Connexin30.3 is expressed in mouse embryonic stem cells and is responsive to leukemia inhibitory factor

The expression of 19 connexin (Cx) isoforms was observed in the mouse embryonic stem (ES) cell line, EB3. Their expression patterns could be classified into either pluripotent state-specific, differentiating stage-specific, or non-specific Cxs. We focused on Cx30.3 as typical of the first category. Cx30.3 was pluripotent state-specific and upregulated by leukemia inhibitory factor (LIF), a specific cytokine that maintains the pluripotent state of ES cell, via a Jak signaling pathway. Cx30.3 protein was localized to both the cell membrane and cytosol. The dynamic movement of Cx30.3 in the cell membrane was suggested by the imaging analysis by means of overexpressed Cx30.3-EGFP fusion protein. The cytosolic portion was postulated to be a ready-to-use Cx pool. The Cx30.3 expression level in ES cell colonies dramatically decreased immediately after their separation into single cells. It was suggested that mRNA for Cx30.3 and Cx30.3 protein might be decomposed more rapidly than mRNA for Cx43 and Cx43 protein, respectively. These indicate possible involvement of Cx30.3 in the rapid formation and/or decomposition of gap junctions; implying a functional relay between Cx30.3 and other systems such as adhesion proteins.


Results
Dynamic expression patterns of Cx isoforms during growth stage from the pluripotent state to an early stage of differentiation. Among 20 Cx isoforms in mouse genome, the gene expressions of 19 isoforms were detected in the pluripotent state of EB3 cells (Fig. 1a) and 15 of them showed the gene expression also in an early stage of differentiation that was defined as the stage after the culture for 6 d in the medium containing no LIF (LIF(− ) medium) (Fig. 1b). The gene expression levels in both stages were same or markedly different. Ten isoforms such as Cx29, Cx32, and Cx43 were the former case. The dynamic changes of the latter case plus one (Cx33) of the former case were analyzed by qRT-PCR (Fig. 1c). Respective genes showed three different expression patterns: (1) higher expression in the pluripotent state than in the early stage of differentiation (Cx30.3, Cx45), (2) lower expression in the pluripotent state than in the early stage of differentiation (Cx26, Cx30), or (3) constant expression throughout the time period (Cx33), or a decrease-then-increase mode of expression (Cx31). Of these 6 isoforms, we focused on Cx30.3 because its expression behavior was thought to be predominantly associated with the pluripotent state.

Expression of Cx30.3 as protein determined by western blot analysis. Western blot analysis
revealed that Cx30.3 protein was expressed when cells were in the pluripotent state (Fig. 2a,b). The quantity of Cx30.3 protein decreased during culture in LIF(− ) medium for 2 d. The expression profile of Cx30.3 protein was consistent with its transcription activity profile (Fig. 1c, Cx30.3).
Then the test sample was fractionated by ultracentrifugation to analyze whether Cx30.3 protein was located in cell membrane or cytosol. The cytosol fraction was not contaminated with cell membrane fraction as supported by the result of α 1 Na + -K + ATPase, a cell membrane marker. As depicted in Fig. 2c, Cx30.3 was localized not only in the membrane protein fraction but also in the cytosol fraction. LIF to Cx30.3 signaling pathway. According to a former ref. 30, the LIF signaling pathway involved Jak-Stat3, PI3 kinase-Akt, and Grb2-MAP kinase pathways. Klf4 and Tbx2 were the next downstream factors of Stat3 and Akt, respectively and upregulated. Tbx2 was also the next downstream factor of MAP kinase, though it was downregulated. The Jak-Stat pathway could be downregulated by the removal of LIF and then re-activated by the re-addition of LIF. In contrast, such a re-activation was not observed with the PI3 kinase-Akt pathway. Here we investigated the involvement of Cx30.3 in these pathways using Klf4 and Tbx2 as specific markers of respective pathways.
