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cx43 ( gja1 ) sirna sc-60008  (Santa Cruz Biotechnology)


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    Structured Review

    Santa Cruz Biotechnology cx43 ( gja1 ) sirna sc-60008
    a Successful knockdown of Sp1 by transfection of primary astrocytes with Sp1 <t>siRNA</t> assessed by immunoblotting (t 4 = 4.548, * p = 0.01 at 24 h, and t 4 = 4.548, * p = 0.009 at 48 h siRNA vs. scrRNA). Representative bands are shown below. b Effects of Sp1 knockdown on OXT-induced prolongation of the longest primary process. Primary astrocytes were transfected with either Sp1 siRNA or a control oligonucleotide (scrRNA) 48 h hrs prior to OXT exposure (500 nM, 10 min). (Interaction: F 1,1106 = 16.51, p < 0.001; * p = 0.012 scrRNA/Veh vs. scrRNA/OXT; * p = 0.031 for siRNA/Veh vs. siRNA/OXT). c Effects of Sp1 knockdown on Gem mRNA expression (t 9 = 2.818, * p = 0.02 vs. scrRNA). d Representative images from Sp1 siRNA or scrRNA-transfected astrocytes subsequently exposed to OXT (500 nM, 10 min) and stained for GFAP (green), DAPI (blue) and Gem (red) showing Sp1-dependency of OXT-induced Gem expression. Scale bar = 10 µm. Quantification of above threshold (Gem+) cells was defined as single cells marked by DAPI/GFAP staining displaying at least one maximum of above threshold Gem fluorescence (interaction: F 1,832 = 22.32, p < 0.001; *** p < 0.001 scrRNA/Veh vs. scrRNA/OXT; scale bar = 30 µm). e Targeting of astrocytes within the rat PVN with AAV6-GFAP::shRNA vectors to induce either Oxtr ( Oxtr shRNA) or Gem ( Gem shRNA) knockdown in a cell type-specific manner. Illustration adapted from Paxinos and Watson (2006) to show anatomical structures of the rat PVN. Representative image taken from the PVN of rats that received bilateral microinfusions of either Oxtr - or Gem- knockdown vector (280 nl per side) 21 d before. Successful transfection is indicated by the expression of the fluorescent reporter mCherry, while cell-type specificity is indicated by double positive immunostaining for mCherry and GFAP (scale bar = 20 µm). f Quantification of perturbance and specificity of AAV6-GFAP::shRNA constructs (n = 3). g Percentage of time spent in open arms of the EPM of rats pre-treated with either Oxtr shRNA or Gem shRNA for astrocyte-specific knockdown of OXTR or Gem, or with scrRNA within the left and right PVN, and locally infused with either Veh or OXT (0.01 nmol/0.5 µl) 10 min prior testing (genotype F 2 , 37 = 0.443, p = 0.639; treatment F 1 , 37 = 0.021, p = 0.885, * p = 0.045 scr/Veh vs. scr/OXT, * p = 0.030 shRNA/Veh vs. shRNA/OXT; n = 6–11/group). h Time spent in the light box of the LDB of rats treated as described above (g), (genotype F 2 , 41 = 3,965, p = 0.027; treatment F 1 , 41 = 6.165, p = 0.017, * p = 0.016 scr/Veh vs. scr/OXT; n = 6–11/group). Data represent mean relative or absolute values +/−SEM.
    Cx43 ( Gja1 ) Sirna Sc 60008, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/cx43+sirna/anti+cx43/pmc12092269-49-20-28
    Average 90 stars, based on 1 article reviews
    cx43 ( gja1 ) sirna sc-60008 - by Bioz Stars, 2026-10
    90/100 stars

    Images

    1) Product Images from "OXTR-mediated signaling in astrocytes contributes to anxiolysis"

    Article Title: OXTR-mediated signaling in astrocytes contributes to anxiolysis

    Journal: Molecular Psychiatry

    doi: 10.1038/s41380-024-02870-5

    a Successful knockdown of Sp1 by transfection of primary astrocytes with Sp1 siRNA assessed by immunoblotting (t 4 = 4.548, * p = 0.01 at 24 h, and t 4 = 4.548, * p = 0.009 at 48 h siRNA vs. scrRNA). Representative bands are shown below. b Effects of Sp1 knockdown on OXT-induced prolongation of the longest primary process. Primary astrocytes were transfected with either Sp1 siRNA or a control oligonucleotide (scrRNA) 48 h hrs prior to OXT exposure (500 nM, 10 min). (Interaction: F 1,1106 = 16.51, p < 0.001; * p = 0.012 scrRNA/Veh vs. scrRNA/OXT; * p = 0.031 for siRNA/Veh vs. siRNA/OXT). c Effects of Sp1 knockdown on Gem mRNA expression (t 9 = 2.818, * p = 0.02 vs. scrRNA). d Representative images from Sp1 siRNA or scrRNA-transfected astrocytes subsequently exposed to OXT (500 nM, 10 min) and stained for GFAP (green), DAPI (blue) and Gem (red) showing Sp1-dependency of OXT-induced Gem expression. Scale bar = 10 µm. Quantification of above threshold (Gem+) cells was defined as single cells marked by DAPI/GFAP staining displaying at least one maximum of above threshold Gem fluorescence (interaction: F 1,832 = 22.32, p < 0.001; *** p < 0.001 scrRNA/Veh vs. scrRNA/OXT; scale bar = 30 µm). e Targeting of astrocytes within the rat PVN with AAV6-GFAP::shRNA vectors to induce either Oxtr ( Oxtr shRNA) or Gem ( Gem shRNA) knockdown in a cell type-specific manner. Illustration adapted from Paxinos and Watson (2006) to show anatomical structures of the rat PVN. Representative image taken from the PVN of rats that received bilateral microinfusions of either Oxtr - or Gem- knockdown vector (280 nl per side) 21 d before. Successful transfection is indicated by the expression of the fluorescent reporter mCherry, while cell-type specificity is indicated by double positive immunostaining for mCherry and GFAP (scale bar = 20 µm). f Quantification of perturbance and specificity of AAV6-GFAP::shRNA constructs (n = 3). g Percentage of time spent in open arms of the EPM of rats pre-treated with either Oxtr shRNA or Gem shRNA for astrocyte-specific knockdown of OXTR or Gem, or with scrRNA within the left and right PVN, and locally infused with either Veh or OXT (0.01 nmol/0.5 µl) 10 min prior testing (genotype F 2 , 37 = 0.443, p = 0.639; treatment F 1 , 37 = 0.021, p = 0.885, * p = 0.045 scr/Veh vs. scr/OXT, * p = 0.030 shRNA/Veh vs. shRNA/OXT; n = 6–11/group). h Time spent in the light box of the LDB of rats treated as described above (g), (genotype F 2 , 41 = 3,965, p = 0.027; treatment F 1 , 41 = 6.165, p = 0.017, * p = 0.016 scr/Veh vs. scr/OXT; n = 6–11/group). Data represent mean relative or absolute values +/−SEM.
    Figure Legend Snippet: a Successful knockdown of Sp1 by transfection of primary astrocytes with Sp1 siRNA assessed by immunoblotting (t 4 = 4.548, * p = 0.01 at 24 h, and t 4 = 4.548, * p = 0.009 at 48 h siRNA vs. scrRNA). Representative bands are shown below. b Effects of Sp1 knockdown on OXT-induced prolongation of the longest primary process. Primary astrocytes were transfected with either Sp1 siRNA or a control oligonucleotide (scrRNA) 48 h hrs prior to OXT exposure (500 nM, 10 min). (Interaction: F 1,1106 = 16.51, p < 0.001; * p = 0.012 scrRNA/Veh vs. scrRNA/OXT; * p = 0.031 for siRNA/Veh vs. siRNA/OXT). c Effects of Sp1 knockdown on Gem mRNA expression (t 9 = 2.818, * p = 0.02 vs. scrRNA). d Representative images from Sp1 siRNA or scrRNA-transfected astrocytes subsequently exposed to OXT (500 nM, 10 min) and stained for GFAP (green), DAPI (blue) and Gem (red) showing Sp1-dependency of OXT-induced Gem expression. Scale bar = 10 µm. Quantification of above threshold (Gem+) cells was defined as single cells marked by DAPI/GFAP staining displaying at least one maximum of above threshold Gem fluorescence (interaction: F 1,832 = 22.32, p < 0.001; *** p < 0.001 scrRNA/Veh vs. scrRNA/OXT; scale bar = 30 µm). e Targeting of astrocytes within the rat PVN with AAV6-GFAP::shRNA vectors to induce either Oxtr ( Oxtr shRNA) or Gem ( Gem shRNA) knockdown in a cell type-specific manner. Illustration adapted from Paxinos and Watson (2006) to show anatomical structures of the rat PVN. Representative image taken from the PVN of rats that received bilateral microinfusions of either Oxtr - or Gem- knockdown vector (280 nl per side) 21 d before. Successful transfection is indicated by the expression of the fluorescent reporter mCherry, while cell-type specificity is indicated by double positive immunostaining for mCherry and GFAP (scale bar = 20 µm). f Quantification of perturbance and specificity of AAV6-GFAP::shRNA constructs (n = 3). g Percentage of time spent in open arms of the EPM of rats pre-treated with either Oxtr shRNA or Gem shRNA for astrocyte-specific knockdown of OXTR or Gem, or with scrRNA within the left and right PVN, and locally infused with either Veh or OXT (0.01 nmol/0.5 µl) 10 min prior testing (genotype F 2 , 37 = 0.443, p = 0.639; treatment F 1 , 37 = 0.021, p = 0.885, * p = 0.045 scr/Veh vs. scr/OXT, * p = 0.030 shRNA/Veh vs. shRNA/OXT; n = 6–11/group). h Time spent in the light box of the LDB of rats treated as described above (g), (genotype F 2 , 41 = 3,965, p = 0.027; treatment F 1 , 41 = 6.165, p = 0.017, * p = 0.016 scr/Veh vs. scr/OXT; n = 6–11/group). Data represent mean relative or absolute values +/−SEM.

