Fig 2: (a) Expression of Cx32, Cx26, Cx30 and Cx43 in normal cervix and CaCx samples. Expression of Cx26, Cx30 and Cx43 was decreased in the cancer samples relative to control, while expression of Cx32 was markedly increased. (b) Western blots showing that expression of expression of Cx32 correlated with increased FIGO score. Data are shown for five samples in each category (c) Immunohistochemistry showing that Cx32 and Cx43 in normal cervix tissue were localized to junctional regions. In CaCx cells, Cx32 aberrantly aggregated in para-nuclear cytoplasm and Cx43 was rarely expressed. (Scale bar: 20 µm). *P<0.05

Fig 3: (A,B): Connexin-43 (Cx43) and Glial Fibrillary Acidic Protein (GFAP) mRNA expression (normalized with Glyceraldehyde-3-phosphate dehydrogenase gene, GAPDH) was assessed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) from the hypothalamus (A) and dorsal vagal complex (DVC) (B) of control ad libitum (n = 14), 24 h fasted (n = 13), 24 h fasted plus 2 h refeed (n = 8) or plus 4 h refeed (n = 8) mice. (C): Representative Western blot analysis of phosphorylated Cx43 (pS368) and Cx43 expression in the hypothalamus of control ad libitum (n = 5) and 24 h fasted (n = 5) mice. (D,E): Cx43 and GFAP mRNA expressions (normalized with GAPDH) were assessed by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) from the hypothalamus (D) and dorsal vagal complex (DVC) (D) of Normal Chow (NC) (n = 9) and High Fat Diet (HFD) fed (n = 9) mice. (F): Representative Western blot analysis and quantification of phosphorylated Cx43 (pS368) and Cx43 expression in the hypothalamus of NC (n = 9) and HFD-fed (n = 9) mice. A two-way analysis of variance test was performed between different experimental groups * p < 0.05 and *** p < 0.001 significantly different from control ad libitum. #p < 0.05 and ##p < 0.001 significantly different from 24 h fasted mice. ns: no significant difference.

Fig 4: AS HBFs selectively modulate the invasion of A549 cells. (A) A549 cells were grown to confluence with NA HBFs (left-hand panel) or AS HBFs (right-hand panel) in 2-well silicone inserts and co-incubated for 48 h following diaphragm removal prior to fixation and immunostaining for a-SMA. Expression of a-SMA was visualized by immunofluorescence and cytofluorimetry, respectively. Scale bar, 100 µm; magnification, ×200. Inserts present A549 cells within the HBF monolayer. (B) Cells were cultivated as in (A) and intracellular localization of Snail-1/Cx43 and co-localization of Snail-1/DNA were visualized with immunofluorescence and cytofluorimetry, respectively (left axes: DNA; right axes: Snail-1). Photomicrographs in (A) and (B) present panoramic till scans of the interfaces between HBF and A549 monolayers obtained by the composition of two rows of succeeding images (7 pictures in a row). Scale bars, 100 µm. (C) NA HBFs and AS HBFs were cultivated in the presence of the media conditioned by ‘separated’ NA HBF/A549 and AS HBF/A549 co-cultures, respectively, for 48 h and immunostained against a-SMA, Snail-1 and Cx43. Scale bars, 50 µm; magnification, ×630. (D) A549 cells were seeded onto microporous membrane, placed in the wells filled with the media conditioned by A549/HBF co-cultures, and allowed to transmigrate for 48 h. The number of the transmigrated cells were counted following 24 h. Data are presented as the mean ± standard error of the mean of 3 independent experiments. *P<0.05 vs. A549 parameters in control medium. Note the infiltration of AS HBFs into A549 monolayers, accompanied by the chemotactic activity of the media from ‘open’ AS HBF/A549 co-cultures and by the presence of scattered A549 within AS HBF continua. HBFs, human bronchial fibroblasts; AS, asthmatic donors; NA, non-asthmatic donors; a-SMA, a-smooth muscle actin; Cx43, connexin43; CM, conditional media.

Fig 5: Cardiac-specific overexpression of Cisd2 at a late-life stage rescues the aging hearts of mice from age-related cardiac fibrosis and intercalated disc (ICD) defects. (A) Age-related cardiac fibrosis can be seen to be ameliorated by enhancing the expression level of Cisd2 in the aging heart. Representative immunofluorescence images of heart sections from the three groups of mice (3-mo WT, 26-mo WT, and 26-mo Cisd2 icOE) stained with antibodies against collagen I (green) and wheat germ agglutinin (red) to identify the cell membrane, with rhodamine phalloidin—to identify actin (purple), and with DAPI—to identify the cardiomyocytic nucleus. Age-related cardiac fibrosis is obvious in the 26-mo WT mice (A2). However, overexpression of cardiac Cisd2 appears to reduce cardiac fibrosis (A3). (B,C) Overexpression of Cisd2 reverses the age-related defects affecting the ICD in the aging heart. Representative immunofluorescence images of heart sections from the three groups of mice (3-mo WT, 26-mo WT, and 26-mo Cisd2 icOE) stained with antibodies against Cx43 (green) was used to localize gap junctions, against pan-cadherin (red)—to localize the ICDs, and against a-actinin (purple)—to stain muscle fibers. The sections were also stained with Hoechst (blue) to identify the nuclei. Cx43 is well-co-localized with pan-cadherin in hearts of the 3-mo WT mice (B1). However, lateralization and loss of co-localization of Cx43 with pan-cadherin were found in hearts of the 26-mo WT mice (B2). White arrows indicate lateralization of gap junctions. Maldistribution of desmosomes was found in the hearts of 26-mo WT mice (C). Co-localization of desmoplakin (green) with pan-cadherin (C1) was found within desmosomes of the 3-mo WT mice, and this was lost in hearts of the 26-mo WT mice (C2). Remarkably, defects in the ICD were reversed by cardiac-specific overexpression of Cisd2 (B3,C3).