Journal: Journal of Traditional Chinese Medicine

Article Title: Efficacy of Bushenjianpi prescription on autoimmune premature ovarian failure in mice

doi: 10.1016/S0254-6272(17)30321-7

Figure Lengend Snippet: Figure 3 Immunohistochemical analysis of the distinct presence of Cx43 and BMP-15 in the oophorons of each group (× 200) A-F: Cx43; G-L: BMP-15. A, G: control group; B, H: model group; C, I: positive group; D, J: low dose of BSJPP group; E, K: moderate dose of BSJPP group; F, L: high dose of BSJPP group. Positive group treated with premarin (0.03 mg/kg) once daily for 90 d. Con- trol and model group received the same volume of distilled water. Low (8.1 mg/kg), moderate (16.2 mg/kg, the dose used in vivo in human studies), and high (32.4 mg/kg) dose of BSJPP groups treated by gastrogavage once daily for 90 d. Immunohistochemi- cal analysis clearly revealed the presence of Cx43 and BMP-15 in the oophorons of control mice and those treated with BSJPP (D, E) and premarin compared with the model group. BSJPP: Bushenjianpi prescription; Cx43: connexin 43.

Article Snippet: Anti-connexin 43 (Cx43) antibody (product lot No. BA1727) and anti-bone morphogenetic protein 15 (BMP-15) antibody (product lot No. BA2018) were purchased from Boster Biological Engineering (Wuhan, China).

Techniques: Immunohistochemical staining, Control, In Vivo

Journal: Journal of Traditional Chinese Medicine

Article Title: Efficacy of Bushenjianpi prescription on autoimmune premature ovarian failure in mice

doi: 10.1016/S0254-6272(17)30321-7

Figure Lengend Snippet: Figure 4 Expression Cx43 and BMP-15 mRNA in the ovaries of mice in each group. 1: control group; 2: model group; 3: positive group; 4: low dose of BSJPP group; 5: moderate dose of BSJPP group; 6: high dose of BSJPP group. Positive group treated with pre- marin (0.03 mg/kg) once daily for 90 d. Control and model group received the same volume of distilled water. Low (8.1 mg/kg), moderate (16.2 mg/kg, the dose used in vivo in human studies), and high (32.4 mg/kg) dose of BSJPP groups treated by gastrogavage once daily for 90 d. Compared with the model group, significantly upregulated expression of both Cx43 and BMP-15 was detected in the control group and the groups treated with BSJPP (M and L groups). BSJPP: Bushenjianpi prescription; Cx43: connexin 43.

Article Snippet: Anti-connexin 43 (Cx43) antibody (product lot No. BA1727) and anti-bone morphogenetic protein 15 (BMP-15) antibody (product lot No. BA2018) were purchased from Boster Biological Engineering (Wuhan, China).

Techniques: Expressing, Control, In Vivo

Journal: Cell Death and Differentiation

Article Title: Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis

doi: 10.1038/s41418-019-0277-x

Figure Lengend Snippet: S282 mutation with alanine induced cardiomyocyte death. a, b Representative images of NRVMs at 36 h after transfection with virus carrying rat Cx43-wt, S279A or S282A gene (20 m.o.i. for each) recorded by microscope (a) and time-lapse cell death curve detected by YOYO-1 probe (b). NRVM death with time-lapse was collected from 16 fields in each well, and three wells with subconfluent NRVMs obtained from 40 rat ventricles were assigned for each treatment (Videos 1, 2). c–e Measured by confocal microscopy, representative images of NRVMs transfected with different mutants for 24 h, and then stained with antibody against Cx43, or loaded with Fluo4 to detect Ca2+ transients or Lucifer Yellow (LY) to detect intercellular communication, as indicated (c). Nucleus was stained with Hochest33258. Scale bar: 10 μm. Typical traces of Ca2+ transients illustrate that S282A cells had uncoupled transients (d) and decreased LY uptake (e). *P < 0.05, **P < 0.01, unpaired two-tailed Student’s t test, n = 4–6 independent determinations for each group as indicated

Article Snippet: Antibodies used were as follows: rabbit anti-Fas, anti-N-Cad and anti-cytochrome C (Abcam), rabbit anti-pS282-Cx43 and anti-pS279-Cx43 (Biobyt), rabbit anti-Cox IV, anti-p-p38 MAPK (Thr180/Tyr182), anti-p38 MAPKα, anti-pS368-Cx43, anti-HA, anti-pS262-Cx43 and anti-Cx43 (Santa Cruz), and rabbit anti-ZO-1 (Proteintech), mouse anti-GAPDH and anti-β-actin (ZSGB-BIO), mouse anti-TNFR1, anti-caspase-8 and anti-FADD (Santa Cruz), and goat anti-Cx43 (Acris) and anti-DR5 (Santa Cruz).