    Techniques Used: Knockdown, Transfection, Western Blot, Control, Expressing, Staining, Fluorescence, shRNA, Plasmid Preparation, Immunostaining, Construct

    a , b Gem siRNA knockdown prevents OXT-induced prolongation of primary processes ( a ; interaction: F 1,405 = 10.58, p < 0.001; ** p = 0.008 scRNA/Veh vs. scRNA/OXT; * p = 0.016 siRNA/OXT vs. scrRNA/Veh) and increase in primary process number ( b ; interaction: F 1,491 = 3.204, p = 0.074; p = 0.09 for scRNA/Veh vs. scRNA/OXT; *** p < 0.001 siRNA-pretreated vs. scRNA-pretreated/OXT astrocytes). Cells were transfected with either Gem siRNA or a control oligonucleotide (scrRNA) 48 h prior to OXT (500 nM, 10 min). Representative Western blot bands of successful knockdown (scRNA/Veh vs. siRNA/Veh) are shown below. c Length of longest primary process after exposure to the ROCK-inhibitor y-27632 (1 µM, 30 min) in primary astrocytes transfected with Gem siRNA ( U = 5936, ** p = 0.009). d Phalloidin/F-actin immunofluorescence after Gem knockdown and subsequent OXT exposure (500 nM, 10 min; interaction: F 1,37 = 0.588, p = 0.448; ** p = 0.003 scrRNA/Veh vs. scrRNA/OXT, p = 0.063 siRNA/Veh vs. siRNA/OXT). e Relative phosphorylation of MYPT at Thr696 following Gem knockdown and subsequent OXT stimulation (interaction: F 1,12 = 15.42, p = 0.006; ** p = 0.003 siRNA/Veh vs. siRNA/OXT). Representative Western blot bands are shown below. f Absolute area of lucifer yellow diffusion (dye spread experiments) after Gem knockdown and subsequent OXT exposure (interaction: F 1,59 = 9.424, p = 0.003; * p = 0.041 scrRNA/Veh vs. scrRNA/OXT). g Gem protein levels in primary astrocytes transfected with either EGFP control plasmid or Gem overexpression (OE) plasmid indicating successful Gem OE after 72 h (t 8 = 3.137, * p = 0.014). h – j Effects of Gem OE on the cytoskeleton of astrocytes reflected by increased process length ( h ; interaction: F 1,354 = 23.64, p < 0.001; *** p < 0.001 EGFP/Veh vs. Gem OE/Veh and vs. EGFP/OXT) and process number ( J; U = 1881, ** p = 0.002). Representative images taken from EGFP-expressing control or Gem OE cells stained for DAPI (blue) and GFAP (red) ( I ; scale bar = 50 µm). k Representative images of phalloidin staining in EGFP vs. Gem OE -transfected cells. Scale bar = 20 µm. l Phalloidin/F-actin immunofluorescence intensity in EGFP vs. Gem OE -transfected cells (t 14 = 4.467, *** p < 0.001). m pMYPT/Thr696 phosphorylation levels reflecting ROCK activity in EGFP vs. Gem OE -expressing cells (t 8 = 2.792, * p = 0.026). n Representative images of scrape-loading dye transfer experiments in EGFP vs. Gem OE -transfected cells. Scale bar = 100 µm; biocytin-AlexaFluor594 streptavidin staining pseudocolored in green. o Degree of GJIC in EGFP vs. Gem OE -transfected cells followed by stimulation with OXT (500 nM, 10 min) (interaction: F 1,44 = 7.450, p = 0.009; *** p < 0.001 EGFP/Veh vs. Gem OE/Veh and vs. Gem OE/OXT; * p = 0.018 EGFP/Veh vs. EGFP/OXT). Data represent mean relative or absolute values +/−SEM for normally distributed data and median +/−min/max values for non-normally distributed data.
    Figure Legend Snippet: a , b Gem siRNA knockdown prevents OXT-induced prolongation of primary processes ( a ; interaction: F 1,405 = 10.58, p < 0.001; ** p = 0.008 scRNA/Veh vs. scRNA/OXT; * p = 0.016 siRNA/OXT vs. scrRNA/Veh) and increase in primary process number ( b ; interaction: F 1,491 = 3.204, p = 0.074; p = 0.09 for scRNA/Veh vs. scRNA/OXT; *** p < 0.001 siRNA-pretreated vs. scRNA-pretreated/OXT astrocytes). Cells were transfected with either Gem siRNA or a control oligonucleotide (scrRNA) 48 h prior to OXT (500 nM, 10 min). Representative Western blot bands of successful knockdown (scRNA/Veh vs. siRNA/Veh) are shown below. c Length of longest primary process after exposure to the ROCK-inhibitor y-27632 (1 µM, 30 min) in primary astrocytes transfected with Gem siRNA ( U = 5936, ** p = 0.009). d Phalloidin/F-actin immunofluorescence after Gem knockdown and subsequent OXT exposure (500 nM, 10 min; interaction: F 1,37 = 0.588, p = 0.448; ** p = 0.003 scrRNA/Veh vs. scrRNA/OXT, p = 0.063 siRNA/Veh vs. siRNA/OXT). e Relative phosphorylation of MYPT at Thr696 following Gem knockdown and subsequent OXT stimulation (interaction: F 1,12 = 15.42, p = 0.006; ** p = 0.003 siRNA/Veh vs. siRNA/OXT). Representative Western blot bands are shown below. f Absolute area of lucifer yellow diffusion (dye spread experiments) after Gem knockdown and subsequent OXT exposure (interaction: F 1,59 = 9.424, p = 0.003; * p = 0.041 scrRNA/Veh vs. scrRNA/OXT). g Gem protein levels in primary astrocytes transfected with either EGFP control plasmid or Gem overexpression (OE) plasmid indicating successful Gem OE after 72 h (t 8 = 3.137, * p = 0.014). h – j Effects of Gem OE on the cytoskeleton of astrocytes reflected by increased process length ( h ; interaction: F 1,354 = 23.64, p < 0.001; *** p < 0.001 EGFP/Veh vs. Gem OE/Veh and vs. EGFP/OXT) and process number ( J; U = 1881, ** p = 0.002). Representative images taken from EGFP-expressing control or Gem OE cells stained for DAPI (blue) and GFAP (red) ( I ; scale bar = 50 µm). k Representative images of phalloidin staining in EGFP vs. Gem OE -transfected cells. Scale bar = 20 µm. l Phalloidin/F-actin immunofluorescence intensity in EGFP vs. Gem OE -transfected cells (t 14 = 4.467, *** p < 0.001). m pMYPT/Thr696 phosphorylation levels reflecting ROCK activity in EGFP vs. Gem OE -expressing cells (t 8 = 2.792, * p = 0.026). n Representative images of scrape-loading dye transfer experiments in EGFP vs. Gem OE -transfected cells. Scale bar = 100 µm; biocytin-AlexaFluor594 streptavidin staining pseudocolored in green. o Degree of GJIC in EGFP vs. Gem OE -transfected cells followed by stimulation with OXT (500 nM, 10 min) (interaction: F 1,44 = 7.450, p = 0.009; *** p < 0.001 EGFP/Veh vs. Gem OE/Veh and vs. Gem OE/OXT; * p = 0.018 EGFP/Veh vs. EGFP/OXT). Data represent mean relative or absolute values +/−SEM for normally distributed data and median +/−min/max values for non-normally distributed data.

    Techniques Used: Knockdown, Transfection, Control, Western Blot, Immunofluorescence, Phospho-proteomics, Diffusion-based Assay, Plasmid Preparation, Over Expression, Expressing, Staining, Activity Assay

    a Representative images taken from mouse acute hippocampal slices treated with either 500 nM OXT or Veh for 10 min. Scale bar = 10 µm. b – d Domain area, process length and process number of astrocytes in acute hippocampal slice preparations from either wildtype (WT; b ), Cx30 knockout ( c ) or Cx43 knockout ( d ) mice treated with OXT or Veh (treatment: domain area: t 14 = 2.016, p = 0.063 WT; t 19 = 3.900, *** p = 0.001 Cx30KO; process length: U = 6, * p = 0.02 WT; t 22 = 2.183, * p = 0.04 Cx30KO; process number: t 13 = 2.321, * p = 0.037 WT; t 19 = 3.456, ** p = 0.003 Cx30KO). e Validation of successful Cx43 knockdown in rat primary astrocytes by means of immunoblotting (t 7 = 4.319, ** p = 0.004). Representative Western blot bands are shown below. f Quantification of longest primary process in cells transfected with either Gja1 siRNA or a control oligonucleotide (scrRNA) and subsequent administration of OXT (interaction: F 1,680 = 3.617, p = 0.058; ** p = 0.01 scrRNA/Veh vs. scrRNA/OXT). g No effect of Cx43 knockdown on Gem protein quantities. Representative Western blot bands are shown below. h Effect of Cx43 knockdown on phosphorylation of Ezrin/Thr576 reflecting an increase in active Ezrin (t 11 = 2.438, * p = 0.033). Representative Western blot bands are shown below. i Correlation of Cx43 level in Gja1 siRNA transfected cells with Ezrin phosphorylation ( p = 0.046, r 2 = 0.582). Data represent mean relative or absolute values +/−SEM.
    Figure Legend Snippet: a Representative images taken from mouse acute hippocampal slices treated with either 500 nM OXT or Veh for 10 min. Scale bar = 10 µm. b – d Domain area, process length and process number of astrocytes in acute hippocampal slice preparations from either wildtype (WT; b ), Cx30 knockout ( c ) or Cx43 knockout ( d ) mice treated with OXT or Veh (treatment: domain area: t 14 = 2.016, p = 0.063 WT; t 19 = 3.900, *** p = 0.001 Cx30KO; process length: U = 6, * p = 0.02 WT; t 22 = 2.183, * p = 0.04 Cx30KO; process number: t 13 = 2.321, * p = 0.037 WT; t 19 = 3.456, ** p = 0.003 Cx30KO). e Validation of successful Cx43 knockdown in rat primary astrocytes by means of immunoblotting (t 7 = 4.319, ** p = 0.004). Representative Western blot bands are shown below. f Quantification of longest primary process in cells transfected with either Gja1 siRNA or a control oligonucleotide (scrRNA) and subsequent administration of OXT (interaction: F 1,680 = 3.617, p = 0.058; ** p = 0.01 scrRNA/Veh vs. scrRNA/OXT). g No effect of Cx43 knockdown on Gem protein quantities. Representative Western blot bands are shown below. h Effect of Cx43 knockdown on phosphorylation of Ezrin/Thr576 reflecting an increase in active Ezrin (t 11 = 2.438, * p = 0.033). Representative Western blot bands are shown below. i Correlation of Cx43 level in Gja1 siRNA transfected cells with Ezrin phosphorylation ( p = 0.046, r 2 = 0.582). Data represent mean relative or absolute values +/−SEM.