EB3 cells were cultured in LIF(− ) medium for 21 h to cease the LIF signal and then the medium was replaced by LIF(+ ) medium. Cx30.3 could be re-activated by the re-addition of LIF in a more remarkably than Klf4 Scientific RepoRts | 7:42403 | DOI: 10.1038/srep42403 (Fig. 3a). However, neither Tbx2 nor Nanog could be re-activated. Then we investigated the effect of Jak inhibitor on the re-activation of Cx30.3 and also on the re-activation of Klf4 as a positive control. After the culture in LIF(− ) medium for 21 h and then in LIF(− ) medium containing Jak inhibitor for 1 h, the medium was replaced by LIF(+ ) medium containing Jak inhibitor. Consequently, both Cx30.3 and Klf4 could be re-activated without the inhibitor, while being inhibited completely with the inhibitor (Fig. 3b). Therefore, Cx30.3 was speculated as a downstream factor branching from Jak. This LIF to Cx30.3 signaling pathway, however, did not link to Nanog.
Growth stage dependence of re-activation of Cx30.3 by re-addition of LIF. Re-activation by re-addition of LIF was regarded as a characteristic property of the LIF to Jak-Stat3 signaling pathway 30 . Then we investigated whether such a property could be maintained only in the pluripotent state or even after differentiation. Experimental protocol is shown in Fig. 3c. According to the mode-1, the result of Fig. 3d-i was obtained. During culture of EB3 cells in LIF(− ) medium for 2 d, Cx30.3 expression levels decreased to 10 times lower than that of control cells cultured in LIF(+ ) medium for 3 d. After the medium was subsequently replaced by LIF(+ ) medium, however, the Cx30.3 expression level immediately increased to a higher level than that of the control within 1 d (Fig. 3d-i, result of "3 d"). In the mode-2, culture in LIF(− ) medium was continued for 3 d and then the medium was replaced by LIF(+ ) medium. The Cx30.3 expression could be re-activated and its level became rapidly 10 times higher than that of 3rd day within 1 d ( Fig. 3d-ii, results of "3 d" and "4 d"). However later addition of LIF according to the mode-3 or mode-4 was not effective to the re-activation of Cx30.3 ( Fig. 3d-iii,-iv).  Table S2 for primer sets. mean ± SD for n = 3. (c) Typical examples of dynamic gene expression patterns analyzed by qRT-PCR. C: control, cultured in LIF(+ ) medium for 3 d. nd: cultured in LIF(− ) medium for n d. mean ± SD for n = 3. Refer to Table S2  The empirical criteria of the culture condition for maintaining pluripotent state or initiating differentiation is as follows (personal communication). The culture in LIF(− ) medium for longer than 3d is the criteria for the differentiation without reversible turning to the pluripotent state even in the LIF(+ ) medium, though no scientific reason of 3 d has not been clarified. According to this criteria, EB3 cells cultured in LIF(− ) medium for no longer than 3 d are thought to be mostly still at the pluripotent state. The re-activation of Cx30.3 occurred only when the cells were staying at this pluripotent state.

Regulation of pluripotency-and differentiation-associated genes by Cx30.3. If the LIF to Cx30.3
signaling pathway links to pluripotency-associated genes such as Oct3/4 and Rex1, the pluripotent state may be maintained by the overexpression of Cx30.3 alone. Then we investigated its possibility by the culture of Cx30.3 overexpressing EB3 cells in LIF(− ) medium. At first we confirmed that the Cx30.3 expression in the Cx30.3 overexpressing EB cells could maintain its sufficiently high level throughout 6 d even in the LIF(− ) medium (Fig. 3e). Under this condition, however, neither Oct3/4 nor Rex1 showed any remarkable response ( Fig. 3f- On the other hand, we suspected that the Cx30.3 overexpression might contribute to the maintenance of pluripotent state by the downregulation of differentiation-associated genes such as Cdx2 and Gata4. As depicted in Fig. 3f-iii, the expression of Cdx2 alone was suppressed slightly at pluripotent state. Both genes were upregulated rather than suppressed after the initiation of differentiation ( Fig. 3f-iii,iv, at 4 d and 5 d).