Techniques: Mutagenesis, Transfection, Microscopy, Confocal Microscopy, Staining, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis

doi: 10.1038/s41418-019-0277-x

Figure Lengend Snippet: Cx43 expression and distribution in NRVMs transfected with different mutants. a–d NRVMs were transfected with virus carrying Cx43-wt, S279A or S282A gene (20 m.o.i. for each) for 24 h and lysed by RIPA (a, b) or RIPA + junctional/non-junctional isolation treatment (c, d). The lysates were analyzed by Western blot (a, c), and the fold changes in phosphorylated S282 (pS282), pS262, pS368 and Cx43 abundances in relative to those of vector cells (after normalized with GAPDH or β-actin) were thereby detected (b, d). Junctional and non-junctional fractions were referred to cell-membrane and cytosolic fractions to detect Cx43 abundance distributed in both regions (see Methods), respectively, and no difference in their pattern between the two regions was found among the different treatments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed Student’s t test, n = 4–5 independent experiments for each group as indicated

Article Snippet: Antibodies used were as follows: rabbit anti-Fas, anti-N-Cad and anti-cytochrome C (Abcam), rabbit anti-pS282-Cx43 and anti-pS279-Cx43 (Biobyt), rabbit anti-Cox IV, anti-p-p38 MAPK (Thr180/Tyr182), anti-p38 MAPKα, anti-pS368-Cx43, anti-HA, anti-pS262-Cx43 and anti-Cx43 (Santa Cruz), and rabbit anti-ZO-1 (Proteintech), mouse anti-GAPDH and anti-β-actin (ZSGB-BIO), mouse anti-TNFR1, anti-caspase-8 and anti-FADD (Santa Cruz), and goat anti-Cx43 (Acris) and anti-DR5 (Santa Cruz).

Techniques: Expressing, Transfection, Isolation, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis

doi: 10.1038/s41418-019-0277-x

Figure Lengend Snippet: Expression of S282 mutant with alanine activated Fas-apoptotic pathway. a, b Apoptosis in NRVMs transfected with virus carrying Cx43-wt, S279A or S282A gene for 28 h (20 m.o.i. for each), respectively, was detected by annexin V-FITC/PI assay using flow cytometry (a) and caspase-3 ELISA kit (b), n = 4 independent determinations for each group. c Caspase-8 activity in these transfected NRVMs was determined by Western blot with a specific antibody against caspase-8, n = 3 independent determinations for each group. d, e Transfection-time-dependent activation of Fas in NRVMs with S282A mutant was analyzed by Western blot, and fold changes of Fas in these cells after normalized with GAPDH, respectively, in relative to those of control cells were thereby detected (d). The activation of caspase-3 by S282A was significantly inhibited by soluble Fas (sFas, 3 μg/60-mm dish, added to the dish when virus was added) (e), n = 3 independent determinations for each group. f The levels of FADD, DR5, and TNFR were determined by Western blot in cells transfected with S282A or Cx43-wt for 28 h and the fold increases in their abundances in relative to those of control (after normalized with GAPDH) were thereby detected, n = 3–4 independent determinations for each group as indicated. *P < 0.05, **P < 0.01, NS: not significant, unpaired two-tailed Student’s t test or one-way ANOVA (e)

Article Snippet: Antibodies used were as follows: rabbit anti-Fas, anti-N-Cad and anti-cytochrome C (Abcam), rabbit anti-pS282-Cx43 and anti-pS279-Cx43 (Biobyt), rabbit anti-Cox IV, anti-p-p38 MAPK (Thr180/Tyr182), anti-p38 MAPKα, anti-pS368-Cx43, anti-HA, anti-pS262-Cx43 and anti-Cx43 (Santa Cruz), and rabbit anti-ZO-1 (Proteintech), mouse anti-GAPDH and anti-β-actin (ZSGB-BIO), mouse anti-TNFR1, anti-caspase-8 and anti-FADD (Santa Cruz), and goat anti-Cx43 (Acris) and anti-DR5 (Santa Cruz).