    Techniques Used: Knock-Out, Biomarker Discovery, Knockdown, Western Blot, Transfection, Control, Phospho-proteomics

    Related Articles

    Transfection:

    Article Title: High Glucose Alters Cx43 Expression and Gap Junction Intercellular Communication in Retinal Müller Cells: Promotes Müller Cell and Pericyte Apoptosis
    Article Snippet: .. Cell Transfection With Cx43 siRNA or Cx43 Plasmid To determine the effect of reduced Cx43 expression on communication between Müller cells and pericytes, rMC-1 was transfected with Cx43 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or scramble siRNA (Qiagen, Valencia, CA, USA) as a negative control in the presence of 8 μM transfection reagent (Lipofectin; Invitrogen, Carlsbad, CA, USA). ..

    Article Title: High Glucose Alters Cx43 Expression and Gap Junction Intercellular Communication in Retinal Müller Cells: Promotes Müller Cell and Pericyte Apoptosis
    Article Snippet: .. To determine the effect of reduced Cx43 expression on communication between Müller cells and pericytes, rMC-1 was transfected with Cx43 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or scramble siRNA (Qiagen, Valencia, CA, USA) as a negative control in the presence of 8 μM transfection reagent (Lipofectin; Invitrogen, Carlsbad, CA, USA). ..

    Article Title: Prevention of cisplatin-induced ototoxicity by the inhibition of gap junctional intercellular communication in auditory cells.
    Article Snippet: to cell membranes in heI-OC1 cells.. Interestingly, we found that the inhibition of gap junction activity reduced cisplatin-induced apoptosis of auditory hair cells.. Cx43 siRNaor 18α-Ga-treated heI-OC1 cells showed higher cell viability compared to control heI-OC1 cells during cisplatin treatment; this was also supported by fluorescence recovery after photobleaching studies.

    Article Title: Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress.
    Article Snippet: .. To assess the effect of connexin expression on the formation of myoendothelial gap junctions and HCASMC phenotypic switching, HCAECs were transfected with Cx40 and Cx43 siRNA (catalog no. sc-29276 and sc-43078, respectively; Santa Cruz Biotechnology) or Cx40 DNA (catalog no. sc-401031-ACT; Santa Cruz Biotechnology) according to the manufacturer’s instructions and co-cultured in contact with HCASMCs. .. Proteins were extracted as described previously [26], separated by 10% SDS-PAGE, electrophoretically transferred to PVDF membranes, and probed with the following primary antibodies: anti-MYH15 (to detection MHC; catalog no. sc-103055), anti-Cx40 (catalog no. sc-365107), anti-Cx43 (catalog no. sc271837), and anti-GAPDH (catalog no. sc-365062) (all 1:500 dilution; Santa Cruz Biotechnology), and anti-H1-calponin (catalog no. C2687; Sigma).

    Plasmid Preparation:

    Article Title: High Glucose Alters Cx43 Expression and Gap Junction Intercellular Communication in Retinal Müller Cells: Promotes Müller Cell and Pericyte Apoptosis
    Article Snippet: .. Cell Transfection With Cx43 siRNA or Cx43 Plasmid To determine the effect of reduced Cx43 expression on communication between Müller cells and pericytes, rMC-1 was transfected with Cx43 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or scramble siRNA (Qiagen, Valencia, CA, USA) as a negative control in the presence of 8 μM transfection reagent (Lipofectin; Invitrogen, Carlsbad, CA, USA). ..

    Expressing:

    Article Title: High Glucose Alters Cx43 Expression and Gap Junction Intercellular Communication in Retinal Müller Cells: Promotes Müller Cell and Pericyte Apoptosis
    Article Snippet: .. Cell Transfection With Cx43 siRNA or Cx43 Plasmid To determine the effect of reduced Cx43 expression on communication between Müller cells and pericytes, rMC-1 was transfected with Cx43 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or scramble siRNA (Qiagen, Valencia, CA, USA) as a negative control in the presence of 8 μM transfection reagent (Lipofectin; Invitrogen, Carlsbad, CA, USA). ..

    Article Title: High Glucose Alters Cx43 Expression and Gap Junction Intercellular Communication in Retinal Müller Cells: Promotes Müller Cell and Pericyte Apoptosis
    Article Snippet: .. To determine the effect of reduced Cx43 expression on communication between Müller cells and pericytes, rMC-1 was transfected with Cx43 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or scramble siRNA (Qiagen, Valencia, CA, USA) as a negative control in the presence of 8 μM transfection reagent (Lipofectin; Invitrogen, Carlsbad, CA, USA). ..

    Article Title: Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress.
    Article Snippet: .. To assess the effect of connexin expression on the formation of myoendothelial gap junctions and HCASMC phenotypic switching, HCAECs were transfected with Cx40 and Cx43 siRNA (catalog no. sc-29276 and sc-43078, respectively; Santa Cruz Biotechnology) or Cx40 DNA (catalog no. sc-401031-ACT; Santa Cruz Biotechnology) according to the manufacturer’s instructions and co-cultured in contact with HCASMCs. .. Proteins were extracted as described previously [26], separated by 10% SDS-PAGE, electrophoretically transferred to PVDF membranes, and probed with the following primary antibodies: anti-MYH15 (to detection MHC; catalog no. sc-103055), anti-Cx40 (catalog no. sc-365107), anti-Cx43 (catalog no. sc271837), and anti-GAPDH (catalog no. sc-365062) (all 1:500 dilution; Santa Cruz Biotechnology), and anti-H1-calponin (catalog no. C2687; Sigma).

    Negative Control:

    Article Title: High Glucose Alters Cx43 Expression and Gap Junction Intercellular Communication in Retinal Müller Cells: Promotes Müller Cell and Pericyte Apoptosis
    Article Snippet: .. Cell Transfection With Cx43 siRNA or Cx43 Plasmid To determine the effect of reduced Cx43 expression on communication between Müller cells and pericytes, rMC-1 was transfected with Cx43 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or scramble siRNA (Qiagen, Valencia, CA, USA) as a negative control in the presence of 8 μM transfection reagent (Lipofectin; Invitrogen, Carlsbad, CA, USA). ..

    Article Title: High Glucose Alters Cx43 Expression and Gap Junction Intercellular Communication in Retinal Müller Cells: Promotes Müller Cell and Pericyte Apoptosis
    Article Snippet: .. To determine the effect of reduced Cx43 expression on communication between Müller cells and pericytes, rMC-1 was transfected with Cx43 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or scramble siRNA (Qiagen, Valencia, CA, USA) as a negative control in the presence of 8 μM transfection reagent (Lipofectin; Invitrogen, Carlsbad, CA, USA). ..

    Article Title: Combining TGF-β signal inhibition and connexin43 silencing for iPSC induction from mouse cardiomyocytes
    Article Snippet: CMs were seeded at 2 × 10 5 cells in a 6-cm dish coated with 0.1% gelatin (Sigma) in DMEM supplemented with 10% FBS. .. On day 7, the CMs were switched from DMEM medium to normal mESC growth media (GIBCO) supplemented with 100 U ml −1 leukemia inhibitory factor (LIF, Millipore) containing either SB-431542 (10 μM) (Tocris), Cx43 siRNA (sense: 5′-CAA UUC UUC UUG CCG CAA TT-3′; antisense: 5′-UUG CGG CAA GAA GAA UUG TT-3′) (100 nM) or both compounds, and as controls, the medium containing either SB-431542 (10 μM), nonsense control siRNA (sense: 5′-AAU UCU CCG AAC GUG UCA CGT-3′; antisense: 5′-GUG ACA CGU UCG GAG AAU UTT-3′) (100 nM) or both compounds, and/or both SB-431542 (10 μM) and Cx40 siRNA (Santa Cruz, sc-43079) (100 nM), which was used as a negative control of Cx43 siRNA. .. To confirm the effect of gap junction protein inhibitors on reprogramming of siPSCs from CMs, the medium contained both SB-431542 (10 μM) and Octanol (Sigma-Aldrich) (500 μM) or Doxyl stearic acid (DSA) (Sigma-Aldrich) (50 μM).

    other:

    Article Title: Exendin-4, a glucagon-like peptide receptor agonist, facilitates osteoblast differentiation via connexin43.
    Article Snippet: Purpose To investigate whether exendin-4 (Ex-4), a glucagon-like peptide 1 receptor (GLP-1R) agonist, affects connexin 43 (Cx43) expression in osteoblasts, and determine the specific mechanism underlying Cx43 modulation by Ex-4.. Methods Osteoblast-like MC3T3-E1 cells were treated with Ex-4 with or without GLP-1R antagonist.. We assessed Cx43 expression using RT-PCR, western blotting, and confocal microscopy; visualized intercellular communication using Lucifer yellow dye transfer assay; evaluated osteoblast differentiation using alkaline phosphatase and Alizarin red S (ARS) staining.

    Article Title: High glucose induces dysfunction of airway epithelial barrier through down-regulation of connexin 43.
    Article Snippet: The airway epithelium is a barrier to the inhaled antigens and pathogens.. Connexin 43 (Cx43) has been found to play critical role in maintaining the function of airway epithelial barrier and be involved in the pathogenesis of the diabetic retinal vasculature, diabetes nephropathy and diabetes skin.. Hyperglycemia has been shown to be an independent risk factor for respiratory infections.