In summary the effects of Cx30.3 overexpression, if any, on the maintenance of pluripotent state were not enough to be alternative to LIF.  Fig. 4-a1,a2). The distribution in cytosol was observable more clearly in a cluster. This suggests that a large number of Cx30.3 protein might be stored in some area of cytosol. On the other hand, Cx43 was distributed dominantly in cell membrane ( Fig. 4-a3,a4). Another noticeable   point was that large fluorescent spots were observed only in Cx43 images. Such spots were speculated as gap junctional plaques. In contrast, no signal was observed in the control ( Fig. 4-a5).

Dynamic localization behavior of Cx30.3 protein.
The life cycle of Cx protein is roughly composed of two processes, i.e. the incorporation in cell membrane to form gap junctions and the removal of gap junctions by internalization. The dynamic behavior of Cx30.3 in these processes were investigated.
Cx30.3 protein labelled with EGFP at C-terminal was overexpressed in EB3 cells. Intense fluorescent spots were observed at the cell-cell contact region (Fig. 4b), which were ascribed to gap junction plaques. At the same time, less intense fluorescent small spots were distributed in the cytosol. Those were thought to be hexamers of Cx30.3 on the way to cell membrane. Time-lapse measurement at every 120 s for 3480 s revealed that Cx30.3-EGFP appeared, moved, or disappeared rapidly (Fig. S1). The image data captured at 0, 960, and 1440 s are displayed in Fig. 4c. A fluorescent spot indicated by a white arrow was supposedly a gap junction plaque. This plaque moved outward about 3 μ m within 960 s and then seemed to stay there for successive 480 s. During the latter period, the fluorescent intensity increased, suggesting 3 dimensional movement of the plaque in the cell-cell contact membrane. On the other hand, blue arrows indicate the appearance of new fluorescent spots that were supposedly hemi-channels. These spots disappeared within 480 s possibly by internalization and decomposition.
A more drastic disappearance of Cx30.3 hemi-channels was observed in large colonies of EB3 cells. Cx30.3-EGFP was principally distributed at cell-cell contact membrane region but no fluorescent spot was observed at the outermost region (Fig. 4d). Such a phenomenon was not observed with Cx26-EGFP overexpressing EB3 cells (unpublished data).
Half-life of overexpressed Cx protein estimated by dynamic imaging analysis. According to a former review 31 , the half-lives of Cx proteins such as Cx43, Cx32, Cx46, and Cx26 ranged 1-5 h. The Cx half-life is influenced by the cell culture conditions. For example, the half-life of Cx43 in gap junction plaques in cultured cells of corneal endothelium increased in response to an acute stressor such as genotoxic stress 32 , suggesting a temporal stabilization of gap junctional intercellular communication under a stress condition. The stabilization remarked in this context, however, was only a small change of the half-life from 1-2 h to 3-4 h. More recently, in the relevance to skin health and hearing loss, Cx30 was found to be unusually stable with a half-life longer than 12 h 33 . On the other hand, the analysis of plaques by fluorescence recovery after photobleaching (FRAP) revealed much more rapid diffusion behavior of Cx molecules within plaque structures. Cx26 and Cx30 expressed in HeLa cells diffused within 30 s, while Cx43 remained persistently immobile for more than 2 min 34 .
Taking these into consideration, the disappearance of Cx30.3 spots (Fig. 4c-blue arrows) should reflect the internalization or decomposition and not the diffusion within the plaque. From this result, the half-life of Cx30.3 was estimated as 240 s that was much shorter than those of other isoforms (1-5 h). In this case, however, Cx30.3 spots were thought to be hemi-channels and this should be a reason why such a short half-life was observed. These suggested that the removal of gap junctions and/or hemi-channels could be promoted by a signal of the decrease or loss of cell-cell contact membrane region.