Techniques: Expressing, Mutagenesis, Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Activation Assay, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis

doi: 10.1038/s41418-019-0277-x

Figure Lengend Snippet: Cx43 S282A+/− mice exhibited arrhythmias and cardiomyocyte apoptosis. a Neonatal lethality (24/81) occurred in heterozygous mice (Het) between day 9 and 21 after birth, while no death was found in wild-type mice (WT). b Typical surface ECG recordings show different changes in Het; generally normal ECG, premature ventricular beat (PVB) and ventricular tachycardia (VT), while no arrhythmia was found in WT. Thereby, three groups: Het(1), Het(2) and Het(3) were divided according to the severity of arrhythmias. c Representative photos of animals and hearts from WT, Het(2) and Het(3) groups, and comparisons of the heart weight (HW) and ratio of heart/body weight (BW) between WT and Het groups, n = 8–12 mice for each group as indicated. d Hearts from different groups stained with H&E showed no obvious morphological change, except for a noticeable increase in the extracellular space in Het(2) and (3) ventricles. Scale bar: 20 μm, n = 5 animals for each group. All the animals were detected between day 10 and 14 after birth. *P < 0.05, NS not significant, unpaired two-tailed Student’s t test

Article Snippet: Antibodies used were as follows: rabbit anti-Fas, anti-N-Cad and anti-cytochrome C (Abcam), rabbit anti-pS282-Cx43 and anti-pS279-Cx43 (Biobyt), rabbit anti-Cox IV, anti-p-p38 MAPK (Thr180/Tyr182), anti-p38 MAPKα, anti-pS368-Cx43, anti-HA, anti-pS262-Cx43 and anti-Cx43 (Santa Cruz), and rabbit anti-ZO-1 (Proteintech), mouse anti-GAPDH and anti-β-actin (ZSGB-BIO), mouse anti-TNFR1, anti-caspase-8 and anti-FADD (Santa Cruz), and goat anti-Cx43 (Acris) and anti-DR5 (Santa Cruz).

Techniques: Staining, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis

doi: 10.1038/s41418-019-0277-x

Figure Lengend Snippet: Cx43 S282A+/− mice exhibited myocardium apoptosis and fibrosis. a–c Mouse ventricles from three groups were stained with antibody against Cx43 or with SIRIUS Red, MASSON or TUNEL as indicated. Nucleus was stained with Hochest33342. Scale bar: 75 μm for Cx43 staining and 20 μm for other images (a). The analysis data for SIRIUS Red-positive signal (b) and TUNEL-positive cells (c) were displayed, n = 5–6 animals for each group as indicated. d Caspase-8/3 activities determined by ELISA kits, respectively, illustrated significant activations of caspase-8/3 in both Het(2) and Het(3) mice, n = 8 mice for each group. Note that Het(3) mice showed severer apoptosis and fibrosis than Het(2) mice. **P < 0.01, unpaired two-tailed Student’s t test

Article Snippet: Antibodies used were as follows: rabbit anti-Fas, anti-N-Cad and anti-cytochrome C (Abcam), rabbit anti-pS282-Cx43 and anti-pS279-Cx43 (Biobyt), rabbit anti-Cox IV, anti-p-p38 MAPK (Thr180/Tyr182), anti-p38 MAPKα, anti-pS368-Cx43, anti-HA, anti-pS262-Cx43 and anti-Cx43 (Santa Cruz), and rabbit anti-ZO-1 (Proteintech), mouse anti-GAPDH and anti-β-actin (ZSGB-BIO), mouse anti-TNFR1, anti-caspase-8 and anti-FADD (Santa Cruz), and goat anti-Cx43 (Acris) and anti-DR5 (Santa Cruz).

Techniques: Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis

doi: 10.1038/s41418-019-0277-x

Figure Lengend Snippet: Cx43 S282A+/− mice exhibited an activation of Fas/FADD pathway. a–d Lysates from ventricles of WT and Het(1)–(3) (a) and from brain, liver and bone tissues of WT and Het(3) mice (b) were analyzed by Western blot with specific antibodies as indicated, respectively, and fold changes in pS282 and Cx43 (c) and Fas/FADD (d) in relative to those in WT ventricles (after normalized with GAPDH) were thereby detected, n = 8 mice for each group in all the panels, except for five mice for each group in b. e Comparing the values of pS282, Cx43, FADD, TUNEL positive cells (from Fig. 5c) and p-p38/p38 MAPK (from Fig. 7b, d) among the ventricles from different groups showed consistence in the activation of apoptosis pathway (p38/FADD), which was correlated with the level of pS282 but not Cx43 expression. **P < 0.01, ***P < 0.001, NS not significant, unpaired two-tailed Student’s t test

Article Snippet: Antibodies used were as follows: rabbit anti-Fas, anti-N-Cad and anti-cytochrome C (Abcam), rabbit anti-pS282-Cx43 and anti-pS279-Cx43 (Biobyt), rabbit anti-Cox IV, anti-p-p38 MAPK (Thr180/Tyr182), anti-p38 MAPKα, anti-pS368-Cx43, anti-HA, anti-pS262-Cx43 and anti-Cx43 (Santa Cruz), and rabbit anti-ZO-1 (Proteintech), mouse anti-GAPDH and anti-β-actin (ZSGB-BIO), mouse anti-TNFR1, anti-caspase-8 and anti-FADD (Santa Cruz), and goat anti-Cx43 (Acris) and anti-DR5 (Santa Cruz).