    Knockdown:

    Article Title: Prevention of cisplatin-induced ototoxicity by the inhibition of gap junctional intercellular communication in auditory cells.
    Article Snippet: to cell membranes in heI-OC1 cells.. Interestingly, we found that the inhibition of gap junction activity reduced cisplatin-induced apoptosis of auditory hair cells.. Cx43 siRNaor 18α-Ga-treated heI-OC1 cells showed higher cell viability compared to control heI-OC1 cells during cisplatin treatment; this was also supported by fluorescence recovery after photobleaching studies.

    Gene Expression:

    Article Title: Prevention of cisplatin-induced ototoxicity by the inhibition of gap junctional intercellular communication in auditory cells.
    Article Snippet: to cell membranes in heI-OC1 cells.. Interestingly, we found that the inhibition of gap junction activity reduced cisplatin-induced apoptosis of auditory hair cells.. Cx43 siRNaor 18α-Ga-treated heI-OC1 cells showed higher cell viability compared to control heI-OC1 cells during cisplatin treatment; this was also supported by fluorescence recovery after photobleaching studies.

    Control:

    Article Title: Combining TGF-β signal inhibition and connexin43 silencing for iPSC induction from mouse cardiomyocytes
    Article Snippet: CMs were seeded at 2 × 10 5 cells in a 6-cm dish coated with 0.1% gelatin (Sigma) in DMEM supplemented with 10% FBS. .. On day 7, the CMs were switched from DMEM medium to normal mESC growth media (GIBCO) supplemented with 100 U ml −1 leukemia inhibitory factor (LIF, Millipore) containing either SB-431542 (10 μM) (Tocris), Cx43 siRNA (sense: 5′-CAA UUC UUC UUG CCG CAA TT-3′; antisense: 5′-UUG CGG CAA GAA GAA UUG TT-3′) (100 nM) or both compounds, and as controls, the medium containing either SB-431542 (10 μM), nonsense control siRNA (sense: 5′-AAU UCU CCG AAC GUG UCA CGT-3′; antisense: 5′-GUG ACA CGU UCG GAG AAU UTT-3′) (100 nM) or both compounds, and/or both SB-431542 (10 μM) and Cx40 siRNA (Santa Cruz, sc-43079) (100 nM), which was used as a negative control of Cx43 siRNA. .. To confirm the effect of gap junction protein inhibitors on reprogramming of siPSCs from CMs, the medium contained both SB-431542 (10 μM) and Octanol (Sigma-Aldrich) (500 μM) or Doxyl stearic acid (DSA) (Sigma-Aldrich) (50 μM).

    Article Title: Prevention of cisplatin-induced ototoxicity by the inhibition of gap junctional intercellular communication in auditory cells.
    Article Snippet: to cell membranes in heI-OC1 cells.. Interestingly, we found that the inhibition of gap junction activity reduced cisplatin-induced apoptosis of auditory hair cells.. Cx43 siRNaor 18α-Ga-treated heI-OC1 cells showed higher cell viability compared to control heI-OC1 cells during cisplatin treatment; this was also supported by fluorescence recovery after photobleaching studies.



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    a Successful knockdown of Sp1 by transfection of primary astrocytes with Sp1 siRNA assessed by immunoblotting (t 4 = 4.548, * p = 0.01 at 24 h, and t 4 = 4.548, * p = 0.009 at 48 h siRNA vs. scrRNA). Representative bands are shown below. b Effects of Sp1 knockdown on OXT-induced prolongation of the longest primary process. Primary astrocytes were transfected with either Sp1 siRNA or a control oligonucleotide (scrRNA) 48 h hrs prior to OXT exposure (500 nM, 10 min). (Interaction: F 1,1106 = 16.51, p < 0.001; * p = 0.012 scrRNA/Veh vs. scrRNA/OXT; * p = 0.031 for siRNA/Veh vs. siRNA/OXT). c Effects of Sp1 knockdown on Gem mRNA expression (t 9 = 2.818, * p = 0.02 vs. scrRNA). d Representative images from Sp1 siRNA or scrRNA-transfected astrocytes subsequently exposed to OXT (500 nM, 10 min) and stained for GFAP (green), DAPI (blue) and Gem (red) showing Sp1-dependency of OXT-induced Gem expression. Scale bar = 10 µm. Quantification of above threshold (Gem+) cells was defined as single cells marked by DAPI/GFAP staining displaying at least one maximum of above threshold Gem fluorescence (interaction: F 1,832 = 22.32, p < 0.001; *** p < 0.001 scrRNA/Veh vs. scrRNA/OXT; scale bar = 30 µm). e Targeting of astrocytes within the rat PVN with AAV6-GFAP::shRNA vectors to induce either Oxtr ( Oxtr shRNA) or Gem ( Gem shRNA) knockdown in a cell type-specific manner. Illustration adapted from Paxinos and Watson (2006) to show anatomical structures of the rat PVN. Representative image taken from the PVN of rats that received bilateral microinfusions of either Oxtr - or Gem- knockdown vector (280 nl per side) 21 d before. Successful transfection is indicated by the expression of the fluorescent reporter mCherry, while cell-type specificity is indicated by double positive immunostaining for mCherry and GFAP (scale bar = 20 µm). f Quantification of perturbance and specificity of AAV6-GFAP::shRNA constructs (n = 3). g Percentage of time spent in open arms of the EPM of rats pre-treated with either Oxtr shRNA or Gem shRNA for astrocyte-specific knockdown of OXTR or Gem, or with scrRNA within the left and right PVN, and locally infused with either Veh or OXT (0.01 nmol/0.5 µl) 10 min prior testing (genotype F 2 , 37 = 0.443, p = 0.639; treatment F 1 , 37 = 0.021, p = 0.885, * p = 0.045 scr/Veh vs. scr/OXT, * p = 0.030 shRNA/Veh vs. shRNA/OXT; n = 6–11/group). h Time spent in the light box of the LDB of rats treated as described above (g), (genotype F 2 , 41 = 3,965, p = 0.027; treatment F 1 , 41 = 6.165, p = 0.017, * p = 0.016 scr/Veh vs. scr/OXT; n = 6–11/group). Data represent mean relative or absolute values +/−SEM.

    Journal: Molecular Psychiatry

    Article Title: OXTR-mediated signaling in astrocytes contributes to anxiolysis

    doi: 10.1038/s41380-024-02870-5

    Figure Lengend Snippet: a Successful knockdown of Sp1 by transfection of primary astrocytes with Sp1 siRNA assessed by immunoblotting (t 4 = 4.548, * p = 0.01 at 24 h, and t 4 = 4.548, * p = 0.009 at 48 h siRNA vs. scrRNA). Representative bands are shown below. b Effects of Sp1 knockdown on OXT-induced prolongation of the longest primary process. Primary astrocytes were transfected with either Sp1 siRNA or a control oligonucleotide (scrRNA) 48 h hrs prior to OXT exposure (500 nM, 10 min). (Interaction: F 1,1106 = 16.51, p < 0.001; * p = 0.012 scrRNA/Veh vs. scrRNA/OXT; * p = 0.031 for siRNA/Veh vs. siRNA/OXT). c Effects of Sp1 knockdown on Gem mRNA expression (t 9 = 2.818, * p = 0.02 vs. scrRNA). d Representative images from Sp1 siRNA or scrRNA-transfected astrocytes subsequently exposed to OXT (500 nM, 10 min) and stained for GFAP (green), DAPI (blue) and Gem (red) showing Sp1-dependency of OXT-induced Gem expression. Scale bar = 10 µm. Quantification of above threshold (Gem+) cells was defined as single cells marked by DAPI/GFAP staining displaying at least one maximum of above threshold Gem fluorescence (interaction: F 1,832 = 22.32, p < 0.001; *** p < 0.001 scrRNA/Veh vs. scrRNA/OXT; scale bar = 30 µm). e Targeting of astrocytes within the rat PVN with AAV6-GFAP::shRNA vectors to induce either Oxtr ( Oxtr shRNA) or Gem ( Gem shRNA) knockdown in a cell type-specific manner. Illustration adapted from Paxinos and Watson (2006) to show anatomical structures of the rat PVN. Representative image taken from the PVN of rats that received bilateral microinfusions of either Oxtr - or Gem- knockdown vector (280 nl per side) 21 d before. Successful transfection is indicated by the expression of the fluorescent reporter mCherry, while cell-type specificity is indicated by double positive immunostaining for mCherry and GFAP (scale bar = 20 µm). f Quantification of perturbance and specificity of AAV6-GFAP::shRNA constructs (n = 3). g Percentage of time spent in open arms of the EPM of rats pre-treated with either Oxtr shRNA or Gem shRNA for astrocyte-specific knockdown of OXTR or Gem, or with scrRNA within the left and right PVN, and locally infused with either Veh or OXT (0.01 nmol/0.5 µl) 10 min prior testing (genotype F 2 , 37 = 0.443, p = 0.639; treatment F 1 , 37 = 0.021, p = 0.885, * p = 0.045 scr/Veh vs. scr/OXT, * p = 0.030 shRNA/Veh vs. shRNA/OXT; n = 6–11/group). h Time spent in the light box of the LDB of rats treated as described above (g), (genotype F 2 , 41 = 3,965, p = 0.027; treatment F 1 , 41 = 6.165, p = 0.017, * p = 0.016 scr/Veh vs. scr/OXT; n = 6–11/group). Data represent mean relative or absolute values +/−SEM.

    Article Snippet: In order to study the involvement of Cx43, Sp1 or Gem in astrocytic OXTR signaling, astrocytes were transfected with respective siRNA oligonucleotides (Cx43 ( Gja1 ) siRNA, sc-60008, Santa Cruz Biotechnology, Dallas, USA; Sp1 siRNA, Santa Cruz, sc-61895; Gem siRNA, Origene, Rockville, USA, SR507514) or a Gem overexpression plasmid (Supplementary Fig. VectorBuilder) by electroporation (NeonTM Transfection System, ThermoFisher; MPK5000).