Co-localization of overexpressed Cx30.3 and Cx43. Intracellular localization of overexpressing Cx30.3 protein in EB3 cells seemed to be different from those of other Cx isoforms such as Cx26, which was also overexpressed in EB3 cells at pluripotent state (unpublished data). In fact, Cx30.3 alone stayed mostly in cytosol in HeLa cells but its transportation to the cell membrane was promoted by the co-expression with Cx31 35 suggesting their functional close interaction.
Therefore we suspected that Cx30.3 in cell membrane of EB3 cells should be co-localized with other isoforms. Cx43, the most predominant isoform, was thought to be a candidate for such a partner of the co-localization. Consequently the co-localization of Cx30.3-DsRed and Cx43-EGFP was detected at cell-cell contact membrane (Fig. 4e). On the other hand, circular spots of Cx30.3-DsRed were located also in cytosol (Fig. 4e-III). It was not yet analyzed whether these spots were Cx30.3 alone or co-localization with other isoforms than Cx43. Such a co-localization suggested a potential role of Cx30.3 as the pool of Cxs for ready-to-use.

Drastic downregulation of Cx30.3 by the dissociation of colonies into single-cells.
Here we simulated a situation of drastic decrease of cell-cell contact membrane region by enzymatic dissociation of ES cell colonies into single-cells. We predicted that the expression of Cx30.3 should decrease rapidly under this condition. Colonies of pluripotent EB3 cells were treated with trypsin to dissociate them into single-cells and applied to a cell sorter. SSEA1 (stage-specific mouse embryonic antigen) stained cells were also prepared to confirm that the collected cells were pluripotent.
Unstained cells emitted auto-fluorescence ( Fig. 5a-i, P1 fraction) but immunostaining with anti-SSEA1 antibody and then reacted with the second antibody labelled with a fluorescent dye generated cells with higher intensity of fluorescence ( Fig. 5a-ii, P2 fraction). Cells in P1 and P2 fractions (Fig. 5a-ii) were thought to be SSEA1 negative (differentiated) cells and SSEA1 positive (pluripotent) cells, respectively. The Cx30.3 mRNA expression intensities analyzed by qRT-PCR are depicted in Fig. 5b. The expression intensity in single-cells decreased dramatically to as low as 10% of that of colonies.
It is well recognized that LIF(+ ) medium can maintain EB3 cells at the pluripotent state. Under this culture condition, however, some portion of cells are somehow differentiated into a SSEA1 negative state. Under the present experimental condition, SSEA1-positive and negative cells co-existed and the Cx30.3 expression levels of both fractions were almost same. Its level was markedly lower than the control level but sufficiently higher than that of the differentiated cells cultured in the LIF(− ) medium for 6 d. Therefore the marked decrease in the Cx30.3 expression was thought to be caused by the loss of cell-cell contact membrane region and not by the loss of pluripotency.  analysis of mRNA in the presence of a transcription inhibitor, actinomycin D. As depicted in Fig. 5c, the quantity of mRNA for Cx30.3 decreased more rapidly than that for Cx43. In single logarithmic chart, regression lines were obtained by exponential function approximation as follows: y 1 = 0.8039 exp[− 0.061x] and y 2 = 1.0615 exp [− 0.019x] for Cx30. 3 and Cx43, respectively. Here x indicates time in min. From these equations, the half-lives were determined as 11.4 min ( = ln2/0.061) for Cx30.3 and 36.5 min (= ln2/0.019) for Cx43, respectively. To test the significance of slope difference, the regression lines were converted to the following formulas: Y 1 = − 0.0948-0.0265X for Cx30.3 and Y 2 = 0.0259-0.0083X for Cx43. According to the calculation steps summarized in Fig. 5d, t-value for the difference of regression line slopes was determined as 3.903. The degree of freedom was 26 and therefore statistical significance by Student's t-test was p < 0.001.
