Fig 2: Ca2+ oscillations induced by Ca2+-free medium application activate the lipolysis process and correlate with changes in the expression of key genes in white adipose tissue. (A,B) Control (A) white adipocytes and cells with Cx43 gene knockdown using Gja1 siRNA (Cx43-KD, (B)) were exposed to Ca2+-free medium for 1 h and then returned to a CO2 incubator for 24 h. Red line—Ca2+ oscillations in 29 ± 14% and Ca2+ transients (blue curve) in the 47 ± 9% of white adipocytes. In Panel (B)—black curve (+CBX): Ca2+-free medium application was performed with 100 µM CBX. (C) Effects of the Ca2+-free medium on the expression of genes in white adipocytes + CBX and Cx43 gene knockdown cells (Cx43-KD) after 24 h. All values are given as the mean ± SEM. Scrambled—cultures transfected with an siRNA whose sequence differed from that of the siRNA against Cx43. Gene expression level was normalized to the reference gene Gapdh and was presented relative to the control (adipocytes were not exposed to Ca2+-free medium), which was considered as 1 (dashed line). A comparison of the experimental groups with the control was made. The differences between the experimental groups and control are marked with asterisks: * p < 0.05, ** p < 0.01, and *** p < 0.001. n/s—no significant difference. (D) The protein abundance of LIPE and ATGL in control, Cx43-KD, CBX-treated, Ca2+-free-treated, CBX + Ca2+-free-treated and CX43-KD + Ca2+-free-treated white adipocytes. The data were obtained by Western blotting. (E) Relative protein levels of LIPE and ATGL in the experimental groups from Panel (D). The data were presented as the mean ± SD of at least three independent experiments. Glyceraldehyde–3–phosphate dehydrogenase (GAPDH) was used as an internal control for normalization. The differences between the experimental groups are marked with asterisks: * p < 0.05, ** p < 0.01, and *** p < 0.001. (F) Effect of the Ca2+-free medium on the accumulation of lipid droplets in control white adipocytes (Control, without Ca2+-free), CBX-treated cells (CBX) after 1 h of Ca2+-free medium exposure in the control (+Ca2+-free) and the presence of 100 µM CBX (CBX + Ca2+-free), as well as in Cx43 gene knockdown cells (Cx43-KD) and Cx43-KD with Ca2+-free medium exposure (Cx43-KD + Ca2+-free).

Fig 3: Cx43 null mice have reduced motile cilia in ECs.a, b The SCs of P1 pups with the indicated Cx43 genotype were cross-sectioned, double immunostained with anti-Cx43 and anti-ARL13B antibodies (a) or anti-Phospho-Cx43 (Ser386) antibody (b), and counterstained with DAPI. Asterisks indicate the central canal (CC). Images are oriented ventral to the bottom. Scale bars = 20 µm. c, d Quantifications of motile cilia per section in embryos in a and Cx43 signals per section in embyos in b. Mean ± SD. ***P < 0.001 and ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test (n = 3 mice per group; one frame per mouse). ns, not significant. e TEM of the SCs of P1 pups with the indicated Cx43 genotype. Arrowheads indicate cross-sectioned motile cilia protruding to the CC and arrow marks a cilium-like structure embedded in an EC. Regions marked by arrowheads or arrow are magnified in the respective lower panels. f The human spinal cord at C1 level from a cadaver was stained with anti-Cx43 and anti-acetylated-a-tubulin antibodies. The boxed areas are magnified in the respective lower panels. Arrowhead denotes staining of Cx43 and arrow motile cilia. Scale bar = 100 µm. g A proposed model for the maintenance of ependymal motile cilia by the Wnt-PLC-IP3-Connexin-Ca2+ axis.

Fig 4: Dephosphorylated cardiac Connexin 43 and its distribution in myocardium after 14 days of cocaine administration. (A) The protein expressions of total Connexin43 (Cx43) and dephosphorylated Cx43 at Serine 368 (dp-Cx43) in the rat’s left ventricle after cocaine administration. Levels of actin served as the internal control. Band densities of total, phosphorylated, and dephosphorylated Cx43 in the cocaine group relative to the means of the control group. Individual values are presented in black bullets. Each bar represents mean and S.D. *, p < 0.05 (B) PTAH staining of myocardium samples from the cocaine group, scale bar = 250 µm. (D,F) PTAH staining of myocardium samples from the cocaine group, scale bar = 100 µm. (H) PTAH staining of myocardium samples from the control group, scale bar = 100 µm. (C) Immunohistochemistry of dp-Cx43 in left ventricle samples from the cocaine group, scale bar = 250 µm. (E,G) Immunohistochemistry of dp-Cx43 in left ventricle samples from the cocaine group, scale bar = 100 µm. (I) Immunohistochemistry of dp-Cx43 in left ventricle samples from the control group, scale bars = 250 µm.

Fig 5: Effect of connexin and pannexin hemichannels blockers on the generation of Ca2+ signals in white adipocytes due to the application of Ca2+-free medium. (A,B) Effects of connexin (Carbenoxolone (CBX), 100 µM; Octanol, 1 mM; or Gap26, 100 µM) and pannexin (Probenecid (PROB) 1 mM) hemichannels blockers on the Ca2+ oscillation (A) and Ca2+ transients (B) of white adipocytes upon application of Ca2+-free medium. Between the first and second application of the Ca2+-free medium there was a 30 min pause in the Ca2+-dynamics registration (recording). (C) Cell knockdown of the Cx43 hemichannels completely suppresses Ca2+ oscillations and significantly suppresses the amplitude of Ca2+ transients in white adipocytes upon application of a Ca2+-free medium. NE—application of 1 µM norepinephrine, an adrenergic receptor agonist. (D) Effect of the investigated blockers on the amplitude of the Ca2+ oscillations and transients upon repeated Ca2+-free application. The amplitude of the Ca2+ response to the first application of the Ca2+-free medium is taken as 100%. Abbreviations: CBX—Carbenoxolone, 100 µM; Octanol, 1 mM; Proadifen, 100 µM; Gap26, 100 µM; blockers of connexin hemichannels: 10Panx, 100 µM and Probenecid, 1 mM; and blockers of pannexin hemichannels, Cx43-KD; Panx-KD—Cx43 and Panx1 knockdown using Gja1 siRNA and Panx1 siRNA; Scrambled—cells transfected with an siRNA whose sequence differed from that of the siRNA against Cx43 and Panx1; 2 response—the amplitude of Ca2+ responses to the Ca2+-free medium application without inhibitors. The differences between the experimental groups and control are marked with asterisks: ** p < 0.01, *** p < 0.001; n/s—no significant difference.