    Techniques: Knockdown, Transfection, Western Blot, Control, Expressing, Staining, Fluorescence, shRNA, Plasmid Preparation, Immunostaining, Construct

    a , b Gem siRNA knockdown prevents OXT-induced prolongation of primary processes ( a ; interaction: F 1,405 = 10.58, p < 0.001; ** p = 0.008 scRNA/Veh vs. scRNA/OXT; * p = 0.016 siRNA/OXT vs. scrRNA/Veh) and increase in primary process number ( b ; interaction: F 1,491 = 3.204, p = 0.074; p = 0.09 for scRNA/Veh vs. scRNA/OXT; *** p < 0.001 siRNA-pretreated vs. scRNA-pretreated/OXT astrocytes). Cells were transfected with either Gem siRNA or a control oligonucleotide (scrRNA) 48 h prior to OXT (500 nM, 10 min). Representative Western blot bands of successful knockdown (scRNA/Veh vs. siRNA/Veh) are shown below. c Length of longest primary process after exposure to the ROCK-inhibitor y-27632 (1 µM, 30 min) in primary astrocytes transfected with Gem siRNA ( U = 5936, ** p = 0.009). d Phalloidin/F-actin immunofluorescence after Gem knockdown and subsequent OXT exposure (500 nM, 10 min; interaction: F 1,37 = 0.588, p = 0.448; ** p = 0.003 scrRNA/Veh vs. scrRNA/OXT, p = 0.063 siRNA/Veh vs. siRNA/OXT). e Relative phosphorylation of MYPT at Thr696 following Gem knockdown and subsequent OXT stimulation (interaction: F 1,12 = 15.42, p = 0.006; ** p = 0.003 siRNA/Veh vs. siRNA/OXT). Representative Western blot bands are shown below. f Absolute area of lucifer yellow diffusion (dye spread experiments) after Gem knockdown and subsequent OXT exposure (interaction: F 1,59 = 9.424, p = 0.003; * p = 0.041 scrRNA/Veh vs. scrRNA/OXT). g Gem protein levels in primary astrocytes transfected with either EGFP control plasmid or Gem overexpression (OE) plasmid indicating successful Gem OE after 72 h (t 8 = 3.137, * p = 0.014). h – j Effects of Gem OE on the cytoskeleton of astrocytes reflected by increased process length ( h ; interaction: F 1,354 = 23.64, p < 0.001; *** p < 0.001 EGFP/Veh vs. Gem OE/Veh and vs. EGFP/OXT) and process number ( J; U = 1881, ** p = 0.002). Representative images taken from EGFP-expressing control or Gem OE cells stained for DAPI (blue) and GFAP (red) ( I ; scale bar = 50 µm). k Representative images of phalloidin staining in EGFP vs. Gem OE -transfected cells. Scale bar = 20 µm. l Phalloidin/F-actin immunofluorescence intensity in EGFP vs. Gem OE -transfected cells (t 14 = 4.467, *** p < 0.001). m pMYPT/Thr696 phosphorylation levels reflecting ROCK activity in EGFP vs. Gem OE -expressing cells (t 8 = 2.792, * p = 0.026). n Representative images of scrape-loading dye transfer experiments in EGFP vs. Gem OE -transfected cells. Scale bar = 100 µm; biocytin-AlexaFluor594 streptavidin staining pseudocolored in green. o Degree of GJIC in EGFP vs. Gem OE -transfected cells followed by stimulation with OXT (500 nM, 10 min) (interaction: F 1,44 = 7.450, p = 0.009; *** p < 0.001 EGFP/Veh vs. Gem OE/Veh and vs. Gem OE/OXT; * p = 0.018 EGFP/Veh vs. EGFP/OXT). Data represent mean relative or absolute values +/−SEM for normally distributed data and median +/−min/max values for non-normally distributed data.

    Journal: Molecular Psychiatry

    Article Title: OXTR-mediated signaling in astrocytes contributes to anxiolysis

    doi: 10.1038/s41380-024-02870-5

    Figure Lengend Snippet: a , b Gem siRNA knockdown prevents OXT-induced prolongation of primary processes ( a ; interaction: F 1,405 = 10.58, p < 0.001; ** p = 0.008 scRNA/Veh vs. scRNA/OXT; * p = 0.016 siRNA/OXT vs. scrRNA/Veh) and increase in primary process number ( b ; interaction: F 1,491 = 3.204, p = 0.074; p = 0.09 for scRNA/Veh vs. scRNA/OXT; *** p < 0.001 siRNA-pretreated vs. scRNA-pretreated/OXT astrocytes). Cells were transfected with either Gem siRNA or a control oligonucleotide (scrRNA) 48 h prior to OXT (500 nM, 10 min). Representative Western blot bands of successful knockdown (scRNA/Veh vs. siRNA/Veh) are shown below. c Length of longest primary process after exposure to the ROCK-inhibitor y-27632 (1 µM, 30 min) in primary astrocytes transfected with Gem siRNA ( U = 5936, ** p = 0.009). d Phalloidin/F-actin immunofluorescence after Gem knockdown and subsequent OXT exposure (500 nM, 10 min; interaction: F 1,37 = 0.588, p = 0.448; ** p = 0.003 scrRNA/Veh vs. scrRNA/OXT, p = 0.063 siRNA/Veh vs. siRNA/OXT). e Relative phosphorylation of MYPT at Thr696 following Gem knockdown and subsequent OXT stimulation (interaction: F 1,12 = 15.42, p = 0.006; ** p = 0.003 siRNA/Veh vs. siRNA/OXT). Representative Western blot bands are shown below. f Absolute area of lucifer yellow diffusion (dye spread experiments) after Gem knockdown and subsequent OXT exposure (interaction: F 1,59 = 9.424, p = 0.003; * p = 0.041 scrRNA/Veh vs. scrRNA/OXT). g Gem protein levels in primary astrocytes transfected with either EGFP control plasmid or Gem overexpression (OE) plasmid indicating successful Gem OE after 72 h (t 8 = 3.137, * p = 0.014). h – j Effects of Gem OE on the cytoskeleton of astrocytes reflected by increased process length ( h ; interaction: F 1,354 = 23.64, p < 0.001; *** p < 0.001 EGFP/Veh vs. Gem OE/Veh and vs. EGFP/OXT) and process number ( J; U = 1881, ** p = 0.002). Representative images taken from EGFP-expressing control or Gem OE cells stained for DAPI (blue) and GFAP (red) ( I ; scale bar = 50 µm). k Representative images of phalloidin staining in EGFP vs. Gem OE -transfected cells. Scale bar = 20 µm. l Phalloidin/F-actin immunofluorescence intensity in EGFP vs. Gem OE -transfected cells (t 14 = 4.467, *** p < 0.001). m pMYPT/Thr696 phosphorylation levels reflecting ROCK activity in EGFP vs. Gem OE -expressing cells (t 8 = 2.792, * p = 0.026). n Representative images of scrape-loading dye transfer experiments in EGFP vs. Gem OE -transfected cells. Scale bar = 100 µm; biocytin-AlexaFluor594 streptavidin staining pseudocolored in green. o Degree of GJIC in EGFP vs. Gem OE -transfected cells followed by stimulation with OXT (500 nM, 10 min) (interaction: F 1,44 = 7.450, p = 0.009; *** p < 0.001 EGFP/Veh vs. Gem OE/Veh and vs. Gem OE/OXT; * p = 0.018 EGFP/Veh vs. EGFP/OXT). Data represent mean relative or absolute values +/−SEM for normally distributed data and median +/−min/max values for non-normally distributed data.

    Article Snippet: In order to study the involvement of Cx43, Sp1 or Gem in astrocytic OXTR signaling, astrocytes were transfected with respective siRNA oligonucleotides (Cx43 ( Gja1 ) siRNA, sc-60008, Santa Cruz Biotechnology, Dallas, USA; Sp1 siRNA, Santa Cruz, sc-61895; Gem siRNA, Origene, Rockville, USA, SR507514) or a Gem overexpression plasmid (Supplementary Fig. VectorBuilder) by electroporation (NeonTM Transfection System, ThermoFisher; MPK5000).

    Techniques: Knockdown, Transfection, Control, Western Blot, Immunofluorescence, Phospho-proteomics, Diffusion-based Assay, Plasmid Preparation, Over Expression, Expressing, Staining, Activity Assay

    a Representative images taken from mouse acute hippocampal slices treated with either 500 nM OXT or Veh for 10 min. Scale bar = 10 µm. b – d Domain area, process length and process number of astrocytes in acute hippocampal slice preparations from either wildtype (WT; b ), Cx30 knockout ( c ) or Cx43 knockout ( d ) mice treated with OXT or Veh (treatment: domain area: t 14 = 2.016, p = 0.063 WT; t 19 = 3.900, *** p = 0.001 Cx30KO; process length: U = 6, * p = 0.02 WT; t 22 = 2.183, * p = 0.04 Cx30KO; process number: t 13 = 2.321, * p = 0.037 WT; t 19 = 3.456, ** p = 0.003 Cx30KO). e Validation of successful Cx43 knockdown in rat primary astrocytes by means of immunoblotting (t 7 = 4.319, ** p = 0.004). Representative Western blot bands are shown below. f Quantification of longest primary process in cells transfected with either Gja1 siRNA or a control oligonucleotide (scrRNA) and subsequent administration of OXT (interaction: F 1,680 = 3.617, p = 0.058; ** p = 0.01 scrRNA/Veh vs. scrRNA/OXT). g No effect of Cx43 knockdown on Gem protein quantities. Representative Western blot bands are shown below. h Effect of Cx43 knockdown on phosphorylation of Ezrin/Thr576 reflecting an increase in active Ezrin (t 11 = 2.438, * p = 0.033). Representative Western blot bands are shown below. i Correlation of Cx43 level in Gja1 siRNA transfected cells with Ezrin phosphorylation ( p = 0.046, r 2 = 0.582). Data represent mean relative or absolute values +/−SEM.