Decay processes of Cx30.3 and Cx43 proteins. The concentration changing process of Cx30.3 and Cx43 proteins were analyzed by western analysis (Fig. 5e) and then quantified using image J. Though the changing profiles of 3 samples of Cx30.3 were markedly varied, it was observed that Cx30.3 protein exhibited initial increase and successive decrease (Fig. 5f). It should take some time for puromycin to diffuse into cytosol and to inhibit the protein synthesis. During this lag time, Cx30.3 protein might be synthesized by means of Cx30.3 mRNA.
The successive decrease should reflect the decay process of Cx30.3 protein. In contrast, Cx43 protein showed no appreciable change of its quantity. These suggest strongly that Cx43 protein should be much more stable than Cx30.3 protein.

Discussion
Cx30.3 is a promising candidate to elucidate the specific role of Cx in the dramatic change from the pluripotent state to an early stage of differentiation in ES cells. To date, the expression of Cx30.3 has been observed only in differentiated functional tissues and HeLa cells, and its involvement in maintaining the pluripotent state of ES cells has not been described.
Mutations of Cx30.1 and Cx30.3 have been associated with erythrokeratoderma variabilis, a rare disorder of skin cornification 36 . The molecular interaction of Cx30.3 with Cx31 was reported to be associated with the same skin disease 35 . The heteromeric connexons of Cx30.3 and Cx31 proteins can be transported to the cell membrane to form gap junctions in HeLa cells, while Cx30.3 protein alone remains in the cytosol. Simultaneous mutations of Cx26 and Cx30.3 caused autosomal recessive non-syndromic hearing loss in the digenic mode of inheritance 37 . The exposure of rats to steroidal compounds caused a variation in expression levels of nine Cx isoforms, including Cx30.3 in the corpus epididymis 38 . In summary, the involvement of Cx30.3 mutations has so far been reported mostly in skin diseases and hearing loss.
A study reporting that Cx30.3 protein alone could not be transported to the cell membrane in HeLa cells 35 strongly suggested this to be a unique property of Cx30.3. In EB3 cells, however, Cx30.3 protein was localized not only in the cytosol, but also in the cell membrane (Fig. 2c, Figs 4-a1, a2). The co-localization of Cx30.3 and Cx43 (Fig. 4e) suggested their close interaction. We suspected that Cx30.3 might be co-localized in the cell membrane also with other isoforms. In this sense, the cytosolic Cx30.3 protein might be a ready-to-use Cx pool for forming heteromeric connexons or gap junction plaques.
It was not surprising that the expression of Cx30.3 decreased in response to a decrease of the cell-cell contact region. In fact, the expression level of ubiquitously expressed Cx43 in EB3 colonies decreased when cells were dissociated into single cells. However its level was never lower than 50% (unpublished data). In sharp contrast, the expression level of Cx30.3 became as low as 10%. Such a remarkable decrease strongly supports a unique role of Cx30.3 in rapid decrease of gap junctions. The short half-life of Cx30.3 mRNA as well as of Cx30.3 protein was thought to allude to such a role for Cx30.3 by leading to the decomposition of unnecessary mRNA and protein as quickly as possible.
In conclusion, Cx30.3 is a novel isoform that has been assigned as a pluripotent state-specific isoform. It has a potential role in contributing to the quick formation and/or decomposition of gap junctions in EB3 cells. Following on from these findings, the promoter of Cx30.3 needs to be analyzed to clarify why such rapid regulation is essential in the pluripotent state and how the cell-cell contact signal is transduced to Cx30.3. Clarification of the potential role of Cx30.3 as a pluripotent state-specific isoform will provide novel insights into the dynamic, functional networks required for cell-cell contact recognition.
Online methods. ES cell culture. EB3, a clone of feeder-free mouse ES cells, was provided by H. Niwa (Center for Developmental Biology, RIKEN, Kobe, Japan) and cultured at 37 °C in the absence of feeder cells in medium for ES cells (ESM) on gelatin-coated dishes. ESM was composed of GMEM (Sigma, St Louis, MO, USA), 10% fetal calf serum, 1 mM sodium pyruvate, 10 −4 M 2-mercaptoethanol, 1 × non-essential amino acids, and 1,000 U/mL of LIF. ESM containing LIF was designated as LIF(+ ) medium and ESM without LIF was designated as LIF(− ) medium hereinafter.
Preparation of pluripotent and differentiated cells. Pluripotent cells were prepared by culturing EB3 cells in LIF(+ ) medium for 3 d. Differentiated cells were prepared by culturing pluripotent cells in LIF(− ) medium for up to 6 d. The cell lineages involved in this study ranged from pluripotent ES cells, to an approximate early stage of differentiation into endodermal, ectodermal, or trophectodermal cells. A temperature of 37 °C was maintained throughout the culture period.

RNA isolation, reverse transcription PCR (RT-PCR), and quantitative RT-PCR (qRT-PCR).
Total RNA was prepared using ISOGEN II (Nippongene, Tokyo, Japan) according to the manufacturer's instructions. Briefly, 70-80% confluent cells were washed with phosphate buffered saline (PBS) and suspended in 0.8 mL ISOGEN II. The prepared total RNA was then treated with DNase to obtain a purified RNA sample.
Scientific RepoRts | 7:42403 | DOI: 10.1038/srep42403 Two μ g of purified total RNA was mixed with 0.5 μ L of 100 ng/μ L oligo (dT) primers at 70 °C for 10 min and cooled on ice for 1 min. RNA was then converted to cDNA using Super Script II reverse transcriptase (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions.
RT-PCR was performed in GoTaq ® Green Master Mix (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol, using a Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). Amplification conditions were as follows: 94 °C for 3 min, followed by 20-40 cycles of a reaction set (94 °C denaturation for 1 min, 55-65 °C annealing for 1 min, 72 °C elongation for 2 min), with a final incubation at 72 °C for 7 min. Primers used for RT-PCR are listed in supplemental table (Table S1). PCR products were separated by agarose gel electrophoresis (100 V, 30 min) and visualized by staining with ethidium bromide.
Separation of the cell membrane and the cytosol fractions of EB3. EB3 cells growing in each culture dish were washed with PBS. Then 2 mL HEPES buffer solution (20 mM HEPES, 250 mM sucrose, 2 mM EDTA, pH 7.4) was added to the dish to collect cells with a scraper. Adding 3 mL HEPES buffer solution, the cell suspension was sonicated (UR-20P, TOMY SEICO Co. Ltd., Tokyo, Japan) on ice and centrifuged at 2,000 g for 10 min. The supernatant was collected and centrifuged at 12,000 g for 20 min. Then the supernatant was centrifuged again at 180,000 g for 90 min. The supernatant was collected as the cytosol fraction. The precipitate was suspended in a RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.6; Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged at 15,000 g for 20 min. The supernatant was collected as the cell membrane fraction.
Western analysis of Cx30.3 protein. Protein sample solutions were prepared from EB3 cells, their cell membrane fraction, their cytosol fraction, Cx30.3 overexpressing EB3 cells, the kidney and the spleen of a C57BL/6 N mouse. Mouse kidney was positive control, while mouse spleen was the negative control, respectively. The protein concentration was determined using a Pierce ® BCA TM Protein Assay kit (Thermo Fisher Scientific).
A sample solution containing 30-50 μ g protein was mixed with a 1/6 volume of buffer solution containing 0.375 M Tris-HCl (pH 6.8), 93 μ g/mL DTT, 0.12 g/mL SDS, 0.6 mL/mL glycerol, and 0.6 mg/mL bromophenol blue. The mixed solution was then heated at 95 °C for 5 min and proteins were separated by SDS-PAGE at 30 mA.
