Fig 2: Co-localization of Cx43, AKAP95 and DNA in A549 cells (A) Co-localization of Cx43 and AKAP95 in the nucleus. (Aa) Blue fluorescence represents DAPI-labeled nuclei; (Ab) green fluorescence represents FITC-labeled AKAP95; (Ac) red fluorescence represents TXRD-labeled Cx43; (Ad) merged image of Aa and Ab; (Ae) merged image of Aa and Ac; (Af) merged image of Ab and Ac; and (Ag) merged image of Aa, Ab, and Ac; (B) Rows 1–5 are the enlarged images of cells 1–5 in Af. (magnification 200 × for A; 200 × 4 for B). (C) Proximity ligation assay and immunofluorescence detected by Laser scanning confocal microscope (200 × ). Labeling with DAPI is shown in (Ca), and lamin B1 is shown in (Cb). In (Cc), the combination of AKAP95 and Cx43 was labeled with TXRD. Results showed interaction between AKAP95 and Cx43 (red). Both proteins were localized in the nucleus. Panel (Cd) is a merged image of (Ca) and (Cb). Panel (Ce) is a merged image of (Cb) and (Cc). Panel (Cf) is a merged image of (Ca) and (Cc). In panel (Cg) (merged image of Ca, Cb and Cc), cells highlighted as I and II point to the combination of AKAP95 and Cx43 crossing the nuclear membrane. Cells II are enlarged in (Ch) (200 × 4). At the lower right corner (200 × 4 × 4), the junction between complex and membrane was demonstrated by yellow fluorescence(arrow), suggesting movement into the nucleus via the nuclear membrane. (D) is the negative control to C. (Di) is DAPI, (Dj) is Lamin B1, (Dk) is Cx43 primary antibody replace by PBS as a negative control in PLA assays; red fluorescence was not detected. Panel (Dl) is a merged image of (Di), (Dj) and (Dk). (E) We detected the quantitative changes of AKAP95/Cx43 complexes in G1, S, G2, M phases using Olympus software in PLA assays. The number of the detected cells was 37 in G1 phase, 30 in S phase, 13 in G2 phase and 10 in M phase. In addition, there were statistically significant differences of the amount of AKAP95/Cx43 complexes between G1 and S phase, G1 and G2 phase respectively.

Fig 3: Gap junction remodeling and Cx43 reduction in post-menopausal rats. (A) The representative electron micrographs of myocardium from both 6M and 18M rats. Up: Full image of intercalated disk including fasciae adherentes junctions, desmosome and gap junctions. Down: magnified images of gap junctions from dashed boxes of above images. The gap junctions are pointed by arrows. (B) The size of gap junction per intercalated discs. (C) Cx43 protein level in myocardium of 6M and 18M rats. (n=6). (D-E) The representative confocal images and quantitative analysis of Cx43 immunofluorescence activity of 6M and 18M rats. The values in column are normalized to 6M. n=3, * P<0.05 and ** P < 0.01 relative to 6M.

Fig 4: E2 prevented Cx43 repression and gap junction defects induced by miR-23a overexpression in NRVCs. (A) qRT-PCR assay revealed that E2 treatment inhibited the expression of miR-23a in NRVCs. (B) E2 treatment prevented the reduction of Cx43 protein level induced by miR-23a in NRVCs. (C) The size of gap junction per intercalated discs. (D) The representative electron micrographs of NRVCs. Up: Full image of intercalated disk including fasciae adherentes junctions, desmosome and gap junctions. Gap junctions were pointed by arrows in dashed boxes. The gap junctions are pointed by arrows. Down: magnified images of gap junctions from dashed boxes of above images. The gap junctions are pointed by arrows. n=3. * P < 0.05 relative to NC. # P < 0.05 relative to miR-23a transfected NRVCs.

Fig 5: Interaction between Cx43 and AKAP95 in A549 and L-O2 cells at different cell cycle phases.A549 and L-O2 cells were treated with L-mimosine (G1) (75 and 80 µg/mL), aphidicolin (S) (0.8 and 1 µg/mL), nocodazole (G2) (0.6 and 1 µg/mL), and colchicine (M) (0.5 and 0.5 µg/mL) for 24 h. A portion of the cells were subjected to total protein extraction and analysis. (A) Changes in AKAP95 and Cx43 expression determined by Western blot; and (B) statistical analysis of AKAP95 and Cx43 expression levels derived from images acquired by the BioRad Chemi DOC XRS + Imaging System. The gray value of protein bands was scanned using Image Lab5.0, and the ratio of band intensity in the treatment group versus the control group was determined using one-way analysis of variance in SPSS 21.0. * indicates statistically significant difference between groups at p < 0.05. Remaining cells were subjected to cell fractionation. (C) Co-immunoprecipitation assay using anti-Cx43 antibody and Western blot using anti-AKAP95 antibody, when cytoplasm and nuclear protein were separated, a small amount nuclear membarnce protein was ramained in the cytoplasm protein. The experiment was repeated four times.