    Journal: Molecular Psychiatry

    Article Title: OXTR-mediated signaling in astrocytes contributes to anxiolysis

    doi: 10.1038/s41380-024-02870-5

    Figure Lengend Snippet: a Representative images taken from mouse acute hippocampal slices treated with either 500 nM OXT or Veh for 10 min. Scale bar = 10 µm. b – d Domain area, process length and process number of astrocytes in acute hippocampal slice preparations from either wildtype (WT; b ), Cx30 knockout ( c ) or Cx43 knockout ( d ) mice treated with OXT or Veh (treatment: domain area: t 14 = 2.016, p = 0.063 WT; t 19 = 3.900, *** p = 0.001 Cx30KO; process length: U = 6, * p = 0.02 WT; t 22 = 2.183, * p = 0.04 Cx30KO; process number: t 13 = 2.321, * p = 0.037 WT; t 19 = 3.456, ** p = 0.003 Cx30KO). e Validation of successful Cx43 knockdown in rat primary astrocytes by means of immunoblotting (t 7 = 4.319, ** p = 0.004). Representative Western blot bands are shown below. f Quantification of longest primary process in cells transfected with either Gja1 siRNA or a control oligonucleotide (scrRNA) and subsequent administration of OXT (interaction: F 1,680 = 3.617, p = 0.058; ** p = 0.01 scrRNA/Veh vs. scrRNA/OXT). g No effect of Cx43 knockdown on Gem protein quantities. Representative Western blot bands are shown below. h Effect of Cx43 knockdown on phosphorylation of Ezrin/Thr576 reflecting an increase in active Ezrin (t 11 = 2.438, * p = 0.033). Representative Western blot bands are shown below. i Correlation of Cx43 level in Gja1 siRNA transfected cells with Ezrin phosphorylation ( p = 0.046, r 2 = 0.582). Data represent mean relative or absolute values +/−SEM.

    Article Snippet: In order to study the involvement of Cx43, Sp1 or Gem in astrocytic OXTR signaling, astrocytes were transfected with respective siRNA oligonucleotides (Cx43 ( Gja1 ) siRNA, sc-60008, Santa Cruz Biotechnology, Dallas, USA; Sp1 siRNA, Santa Cruz, sc-61895; Gem siRNA, Origene, Rockville, USA, SR507514) or a Gem overexpression plasmid (Supplementary Fig. VectorBuilder) by electroporation (NeonTM Transfection System, ThermoFisher; MPK5000).

    Techniques: Knock-Out, Biomarker Discovery, Knockdown, Western Blot, Transfection, Control, Phospho-proteomics

    ( A ) Gap junction intercellular coupling (GJIC) was measured by a modified ‘parachute assay’ where calcein-loaded donors are dropped onto a monolayer of acceptors of either the same (homocellular) or different (heterocellular) cell type, and calcein transfer is measured as a linear increase in fluorescent acceptor/donor ratio over time. Scale bars are 50 µM. ( B ) GJIC between eutopic ESCs decreased progressively with the disease, reaching significance in Endometriosis III-IV patients. ( C ) Heterocellular ESC-PMC GJIC was induced compared to ESC homocellular coupling and this increased with disease progression to 2–4.5-fold in Endometriosis III-IV patients. ( D–K ) Immunocytochemical staining of Cx43 (red), with cell outlines from phase (yellow) or membrite labeling (blue) superimposed in the lower panels. ESCs alone showed some labeling between cells (arrowheads), but most Cx43 was in intracellular pools ( D–G ). By contrast, in mixed cultures of PMCs with ESCs [labeled with cell tracker green (*)] there is less intracellular Cx43 labeing, and punctate staining of GJs between cells is increased in frequency [Arrowheads: ESC-ESC (green in yellow); PMC-PMC (hollow yellow); ESC-PMC (solid yellow)] ( H–K ). Nuclei are stained with DAPI. Scale bars are 10 μm. Number of repeats for each condition are shown in B and C, with 8–10 patients in each group. Significance based on two-tailed t-tests. Full data in . Figure 3—source data 1. Original data tables and statistical analyses for .

    Journal: eLife

    Article Title: Hypersensitive intercellular responses of endometrial stromal cells drive invasion in endometriosis

    doi: 10.7554/eLife.94778

    Figure Lengend Snippet: ( A ) Gap junction intercellular coupling (GJIC) was measured by a modified ‘parachute assay’ where calcein-loaded donors are dropped onto a monolayer of acceptors of either the same (homocellular) or different (heterocellular) cell type, and calcein transfer is measured as a linear increase in fluorescent acceptor/donor ratio over time. Scale bars are 50 µM. ( B ) GJIC between eutopic ESCs decreased progressively with the disease, reaching significance in Endometriosis III-IV patients. ( C ) Heterocellular ESC-PMC GJIC was induced compared to ESC homocellular coupling and this increased with disease progression to 2–4.5-fold in Endometriosis III-IV patients. ( D–K ) Immunocytochemical staining of Cx43 (red), with cell outlines from phase (yellow) or membrite labeling (blue) superimposed in the lower panels. ESCs alone showed some labeling between cells (arrowheads), but most Cx43 was in intracellular pools ( D–G ). By contrast, in mixed cultures of PMCs with ESCs [labeled with cell tracker green (*)] there is less intracellular Cx43 labeing, and punctate staining of GJs between cells is increased in frequency [Arrowheads: ESC-ESC (green in yellow); PMC-PMC (hollow yellow); ESC-PMC (solid yellow)] ( H–K ). Nuclei are stained with DAPI. Scale bars are 10 μm. Number of repeats for each condition are shown in B and C, with 8–10 patients in each group. Significance based on two-tailed t-tests. Full data in . Figure 3—source data 1. Original data tables and statistical analyses for .

    Article Snippet: In other experiments (data not shown), Cx43 KD was achieved by a 24- hr pre-treatment of the LP9 monolayer with a combination of two siRNAs to Cx43 (10 pmoles/well or 5 nM final concentration) - Ambion Silencer Select in OptiMEM (Gibco, NY) with RNAiMAX (1/100 dilution, Invitrogen/Thermo Fisher), diluted 1:1 with assay media, per manufacturer’s instructions.

    Techniques: Modification, Staining, Labeling, Two Tailed Test

    ( A ) GAP27 peptide: Averaging ESCs from control (black bars, n=3) and endometriosis patients (gray bars, n=6), the invasion was inhibited by a peptide inhibitor of GJ channels, GAP27 (percent Gap Junction Intercellular Coupling (GJIC) compared to untreated shown below each bar). ( B-D ) shRNA: ( B ) Infection of a doxycycline-inducible Cx43 shRNA into endometriosis ESCs or LP9 peritoneal mesothelial cells (PMCs) reduced levels of Cx43 protein (arrow) compared to Laminin controls (doublet at ~66 kD), while expression of DN or wt Cx43 increased Cx43 expression levels. (% of untreated shown below gel) (see and ). ( C ) Cx43 shRNA inhibited GJIC by >90% compared to scrambled shRNA in infected LP9 PMCs (black bars, n=3) and Endometriosis ESCs (Gray bars, n=4). ( D ) Invasiveness was inhibited by ~85% in Cx43 shRNA infected compared to uninfected neighbors, whether expressed in ESCs (n=7), or PMCs (n=2). DN Cx43 inhibited invasiveness by 98% when expressed in ESCs, and 65% when expressed in PMCs, where ~70% of the monolayer was infected. N represents independent tests with different shRNAs, with 10 technical replicates of each. Significance based on two-tailed t-tests. Full data in . Figure 4—source data 1. Original full unannotated image of SDS gel in . Figure 4—source data 2. Annotated image of gel in with labels and bands highlighted. Figure 4—source data 3. Original data table and statistical analysis for .

    Journal: eLife

    Article Title: Hypersensitive intercellular responses of endometrial stromal cells drive invasion in endometriosis

    doi: 10.7554/eLife.94778

    Figure Lengend Snippet: ( A ) GAP27 peptide: Averaging ESCs from control (black bars, n=3) and endometriosis patients (gray bars, n=6), the invasion was inhibited by a peptide inhibitor of GJ channels, GAP27 (percent Gap Junction Intercellular Coupling (GJIC) compared to untreated shown below each bar). ( B-D ) shRNA: ( B ) Infection of a doxycycline-inducible Cx43 shRNA into endometriosis ESCs or LP9 peritoneal mesothelial cells (PMCs) reduced levels of Cx43 protein (arrow) compared to Laminin controls (doublet at ~66 kD), while expression of DN or wt Cx43 increased Cx43 expression levels. (% of untreated shown below gel) (see and ). ( C ) Cx43 shRNA inhibited GJIC by >90% compared to scrambled shRNA in infected LP9 PMCs (black bars, n=3) and Endometriosis ESCs (Gray bars, n=4). ( D ) Invasiveness was inhibited by ~85% in Cx43 shRNA infected compared to uninfected neighbors, whether expressed in ESCs (n=7), or PMCs (n=2). DN Cx43 inhibited invasiveness by 98% when expressed in ESCs, and 65% when expressed in PMCs, where ~70% of the monolayer was infected. N represents independent tests with different shRNAs, with 10 technical replicates of each. Significance based on two-tailed t-tests. Full data in . Figure 4—source data 1. Original full unannotated image of SDS gel in . Figure 4—source data 2. Annotated image of gel in with labels and bands highlighted. Figure 4—source data 3. Original data table and statistical analysis for .

    Article Snippet: In other experiments (data not shown), Cx43 KD was achieved by a 24- hr pre-treatment of the LP9 monolayer with a combination of two siRNAs to Cx43 (10 pmoles/well or 5 nM final concentration) - Ambion Silencer Select in OptiMEM (Gibco, NY) with RNAiMAX (1/100 dilution, Invitrogen/Thermo Fisher), diluted 1:1 with assay media, per manufacturer’s instructions.

    Techniques: Control, shRNA, Infection, Expressing, Two Tailed Test, SDS-Gel

    ( A ) Probing the topological surface of a PMC monolayer using an AFM probe under constant force allows the identification of sites of intercellular contact (where penetration of the probe is maximal). ( B–C ) 3-D reconstructions of the surface of an LP9 PMC monolayer alone ( B ) or in the presence of ESCs which induce the opening of wide gaps ( C ) (Scale bars in μm). ( D ) Penetration depth between PMCs increased more with ESCs from endometriosis than control patients. ( E ) PMC monolayer integrity (i.e. lower penetrance) is reduced by Cx43 shRNA KD and enhanced by Cx43 overexpression. ( F ) In contrast, when ESCs are dropped onto a PMC monolayer, the increased penetrance that is induced is eliminated by the expression of Cx43shRNA or DNCx43 in the PMCs and is enhanced by Cx43 overexpression. Each dot in D-F represents a single image analysis. Significance based on two-tailed t-test. Full data in , and . Figure 5—source data 1. Original data table and statistical analysis table for . Figure 5—source data 2. Original data table and statistical analysis table for . Figure 5—source data 3. Original data table and statistical analysis table for .

    Journal: eLife

    Article Title: Hypersensitive intercellular responses of endometrial stromal cells drive invasion in endometriosis

    doi: 10.7554/eLife.94778

    Figure Lengend Snippet: ( A ) Probing the topological surface of a PMC monolayer using an AFM probe under constant force allows the identification of sites of intercellular contact (where penetration of the probe is maximal). ( B–C ) 3-D reconstructions of the surface of an LP9 PMC monolayer alone ( B ) or in the presence of ESCs which induce the opening of wide gaps ( C ) (Scale bars in μm). ( D ) Penetration depth between PMCs increased more with ESCs from endometriosis than control patients. ( E ) PMC monolayer integrity (i.e. lower penetrance) is reduced by Cx43 shRNA KD and enhanced by Cx43 overexpression. ( F ) In contrast, when ESCs are dropped onto a PMC monolayer, the increased penetrance that is induced is eliminated by the expression of Cx43shRNA or DNCx43 in the PMCs and is enhanced by Cx43 overexpression. Each dot in D-F represents a single image analysis. Significance based on two-tailed t-test. Full data in , and . Figure 5—source data 1. Original data table and statistical analysis table for . Figure 5—source data 2. Original data table and statistical analysis table for . Figure 5—source data 3. Original data table and statistical analysis table for .

    Article Snippet: In other experiments (data not shown), Cx43 KD was achieved by a 24- hr pre-treatment of the LP9 monolayer with a combination of two siRNAs to Cx43 (10 pmoles/well or 5 nM final concentration) - Ambion Silencer Select in OptiMEM (Gibco, NY) with RNAiMAX (1/100 dilution, Invitrogen/Thermo Fisher), diluted 1:1 with assay media, per manufacturer’s instructions.

    Techniques: Control, shRNA, Over Expression, Expressing, Two Tailed Test

    ( A ) Using a constant force of 1 nN, the AFM tip was moved over the peritoneal mesothelial cell (PMC) monolayer progressively further away from a dropped DiI labeled ESC. ( B ) ESCs from an endometriosis III-IV patient showed greater homocellular (solid gray) and induced heterocellular gap junction intercellular coupling (GJIC) with PMCs (striped gray) than those from an endometriosis I-II patient. GJIC of LP9 PMCs was also measured and shown to be decreased by 60% through an expression of Cx43shRNA (black). ( C ) Penetration through the LP9 PMC monolayer decayed with distance from the dropped ESC much faster in the poorly coupled Endo I-II ESCs (gray) than the better coupled Endo III-IV ESCs (black). Penetration of the monolayer was eliminated by KD of Cx43 in PMCs (red). ( D ) Conversely, the decay in penetration of the PMC monolayer induced by Endo III-IV ESC cells (black) was greatly reduced by over-expression of Cx43 in PMCs (green). Full data in . Figure 6—source data 1. Original data sets for .

    Journal: eLife

    Article Title: Hypersensitive intercellular responses of endometrial stromal cells drive invasion in endometriosis

    doi: 10.7554/eLife.94778

    Figure Lengend Snippet: ( A ) Using a constant force of 1 nN, the AFM tip was moved over the peritoneal mesothelial cell (PMC) monolayer progressively further away from a dropped DiI labeled ESC. ( B ) ESCs from an endometriosis III-IV patient showed greater homocellular (solid gray) and induced heterocellular gap junction intercellular coupling (GJIC) with PMCs (striped gray) than those from an endometriosis I-II patient. GJIC of LP9 PMCs was also measured and shown to be decreased by 60% through an expression of Cx43shRNA (black). ( C ) Penetration through the LP9 PMC monolayer decayed with distance from the dropped ESC much faster in the poorly coupled Endo I-II ESCs (gray) than the better coupled Endo III-IV ESCs (black). Penetration of the monolayer was eliminated by KD of Cx43 in PMCs (red). ( D ) Conversely, the decay in penetration of the PMC monolayer induced by Endo III-IV ESC cells (black) was greatly reduced by over-expression of Cx43 in PMCs (green). Full data in . Figure 6—source data 1. Original data sets for .

    Article Snippet: In other experiments (data not shown), Cx43 KD was achieved by a 24- hr pre-treatment of the LP9 monolayer with a combination of two siRNAs to Cx43 (10 pmoles/well or 5 nM final concentration) - Ambion Silencer Select in OptiMEM (Gibco, NY) with RNAiMAX (1/100 dilution, Invitrogen/Thermo Fisher), diluted 1:1 with assay media, per manufacturer’s instructions.

    Techniques: Labeling, Expressing, Over Expression

    ( A ) In healthy patients, when endometrial cells (light brown) encounter a mesothelium (brick red) following arrival in the peritoneum via retrograde menstruation, there is limited GJIC between endometrial stromal cells (ESCs) and peritoneal mesothelial cells (PMCs). ESCs also likely undergo apoptosis. ( B ) In endometriosis, interactions with mesothelial cells trigger Cx43 trafficking to the cell surface and a significant enhancement of GJIC. The increased GJIC mediates the transfer of signals to PMCs (green triangles), which propagate through the mesothelium, inducing disruption of the adhesive and tight junctions between PMCs, facilitating the invasion of the ESCs. There would also be a passage of signals from PMCs to ESCs (purple dots) that induce further changes in ESCs that could promote invasion . EECs (green cells) show minimal invasion alone, but can enhance ESC invasion, and in endometriosis invade with ESCs.

    Journal: eLife

    Article Title: Hypersensitive intercellular responses of endometrial stromal cells drive invasion in endometriosis

    doi: 10.7554/eLife.94778

    Figure Lengend Snippet: ( A ) In healthy patients, when endometrial cells (light brown) encounter a mesothelium (brick red) following arrival in the peritoneum via retrograde menstruation, there is limited GJIC between endometrial stromal cells (ESCs) and peritoneal mesothelial cells (PMCs). ESCs also likely undergo apoptosis. ( B ) In endometriosis, interactions with mesothelial cells trigger Cx43 trafficking to the cell surface and a significant enhancement of GJIC. The increased GJIC mediates the transfer of signals to PMCs (green triangles), which propagate through the mesothelium, inducing disruption of the adhesive and tight junctions between PMCs, facilitating the invasion of the ESCs. There would also be a passage of signals from PMCs to ESCs (purple dots) that induce further changes in ESCs that could promote invasion . EECs (green cells) show minimal invasion alone, but can enhance ESC invasion, and in endometriosis invade with ESCs.

    Article Snippet: In other experiments (data not shown), Cx43 KD was achieved by a 24- hr pre-treatment of the LP9 monolayer with a combination of two siRNAs to Cx43 (10 pmoles/well or 5 nM final concentration) - Ambion Silencer Select in OptiMEM (Gibco, NY) with RNAiMAX (1/100 dilution, Invitrogen/Thermo Fisher), diluted 1:1 with assay media, per manufacturer’s instructions.

    Techniques: Disruption, Adhesive

    Distribution of total Cx43, Cx43 S368 , and Cx43 S325/328/330 in hearts of chagasic mice and uninfected controls. (A, C, E) Heart sections from uninfected mice and (B, D, F) chagasic mice. Samples were stained with WGA (red), cell nuclei stained with DAPI (blue), and with anti-total Cx43, anti-Cx43 S368 , and anti-Cx43 S325/328/330 (green) antibodies; images analyzed by confocal microscopy. Scale bars = 25 µm. White arrows indicate lateralization, while yellow asterisks indicate Cx43 in cytoplasm. WGA, Wheat Germ Agglutinin; Cx43, Connexin 43.

    Journal: Frontiers in Immunology

    Article Title: The effects of inflammation on connexin 43 in chronic Chagas disease cardiomyopathy

    doi: 10.3389/fimmu.2024.1440662

    Figure Lengend Snippet: Distribution of total Cx43, Cx43 S368 , and Cx43 S325/328/330 in hearts of chagasic mice and uninfected controls. (A, C, E) Heart sections from uninfected mice and (B, D, F) chagasic mice. Samples were stained with WGA (red), cell nuclei stained with DAPI (blue), and with anti-total Cx43, anti-Cx43 S368 , and anti-Cx43 S325/328/330 (green) antibodies; images analyzed by confocal microscopy. Scale bars = 25 µm. White arrows indicate lateralization, while yellow asterisks indicate Cx43 in cytoplasm. WGA, Wheat Germ Agglutinin; Cx43, Connexin 43.

    Article Snippet: Heart sections were incubated overnight with anti-total Cx43 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA; SC-9059), anti-Cx43 S368 (1:100; Thermo Fisher Scientific; 48-3000), anti-Cx43 S325/328/330 (1:2000) ( ) and N-cadherin (1:100; Thermo Fisher Scientific; 33-3900) antibodies, at 4°C.

    Techniques: Staining, Confocal Microscopy

    Co-staining with Cx43 and N-Cadherin in hearts of chagasic mice and uninfected controls. (A, B) Non-infected hearts show the presence of Cx43 (green) and N-cadherin (red) in the intercalated disk area; (C, D) chagasic hearts frequently presented N-cad exclusively in intercalated disc areas, with Cx43 dispersed into the cytoplasm. White arrows indicate Cx43 in cytoplasm, while white asterisks indicate intercalated discs with only N-cad expression. N-cad, N-cadherin; Cx43, Connexin 43. Scale bars = 25 µm.