Blotting onto a PVDF membrane was conducted at 300 mA for 3 h at 4 °C. The PVDF membrane was then immersed in 5% skim milk dissolved in Tris-buffered saline (25 mM Tris-HCl, pH 7.5, 0.15 M NaCl) containing 0.1% Tween 20 (TBS-T) for 1 h at 4 °C with gentle shaking. After overnight incubation at 4 °C with rabbit anti-mouse Cx30.3 polyclonal antibody (40-0900, Thermo Fisher Scientific) in 5% skim milk/TBS-T solution with shaking at 40 rpm, the PVDF membrane was washed three times with TBS-T and incubated with anti-rabbit immunoglobulin conjugated to alkaline phosphatase (Promega) for 1 h at 25 °C, with shaking at 40 rpm. The PVDF membrane was subsequently incubated with Western Blue Stabilized Substrate for alkaline phosphatase (Promega) for 5 min at 25 °C.
To re-probe for β -actin, the PVDF membrane was incubated with mouse anti-β -actin antibody conjugated to alkaline phosphatase (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 25 °C for 2 h, and then stained for alkaline phosphatase as described above. The re-probe for α 1 Na + -K + ATPase, a cell membrane marker was conducted with mouse monoclonal anti-α 1 Na + -K + ATPase antibody conjugated to alkaline phosphatase (ab7671, Abcam) in the same manner. Stained image was quantified using Image J (http://imagej.nih.gov/ij/). Fluorescent microscopy. EGFP and DsRed were introduced to visualize overexpressing Cx proteins. PKH26 (Sigma-Aldrich) was used to stain membrane structure. Those fluorescent images were observed with a confocal laser scanning microscope (LSM510, Carl Zeiss Co., Ltd., Jena, Germany) and also with an all-in-one fluorescence microscope (BZ-X700, Keyence Co., Osaka, Japan).
Immunostaining of endogenous Cx30.3 and Cx43 proteins. EB3 cells were cultured in ESM for 24 h. The medium was removed and the cells were washed twice with PBS. The cells were treated with 4% paraformaldehyde on ice for 15 min for fixation. After incubation 3 times in 10 mM glycine-PBS for 5 min, the blocking treatment was conducted by the incubation in 2% gelatin-PBS for 20 min followed by the incubation 3 times with PBS-glycine, and the incubation with 0.1% BSA-PBS for 5 min. And then the cells were reacted with the primary antibody at 37 °C for 40 min in 1% BSA-PBS containing a rabbit anti-mouse Cx30.3 polyclonal antibody (1:500 dilution) (40-0900, Thermo Fisher Scientific) or a rabbit anti-Cx43 polyclonal antibody (1:2000 dilution) (ab11370, Abcam, Cambridge, UK). After the reaction, the blocking treatment was conducted 6 times with 0.1% BSA-PBS for 5 min. Then the cells were reacted with the second antibody at 37 °C for 40 min in 1% BSA-PBS containing an anti-rabbit immunoglobulin antibody labelled with Alexa Fluor 488 (1:300 dilution) (Invitrogen). Finally the cells were washed 6 times with 0.1% BSA-PBS for 5 min. The mixture was then added to culture dishes of 90% confluent EB3 cells and incubated for 4 h. The cells were subsequently cultured at 37 °C for 24 h and then G418 was added to the medium at 1.5 μ g/mL to select cultures over 7 d. After washing with PBS, an appropriate colony was picked with a micro-pipette, transferred to a 10 μ L trypsin solution Scientific RepoRts | 7:42403 | DOI: 10.1038/srep42403 and incubated for 3 min at 25 °C. ESM (100 μ L) was then added to the solution and the cells were re-suspended. The cell suspension was transferred into a well of a 48-well plate to which 500 μ L ESM/well was added beforehand.
