scholar]
  9. Khoo NK, Zhang Y, Bechberger JF, Bond SL, Hum K, Lala PK. 1998. SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. II. Changes in gap junctional intercellular communication. Int J Cancer. 77(3):440–448. [DOI] [PubMed] [Google Scholar]
  10. Lassailly F, Griessinger E, Bonnet D. 2010. “Microenvironmental contaminations” induced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell tracking. Blood. 115(26):5347–5354. [DOI] [PubMed] [Google Scholar]
  11. Lee JY, Choi EJ, Lee J. 2015. A new high-throughput screening-compatible gap junctional intercellular communication assay. BMC Biotechnol. 15:90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Luo H, Wang X, Ge H, Zheng N, Peng F, Fu Y, Tao L, Wang Q. 2019. Inhibition of ubiquitinspecific protease 14 promotes connexin 32 internalization and counteracts cisplatin cytotoxicity in human ovarian cancer cells. Oncol Rep. 42(3):1237–1247. [DOI] [PubMed] [Google Scholar]
  13. McKarns SC, Doolittle DJ. 1992. Limitations of the scrape-loading/dye transfer technique to quantify inhibition of gap junctional intercellular communication. Cell Biol Toxicol. 8(1):89–103. [DOI] [PubMed] [Google Scholar]
  14. Mese G, Richard G, White TW. 2007. Gap junctions: basic structure and function. J Invest Dermatol. 127(11):2516–2524. [DOI] [PubMed] [Google Scholar]
  15. Nishimura T, Dunk C, Lu Y, Feng X, Gellhaus A, Winterhager E, Rossant J, Lye SJ. 2004. Gap junctions are required for trophoblast proliferation in early human placental development. Placenta. 25(7):595–607. [DOI] [PubMed] [Google Scholar]
  16. Otto T, Gellhaus A, Luschen N, Scheidler J, Bendix I, Dunk C, Wolf N, Lennartz K, Koninger A, Schmidt M et al. 2015. Oxygen Sensitivity of Placental Trophoblast Connexins 43 and 46: A Role in Preeclampsia? J Cell Biochem. 116(12):2924–2937. [DOI] [PubMed] [Google Scholar]
  17. Pidoux G, Gerbaud P, Gnidehou S, Grynberg M, Geneau G, Guibourdenche J, Carette D, Cronier L, Evain-Brion D, Malassine A et al. 2010. ZO-1 is involved in trophoblastic cell differentiation in human placenta. Am J Physiol Cell Physiol. 298(6):C1517–1526. [DOI] [PubMed] [Google Scholar]
  18. Shi G, Zheng X, Wu X, Wang S, Wang Y, Xing F. 2019. All-trans retinoic acid reverses epithelial-mesenchymal transition in paclitaxel-resistant cells by inhibiting nuclear factor kappa B and upregulating gap junctions. Cancer Sci. 110(1):379–388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Solan JL, Lampe PD. 2016. Kinase programs spatiotemporally regulate gap junction assembly and disassembly: Effects on wound repair. Semin Cell Dev Biol. 50:40–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Trosko JE. 2011. The gap junction as a “Biological Rosetta Stone”: implications of evolution, stem cells to homeostatic regulation of health and disease in the Barker hypothesis. J Cell Commun Signal. 5(1):53–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wang R, Huang F, Chen Z, Li S. 2015. Downregulation of connexin 32 attenuates hypoxia/reoxygenation injury in liver cells. J Biochem Mol Toxicol. 29(4):189–197. [DOI] [PubMed] [Google Scholar]
  22. Wang SQ, Zhang SW, Zhang CZ, Zhao ZY, Wang YJ. 2017. Connexin 43 enhances oxaliplatin cytotoxicity in colorectal cancer cell lines. Cell Mol Biol (Noisy-le-grand). 63(4):53–58. [DOI] [PubMed] [Google Scholar]
  23. Winterhager E, Kidder GM. 2015. Gap junction connexins in female reproductive organs: implications for women’s reproductive health. Hum Reprod Update. 21(3):340–352. [DOI] [PubMed] [Google Scholar]
  24. Yeo JH, Lee J. 2019. An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay. J Vis Exp.(144). [DOI] [PubMed] [Google Scholar]
  25. Yin X, Feng L, Ma D, Yin P, Wang X, Hou S, Hao Y, Zhang J, Xin M, Feng J. 2018. Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone. J Neuroinflammation. 15(1):97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zhang Y, Tan X, Xue L. 2018. The alpha2-adrenoreceptor agonist dexmedetomidine protects against lipopolysaccharide-induced apoptosis via inhibition of gap junctions in lung fibroblasts. Biochem Biophys Res Commun. 495(1):92–97. [DOI] [PubMed] [Google Scholar]
  27. Ziambaras K, Lecanda F, Steinberg TH, Civitelli R. 1998. Cyclic stretch enhances gap junctional communication between osteoblastic cells. J Bone Miner Res. 13(2):218–228. [DOI] [PubMed] [Google Scholar]
  28. Zong L, Zhu Y, Liang R, Zhao HB. 2016. Gap junction mediated miRNA intercellular transfer and gene regulation: A novel mechanism for intercellular genetic communication. Sci Rep. 6:19884. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES