Journal: Experimental & Molecular Medicine

Article Title: CD38/ADP-ribose/TRPM2-mediated nuclear Ca 2+ signaling is essential for hepatic gluconeogenesis in fasting and diabetes

doi: 10.1038/s12276-023-01034-9

Figure Lengend Snippet: a Glucagon-induced ADPR production after preincubation with oleamide (50 μM) for 30 min. n = 6 independent experiments. *** P < 0.001 and not significant (NS). b Effect of oleamide on glucagon-induced mRNA expression of G6pc and Pck1 . n = 6 indepe n dent experiments. ** P < 0.01, *** P < 0.001. c After isolating nuclei, nucleoplasts were stained with anti-Cx43 antibody (green). Lamin B1 was used as a marker for the inner membrane of the nucleus. The nucleus was stained with DAPI (blue). Scale bar, 5 μm. d The bar graph represents the localization of Cx43. n = 6 independent experiments. *** P < 0.001. e Hepatocytes were transduced with adenoviral vectors expressing Ad-Cx43-NLS or Ad-Cx43 S368A -NLS at an MOI of 50 for 24 h and then assayed for immunoblot analysis for Ad-NLS, Ad-Cx43-NLS, or Ad-Cx43 S368A -NLS and qRT-PCR of G6pc and Pck1 in hepatocytes infected with Cx43 WT or S368A before and after treatment with glucagon. n = 8 independent experiments. ** P < 0.01, *** P < 0.001. f , g Blood glucose levels during fasting ( f ) and after pyruvate challenge ( g ) in mice after treatment with Ad-NLS, Ad-Cx43-NLS, or Ad-Cx43 S368A -NLS. n = 10 mice per group. * P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.05; Ad-NLS vs . Ad-Cx43-NLS, ## P < 0.001; Ad-Cx43-NLS vs . Ad-Cx43 S368A -NLS. h Nucleoplasts were stained with anti-PKCδ antibody (green). Lamin B1 was used as a marker for the inner membrane of the nucleus. The nucleus was stained with DAPI (blue). Scale bar, 5 μm. i Glucagon-induced G6pc and Pck1 mRNA levels. GF109203X (1 μM), Go6976 (1 μM), or rottlerin (3 μM) was preincubated for 30 min. n = 6 independent experiments. ** P < 0.01, *** P < 0.001. j Effects of PKCδ KD on glucagon-induced G6pc and Pck1 gene expression. Immunoblotting of PKCδ and β-actin (upper). n = 6 independent experiments. ** P < 0.01, *** P < 0.001. k Nucleoplasts were incubated with OAG for the indicated times. Immunoblotting for pCx43 (S368), Cx43, and Lamin B1. l NAD and ADPR levels after nucleoplasts were incubated with OAG (100 μM) for 30 sec. Ara-2’-F-NAD (200 nM) was preincubated for 30 min. n = 6 independent experiments. *** P < 0.001. m NAD and ADPR levels in PKCδ-knockdown nucleoplasts. OAG (100 μM) was incubated for 30 sec. Immunoblotting of PKCδ and Lamin B1 (upper). n = 6 independent experiments. ** P < 0.01, *** P < 0.001. Data are represented as the mean ± SEM. Statistics were determined by one-way ANOVA followed by Tukey’s multiple comparison test except d where unpaired t test was used. For confocal images and immunoblotting, representative images of three independent experiments are shown.

Article Snippet: We used antibodies for the following proteins: pCREB (Ser133) (Cell Signaling, 9198), pCaMKII (Cell Signaling, 3361), pCaMKIV (Santa Cruz, sc-28443-R), CREB (Cell Signaling, 9197), CaMKII (Cell Signaling, 3362), CaMKIV (Cell Signaling, 4032), PARP-1 (Santa Cruz, sc-53643), Actin (Merck Millipore, MAB1501), pCx43(Ser368) (Sigma-Aldrich, SAB4300504), Cx43 (Cell Signaling, 3512), Flag (Sigma-Aldrich, F7425), Lamin B1 (Cell Signaling, 12586), PKCδ (Cell Signaling, 9616), TRPM2 (Novus, NB110-81601), Na + K + -ATPase (Novus, NB300-146), Calregulin (Santa Cruz, sc-7431), Nesprin 3 (MyBioSource, MBS2535184), Myc (Invitrogen, 46-0603), CD38 (Santa Cruz, sc-7049), PLCβ1 (Santa Cruz, sc-5291), PLCβ3 (Santa Cruz, sc-133231), PLCβ4 (Santa Cruz, sc-404), PLCδ1 (Santa Cruz, sc-365811), PLCδ3 (Santa Cruz, sc-514912), PLCδ4 (Santa Cruz, sc-373875), PLCγ1 (Santa Cruz, sc-7290), PLCγ2 (Santa Cruz, sc-5283), HSP90 (Cell Signaling, 4874), GAPDH (Santa Cruz, sc-166574), and LAMP-1 (BD Biosciences, 553792).

Techniques: Expressing, Staining, Marker, Transduction, Western Blot, Quantitative RT-PCR, Infection, Incubation

Journal: Experimental & Molecular Medicine

Article Title: CD38/ADP-ribose/TRPM2-mediated nuclear Ca 2+ signaling is essential for hepatic gluconeogenesis in fasting and diabetes

doi: 10.1038/s12276-023-01034-9

Figure Lengend Snippet: a Hepatic ADPR level of m + /db and db/db mice fed and fasted for 19 h. n = 6 independent experiments. *** P < 0.001. b Effect of 8-Br-ADPR on glucagon-induced nuclear Ca 2+ signaling in hepatocytes prepared from m + /db and db/db mice. The time point where 100 nM glucagon (Gcg) is added is indicated by the arrow. n = 20 cells for each condition. c Glucagon-induced mRNA levels of G6pc in hepatocytes prepared from m + /db and db/db mice. Cells were preincubated with 8-Br-ADPR (100 μM) for 30 min before treatment with glucagon (100 nM). n = 6 indepe n dent experiments. *** P < 0.001, # P < 0.05; vehicle vs glucagon, ## P < 0.001; glucagon vs 8-Br-ADPR plus glucagon. d – f Effects of 8-Br-ADPR on the pyruvate tolerance test ( d , n = 5 mice per group), CRE luciferase activity ( e , n = 3 mice per group), and gluconeogenic gene expression ( f , n = 7 mice per group) in db/db mice. 8-Br-ADPR (32 mg/kg) was intravenously administered to mice. ** P < 0.01, *** P < 0.001. g Immunoblotting for pCx43 (S368), Cx43, and β-actin in the livers of m + /db and db/db mice after fasting for 19 h. n = 4 mice per group. Data are represented as the mean ± SEM. Statistics were determined by unpaired t test ( d , f ) or one-way ANOVA followed by Tukey’s multiple comparison test ( a , c ).

Article Snippet: We used antibodies for the following proteins: pCREB (Ser133) (Cell Signaling, 9198), pCaMKII (Cell Signaling, 3361), pCaMKIV (Santa Cruz, sc-28443-R), CREB (Cell Signaling, 9197), CaMKII (Cell Signaling, 3362), CaMKIV (Cell Signaling, 4032), PARP-1 (Santa Cruz, sc-53643), Actin (Merck Millipore, MAB1501), pCx43(Ser368) (Sigma-Aldrich, SAB4300504), Cx43 (Cell Signaling, 3512), Flag (Sigma-Aldrich, F7425), Lamin B1 (Cell Signaling, 12586), PKCδ (Cell Signaling, 9616), TRPM2 (Novus, NB110-81601), Na + K + -ATPase (Novus, NB300-146), Calregulin (Santa Cruz, sc-7431), Nesprin 3 (MyBioSource, MBS2535184), Myc (Invitrogen, 46-0603), CD38 (Santa Cruz, sc-7049), PLCβ1 (Santa Cruz, sc-5291), PLCβ3 (Santa Cruz, sc-133231), PLCβ4 (Santa Cruz, sc-404), PLCδ1 (Santa Cruz, sc-365811), PLCδ3 (Santa Cruz, sc-514912), PLCδ4 (Santa Cruz, sc-373875), PLCγ1 (Santa Cruz, sc-7290), PLCγ2 (Santa Cruz, sc-5283), HSP90 (Cell Signaling, 4874), GAPDH (Santa Cruz, sc-166574), and LAMP-1 (BD Biosciences, 553792).

Techniques: Luciferase, Activity Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION

doi: 10.1101/2023.05.25.542366

Figure Lengend Snippet: A. Representative western blots suggest that neither total connexin43 (Cx43) nor Cx43 phosphorylated at serine-368 (pCx43-S368) significantly change in response to acute exposure to osmotic agents. Lane 7 contains a positive control using hearts exposed to 60 min of room temperature ischemia, a condition shown to upregulate Cx43 pS368. B. Summary data plotted with mean and standard deviation error bars do not reveal significant differences in either total or pCx43-S368 for all conditions (N=3 hearts per condition. p=n.s. via ordinary one-way ANOVA applying Dunnett’s correction). C. Representative fluorescent images of calcein-AM fluorescence in control cultures (N=13 plates, n=220 cells) and cultures exposed to mannitol (N=7 plates, n=116 cells), albumin (N=4 plates, n=77 cells), Dextran 70kDa (N=4 plates, n=102 cells), Dextran 2MDa (N=4 plates, n=95 cells), and carbenoxolone (CBX, N=6 plates, n=110 cells). Measurements were made before bleaching (Pre-bleach), just after bleaching (t=0s), halfway through and at the end of the recovery period (t=150 and 300s, respectively). D. Fluorescence recovery after photobleaching (FRAP) plot of bleached cells during 300s post bleaching. Solid lines represent averages at each point, and shaded areas represent the standard error of the mean. E. Normalized recovery demonstrates that only CBX significantly decreased FRAP relative to control. *p<0.05, nested ANOVA applying Dunnett’s correction with culture plate nested under treatment type.

Article Snippet: Electrophoresis was performed to separate proteins which were then transferred to a PVDF membrane, blocked with 5% bovine serum albumin for 1 hour at room temperature and incubated overnight with a primary antibody against the principal ventricular GJ protein Cx43 phosphorylated at Ser368 (pCx43, 1:1000, #3511S, Cell Signaling Technologies), at 4°C.

Techniques: Western Blot, Positive Control, Standard Deviation, Fluorescence

Journal: bioRxiv

Article Title: EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION

doi: 10.1101/2023.05.25.542366

Figure Lengend Snippet: A. Representative images of ventricular tissue slices perfused with 0.5, 3, and 10kDa fluorescent dyes. Panels demonstrate extracellular diffusion; Cx43 signal is used to identify the intercalated disc (ID), and nuclei are shown alone and with overlay of all signals. B. A region of interest is identified around the Cx43 ID signal and intensity of signal is averaged to identify the peak of the Cx43 signal. The same spatial averaging with 0.5, 3, and 10kDa signal demonstrates a dip in 3 and 10kDa signal with a peak in the 0.5kDa dye demonstrating the 0.5kDa dye permeates the ID, but the other two dyes do not. C. The intensity of the dye fluorescent signal at the peak of the Cx43 signal is normalized to the intensity of signal beyond the ID to reveal that 3 (n=3 images) and 10kDa (n=4 images) fluorescent signal are significantly lower than 0.5kDa (n=7 images) signal intensity. *p<0.05 relative to 0.5kDa, one way ANOVA with Dunnett’s correction.

Article Snippet: Electrophoresis was performed to separate proteins which were then transferred to a PVDF membrane, blocked with 5% bovine serum albumin for 1 hour at room temperature and incubated overnight with a primary antibody against the principal ventricular GJ protein Cx43 phosphorylated at Ser368 (pCx43, 1:1000, #3511S, Cell Signaling Technologies), at 4°C.

Techniques: Diffusion-based Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Plasmalogens Regulate Retinal Connexin 43 Expression and Müller Glial Cells Gap Junction Intercellular Communication and Migration

doi: 10.3389/fcell.2022.864599

Figure Lengend Snippet: Plasmalogen depletion impacts Cx43 expression in primary Müller cells culture. (A) Cx43 protein expression is significantly decreased in siDHAPAT-transfected primary Müller cells. (B) Reverse transcription quantitative PCR (RT-qPCR) indicates that siDHAPAT transfection significantly downregulated Gja1 expression. (C,D) Western-blot analyses show a significant increase in the phosphorylation status of Cx43 on its S368 residue (C) but not on its Y265 residue (D) . n = 6–8 cell cultures. Mann & Whitney test. * p < 0.05; ** p < 0.01. Data shown as mean ± SEM.

Article Snippet: Primary antibodies used were 1:1,000 rabbit anti DHAPAT (ref 14931-1-AP, ProteinTech, United Kingdom), mouse anti-Cx43 at 1:1,000 dilution (ref 610062, BD Biosciences, United States), 1:1,000 mouse anti-pCx43 (S368) (ref 52559, Cell Signaling Technology, Netherlands), 1:1,000 rabbit anti-pCx43 (Y265) (ref ab193373, Abcam, United Kingdom), rabbit anti GFAP (1:1,000 dilution, ref sc-6171-R, Santa Cruz Biotechnology, United States), mouse anti β -tubulin (1:2000 dilution, ref 32–26,000, ThermoFisher) and rabbit anti β-actin (1:2000 dilution, ref A2066, Sigma-Aldrich).