Another set of overexpression vectors for fusion proteins, Cx30.3-EGFP, Cx30.3-DsRed, and Cx43-EGFP, was prepared by inserting a connexin gene into pCMV-gene-EGFP or pCMV-gene-DsRed for use in the investigation of their intracellular localization. The vector products were linearized by treating with ApaLI and introduced into EB3 cells by electroporation using a GENE PULSER II (Nippon Bio-Rad, Tokyo, Japan). Transfected cells were selected as outlined above.
Cx30.3 and Cx43 co-overexpressing cell line. Overexpression vectors for Cx30.3 and Cx43 were simultaneously introduced into EB3 cells. pCMV-Cx30.3-DsRed (2 μ g/250 μ L GMEM) and pCMV-Cx43-EGFP (2 μ g/250 μ L GMEM) were prepared respectively and then mixed with lipofectamine 2000 (10 μ L/250 μ L GMEM). The mixture was stirred gently and stood still for 20 min at 25 °C. The mixture was then added to culture dishes of 90% confluent EB3 cells and incubated for 4 h. The cells were subsequently cultured at 37 °C for 24 h and then G418 was added to the medium at 1.5 μ g/mL to select cultures over 7 d.
Immunostaining of SSEA1. SSEA1 is a pluripotent state marker for mouse cells and was used to confirm that EB3 cells in colonies could maintain the pluripotent state even after dissociation into single-cells. EB3 cells were cultured in a dish with a diameter of 35 mm containing LIF(+ ) medium. After the culture at 37 °C for 3 d, the medium was removed and the cells were washed with PBS. Four mL PBS-EDTA solution was added to the dish and incubated for 5 min. Then cells were separated from the dish bottom using a cell scraper, and transferred into a 15 mL centrifugation tube. After the centrifugation at 1500 rpm × 5 min at 4 °C, the precipitate was suspended in 600 μ L PBS. Cells (10 6 ) in 2 mL of 3% BSA/PBS were added to a centrifugation tube and then centrifuged again as above. The precipitate was suspended in 100 μ L 3% BSA/PBS and rested for 10 min at 25 °C for blocking. To this suspension, mouse anti-SSEA1 antibody (sc-21702, Santa Cruz Biotechnology) was added and incubated for 1 h at 25 °C. The supernatant was removed by centrifugation at 1500 rpm × 5 min at 4 °C and then donkey anti-mouse IgG labelled with Alexa488 (A21202, Thermo Fisher Scientific) was added and cells were incubated for 30 min at 25 °C. After centrifugation at 1500 rpm × 5 min at 4 °C, the precipitate was suspended in 0.5-1.0 mL PBS.
Flow cytometry. A cell suspension was centrifuged at 1500 rpm × 5 min at 4 °C and the precipitate was re-suspended in 0.5-1.0 mL PBS and then filtered with a cell strainer (pore size 35 μ m). The cell suspension was analyzed with a flow cytometer (FACSAria II, BD Biosciences, San Jose, CA, USA). The fluorescence of Alexa488 was detected at 515-545 nm by excitation at 488 nm.
Based on the flow cytogram indicated by the histogram of the number of cells versus the fluorescence intensity, the fractions of control cells emitting auto-fluorescence, SSEA1 negative cells, and SSEA1 positive cells were collected and then applied to qRT-PCR as described above.
Analysis of mRNA half-time. EB3 cells were cultured in LIF(+ ) medium in 6-well plates. The initial cell density was adjusted at 1 × 10 5 cells/well. After 24 h, the medium was replaced by a fresh LIF(+ ) medium containing actinomycin D (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 5 μ g/mL to inhibit transcription. After prescribed times, the medium was removed and the cells were collected by means of ISOGEN II. RNA extraction and qRT-PCR were conducted in the same way as described above.
Statistics. Test sample preparation of mRNA or protein was conducted by one test sample per one dish. In most cases, three test samples were analyzed per one analytical item. One test sample was analyzed twice and the average of the two results was recorded as the value for the test sample. At critical points, two-sided Student's t-test was conducted to estimate statistical significance.