Techniques: Activation Assay, Western Blot, TUNEL Assay, Expressing, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis

doi: 10.1038/s41418-019-0277-x

Figure Lengend Snippet: Activation of p38 MAPK responsible for the cell death in S282 mutated NRVMs and Cx43-S282A+/− mice. a–d Lysates from S282A-transfected NRVMs (a) and ventricles of WT and Het mice (b) were analyzed by Western blot using specific antibody for phosphorylated p38 MAPK (p-p38) or p38 MAPK, and fold increases in p-p38 in cells (c) and ventricles (d) after normalized with p38 MAPK in relative to their counterpart controls were thereby detected, n = 3–4 independent determinations (c) and 8 mice (d) for each group as indicated. e The activation of p38 MAPK was transfection-time-dependent (data from a) and compared with that in Fas activation (data from Fig. 3d) in S282A transfected cells. f–h The inhibitory effect of SB203580 (SB, 10 μM, added at the time of transfection and harvested 28 h after) on Fas/FADD activation (f, g) and the lowered cell viability (h) induced by S282A transfection were detected by Western blot and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, respectively, n = 4 (g) and 8–11 (h) independent determinations for each group as indicated. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed Student’s t test (c, d) and one-way ANOVA (g, h)

Article Snippet: Antibodies used were as follows: rabbit anti-Fas, anti-N-Cad and anti-cytochrome C (Abcam), rabbit anti-pS282-Cx43 and anti-pS279-Cx43 (Biobyt), rabbit anti-Cox IV, anti-p-p38 MAPK (Thr180/Tyr182), anti-p38 MAPKα, anti-pS368-Cx43, anti-HA, anti-pS262-Cx43 and anti-Cx43 (Santa Cruz), and rabbit anti-ZO-1 (Proteintech), mouse anti-GAPDH and anti-β-actin (ZSGB-BIO), mouse anti-TNFR1, anti-caspase-8 and anti-FADD (Santa Cruz), and goat anti-Cx43 (Acris) and anti-DR5 (Santa Cruz).

Techniques: Activation Assay, Transfection, Western Blot, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis

doi: 10.1038/s41418-019-0277-x

Figure Lengend Snippet: Enhanced physical interaction between p38 MAPK and Cx43 in S282A transfected cardiomyocytes and Cx43 S282A+/− hearts. a Representative images of NRVMs transfected with adenovirus and ventricles from WT and Het(2) mice, as indicated, and double-stained with antibodies specific for p38 MAPK (green) and Cx43 (red). The cells indicated with dot irregular shapes and yellow arrows were S282A-negative cells. White arrows point to p38 MAPK colocalized with Cx43 in S282A-positive cell surface and lateral side of the nucleus, and in ventricles of WT and Het(2) mice in enlarged images. Nucleus was stained with Hochest33342, scale bar: 10 μm, n = 3 independent experiments for cell experiment and four hearts for ventricle test in each group. b The quantitative analysis of p38/Cx43 colocalization, represented by Pearson’s correlation coefficient (see Methods), in adenovirus transfected-NRVMs, n = 43–67 cells as indicated, ***P < 0.001, unpaired two-tailed Student’s t test. c Ventricle lysates from WT and Het(2) mice were immunoprecipitated with anti-Cx43 antibody and then Western blotted with anti-p-p38, anti-p38 or anti-FADD antibody, n = 3 independent experiments from 32 mice for each group. d Lysates from vector and Cx43-wt or S282A-transfected cardiomyocytes were immunoprecipitated with anti-Cx43 antibody and then Western blotted with anti-p-p38 or anti-p38 antibody, n = 3 independent experiments for each group

Article Snippet: Antibodies used were as follows: rabbit anti-Fas, anti-N-Cad and anti-cytochrome C (Abcam), rabbit anti-pS282-Cx43 and anti-pS279-Cx43 (Biobyt), rabbit anti-Cox IV, anti-p-p38 MAPK (Thr180/Tyr182), anti-p38 MAPKα, anti-pS368-Cx43, anti-HA, anti-pS262-Cx43 and anti-Cx43 (Santa Cruz), and rabbit anti-ZO-1 (Proteintech), mouse anti-GAPDH and anti-β-actin (ZSGB-BIO), mouse anti-TNFR1, anti-caspase-8 and anti-FADD (Santa Cruz), and goat anti-Cx43 (Acris) and anti-DR5 (Santa Cruz).

Techniques: Transfection, Staining, Two Tailed Test, Immunoprecipitation, Western Blot, Plasmid Preparation