Techniques: Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Residue, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Diazoxide Improves Mitochondrial Connexin 43 Expression in a Mouse Model of Doxorubicin-Induced Cardiotoxicity

doi: 10.3390/ijms19030757

Figure Lengend Snippet: Effect of DOXO or DZX or combined DZX+DOXO treatment on Cx43 ( A ) and pCx43 ( B ) amount. Mice received a single administration (1th group), two administrations (2nd group), or three administrations (3rd group) of DOXO (10 mg/kg; i.p.) or DZX (20 mg/kg; i.p.) or combined DZX+DOXO treatment and Cx43 and pCx43 amount was detected by Western blot analysis of tissue homogenates from mice; GAPDH amount was used as loading control. Values were expressed as mean ± S.E.M. from at least three independent experiments each performed in duplicate. Data were analyzed by Student’s t -test. * p < 0.05 vs. control; # p < 0.05 DZX+DOXO vs. DOXO.

Article Snippet: Membrane strips were blocked for 1h at room temperature in blocking solution (NaCl/TRIS, 0.1% Tween 20 ( v / v ), 5% powdered milk ( w / v ) and then blotted with primary antibody anti-Cx43 (Sigma, 1:8000), anti-pCx43 phosphorylate on Ser368 (Santa Cruz, 1:250), anti-sarco/endoplasmic reticulum Ca2 + -ATP ase (SERCAII; Santa Cruz, 1:250), anti-phospholamban (PLB; Santa Cruz, 1:250), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz, 1:1000, used as loading control) overnight at 4 °C.

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Diazoxide Improves Mitochondrial Connexin 43 Expression in a Mouse Model of Doxorubicin-Induced Cardiotoxicity

doi: 10.3390/ijms19030757

Figure Lengend Snippet: Effect of DOXO or DZX or combined DZX+DOXO treatment on mitochondrial amount of Cx43 ( A ) and mitoCx43 phosphorylated ( B ). Mice received a single administration (1th group), two administrations (2nd group), or three administrations (3rd group) of DOXO (10 mg/kg; i.p.) or DZX (20 mg/kg; i.p.) or combined DZX+DOXO treatment and amounts of mitoCx43 and mitoCx43 phosphorylated (mpCx43) were detected by Western blot analysis into tissue homogenates from mice; TOM20 protein amount was used as loading control. Values were expressed as mean ± S.E.M. from at least three independent experiments each performed in duplicate. Data were analyzed by Student’s t -test. * p <0.05, ** p < 0.005 and *** p < 0.001 vs. control. Representative Western blots of Na + /K + ATPase and Ox-Phos Complex II were used as markers of sarcolemma (SF) and mitochondria (MF), respectively, to demonstrate the purity of the mitochondrial extracts ( C ).

Article Snippet: Membrane strips were blocked for 1h at room temperature in blocking solution (NaCl/TRIS, 0.1% Tween 20 ( v / v ), 5% powdered milk ( w / v ) and then blotted with primary antibody anti-Cx43 (Sigma, 1:8000), anti-pCx43 phosphorylate on Ser368 (Santa Cruz, 1:250), anti-sarco/endoplasmic reticulum Ca2 + -ATP ase (SERCAII; Santa Cruz, 1:250), anti-phospholamban (PLB; Santa Cruz, 1:250), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz, 1:1000, used as loading control) overnight at 4 °C.

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Diazoxide Improves Mitochondrial Connexin 43 Expression in a Mouse Model of Doxorubicin-Induced Cardiotoxicity

doi: 10.3390/ijms19030757

Figure Lengend Snippet: Effect of DOXO or DZX or combined DZX+DOXO treatment on Cx43 localization. C57BL/6j mice received a single administration (1st group), two administrations (2nd group), or three administrations (3rd group). Frozen myocardial tissue sections were stained with Anti-Cx43 (green), TOM20 (red) and nucleus with DAPI (blue) and were determined by Immunofluorescence assay at confocal microscopy for mitoCx43 localization. Scale bar 10 µm.

Article Snippet: Membrane strips were blocked for 1h at room temperature in blocking solution (NaCl/TRIS, 0.1% Tween 20 ( v / v ), 5% powdered milk ( w / v ) and then blotted with primary antibody anti-Cx43 (Sigma, 1:8000), anti-pCx43 phosphorylate on Ser368 (Santa Cruz, 1:250), anti-sarco/endoplasmic reticulum Ca2 + -ATP ase (SERCAII; Santa Cruz, 1:250), anti-phospholamban (PLB; Santa Cruz, 1:250), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz, 1:1000, used as loading control) overnight at 4 °C.

Techniques: Staining, Immunofluorescence, Confocal Microscopy