    Journal: Frontiers in Immunology

    Article Title: The effects of inflammation on connexin 43 in chronic Chagas disease cardiomyopathy

    doi: 10.3389/fimmu.2024.1440662

    Figure Lengend Snippet: Co-staining with Cx43 and N-Cadherin in hearts of chagasic mice and uninfected controls. (A, B) Non-infected hearts show the presence of Cx43 (green) and N-cadherin (red) in the intercalated disk area; (C, D) chagasic hearts frequently presented N-cad exclusively in intercalated disc areas, with Cx43 dispersed into the cytoplasm. White arrows indicate Cx43 in cytoplasm, while white asterisks indicate intercalated discs with only N-cad expression. N-cad, N-cadherin; Cx43, Connexin 43. Scale bars = 25 µm.

    Article Snippet: Heart sections were incubated overnight with anti-total Cx43 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA; SC-9059), anti-Cx43 S368 (1:100; Thermo Fisher Scientific; 48-3000), anti-Cx43 S325/328/330 (1:2000) ( ) and N-cadherin (1:100; Thermo Fisher Scientific; 33-3900) antibodies, at 4°C.

    Techniques: Staining, Infection, Expressing

    Ultrastructural analysis of total Cx43 in intercalated discs. Hearts from uninfected and chagasic control mice (n = 3 per group) were processed for analysis by transmission electron microscopy. Ultrathin sections were incubated with total anti-Cx43 antibody, with labeling revealed by the immunogold technique. (A–C) representative sections from uninfected control mice; (D–F) representative sections from chagasic mice. Red arrows indicate the presence of gap junctions in the intercalated discs; yellow asterisks indicate desmosome; P, plicate; IP, interplicate; M, myofibrils. Scale bars = 0.5 µm (A, B, D, E) , 0.2 µm (C, F) .

    Journal: Frontiers in Immunology

    Article Title: The effects of inflammation on connexin 43 in chronic Chagas disease cardiomyopathy

    doi: 10.3389/fimmu.2024.1440662

    Figure Lengend Snippet: Ultrastructural analysis of total Cx43 in intercalated discs. Hearts from uninfected and chagasic control mice (n = 3 per group) were processed for analysis by transmission electron microscopy. Ultrathin sections were incubated with total anti-Cx43 antibody, with labeling revealed by the immunogold technique. (A–C) representative sections from uninfected control mice; (D–F) representative sections from chagasic mice. Red arrows indicate the presence of gap junctions in the intercalated discs; yellow asterisks indicate desmosome; P, plicate; IP, interplicate; M, myofibrils. Scale bars = 0.5 µm (A, B, D, E) , 0.2 µm (C, F) .

    Article Snippet: Heart sections were incubated overnight with anti-total Cx43 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA; SC-9059), anti-Cx43 S368 (1:100; Thermo Fisher Scientific; 48-3000), anti-Cx43 S325/328/330 (1:2000) ( ) and N-cadherin (1:100; Thermo Fisher Scientific; 33-3900) antibodies, at 4°C.

    Techniques: Control, Transmission Assay, Electron Microscopy, Incubation, Antibody Labeling

    Distribution of total Cx43, Cx43 S368 , and Cx43 S325/328/330 in human cardiac muscle tissue. (A, C, E) Sections of explanted hearts from non-Chagas patients and (B, D, F) sections of explanted hearts from Chagas patients who underwent heart transplantation were analyzed by immunofluorescence. Samples were labeled with WGA (red), cell nuclei with DAPI (blue), and total Cx43 total, Cx43 S368 or Cx43 S325/328/330 (green). (G, H) Co-labeling of Cx43 (green) and N-cadherin (red), and co-localization between Cx43 and N-cadherin (yellow). White arrows indicate the presence of Cx43 outside the intercalated disc area. Scale bars = 50 µm. WGA, Wheat Germ Agglutinin; N-cad, N-cadherin; Cx43, Connexin 43.

    Journal: Frontiers in Immunology

    Article Title: The effects of inflammation on connexin 43 in chronic Chagas disease cardiomyopathy

    doi: 10.3389/fimmu.2024.1440662

    Figure Lengend Snippet: Distribution of total Cx43, Cx43 S368 , and Cx43 S325/328/330 in human cardiac muscle tissue. (A, C, E) Sections of explanted hearts from non-Chagas patients and (B, D, F) sections of explanted hearts from Chagas patients who underwent heart transplantation were analyzed by immunofluorescence. Samples were labeled with WGA (red), cell nuclei with DAPI (blue), and total Cx43 total, Cx43 S368 or Cx43 S325/328/330 (green). (G, H) Co-labeling of Cx43 (green) and N-cadherin (red), and co-localization between Cx43 and N-cadherin (yellow). White arrows indicate the presence of Cx43 outside the intercalated disc area. Scale bars = 50 µm. WGA, Wheat Germ Agglutinin; N-cad, N-cadherin; Cx43, Connexin 43.

    Article Snippet: Heart sections were incubated overnight with anti-total Cx43 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA; SC-9059), anti-Cx43 S368 (1:100; Thermo Fisher Scientific; 48-3000), anti-Cx43 S325/328/330 (1:2000) ( ) and N-cadherin (1:100; Thermo Fisher Scientific; 33-3900) antibodies, at 4°C.

    Techniques: Transplantation Assay, Immunofluorescence, Labeling

    Effect of inflammatory microenvironment on Cx43 distribution in vitro . (A) Experimental in vitro design involving H9c2 cells and iPSC-derived cardiomyocytes, with both cell types stimulated by pro-inflammatory cytokines (IL-1β, TNF, and IFN-γ, 10 ng/ml of each cytokine) for 48 hours for immunofluorescence analysis. (B) Immunofluorescence of H9c2 cells and iPSC-derived cardiomyocytes; (C) Analysis of Cx43 distribution by fluorescence intensity in H9c2 cells and iPSC-derived cardiomyocytes after 48 hours of stimulation with pro-inflammatory cytokines IL-1β, TNF, and IFN-γ. Cells were stained with WGA (red), cell nuclei with DAPI (blue), and total Cx43 with anti-Cx43 antibody (green). Images analyzed by confocal microscopy. Scale bars = 50 µm. WGA, Wheat Germ Agglutinin; Cx43, Connexin 43; IL-1β, Interleukin 1 beta; TNF, Tumor necrosis factor; IFN-γ, Interferon gamma; C, Cytoplasm; N, Cell Nucleus. Created with BioRender.com .

    Journal: Frontiers in Immunology

    Article Title: The effects of inflammation on connexin 43 in chronic Chagas disease cardiomyopathy

    doi: 10.3389/fimmu.2024.1440662

    Figure Lengend Snippet: Effect of inflammatory microenvironment on Cx43 distribution in vitro . (A) Experimental in vitro design involving H9c2 cells and iPSC-derived cardiomyocytes, with both cell types stimulated by pro-inflammatory cytokines (IL-1β, TNF, and IFN-γ, 10 ng/ml of each cytokine) for 48 hours for immunofluorescence analysis. (B) Immunofluorescence of H9c2 cells and iPSC-derived cardiomyocytes; (C) Analysis of Cx43 distribution by fluorescence intensity in H9c2 cells and iPSC-derived cardiomyocytes after 48 hours of stimulation with pro-inflammatory cytokines IL-1β, TNF, and IFN-γ. Cells were stained with WGA (red), cell nuclei with DAPI (blue), and total Cx43 with anti-Cx43 antibody (green). Images analyzed by confocal microscopy. Scale bars = 50 µm. WGA, Wheat Germ Agglutinin; Cx43, Connexin 43; IL-1β, Interleukin 1 beta; TNF, Tumor necrosis factor; IFN-γ, Interferon gamma; C, Cytoplasm; N, Cell Nucleus. Created with BioRender.com .

    Article Snippet: Heart sections were incubated overnight with anti-total Cx43 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA; SC-9059), anti-Cx43 S368 (1:100; Thermo Fisher Scientific; 48-3000), anti-Cx43 S325/328/330 (1:2000) ( ) and N-cadherin (1:100; Thermo Fisher Scientific; 33-3900) antibodies, at 4°C.

    Techniques: In Vitro, Derivative Assay, Immunofluorescence, Fluorescence, Staining, Confocal Microscopy

    Functional in vitro analysis of Cx43: Action Potential Duration and Lucifer Yellow dye transfer. (A) Schematic drawing of functional testing performed on iPSC-derived cardiomyocytes and H9c2 cardiomyocytes, stimulated with pro-inflammatory cytokines to respectively analyze the duration of action potential and dye transfer between neighboring cells. (B) After stimulation, action potential duration (APD) of iPSC-derived cardiomyocytes was determined at 10%, 50% and 90% levels. (C) Lucifer Yellow dye transfer test (green) performed in H9c2 cells (bright field) stimulated with pro-inflammatory cytokines IL-1β, TNF, and IFN-γ for 48 hours. Predominance of dye observed in cells at the margin where scalpel incision was made, while absence of stimulation resulted in dye reaching more distant cells; IL-1β = Interleukin 1 beta; TNF = Tumor necrosis factor; IFN-γ = Interferon gamma; CTRL = Control group; ***P< 0.001 compared to unstimulated cells. Scale bars = 100 μm.

    Journal: Frontiers in Immunology

    Article Title: The effects of inflammation on connexin 43 in chronic Chagas disease cardiomyopathy

    doi: 10.3389/fimmu.2024.1440662

    Figure Lengend Snippet: Functional in vitro analysis of Cx43: Action Potential Duration and Lucifer Yellow dye transfer. (A) Schematic drawing of functional testing performed on iPSC-derived cardiomyocytes and H9c2 cardiomyocytes, stimulated with pro-inflammatory cytokines to respectively analyze the duration of action potential and dye transfer between neighboring cells. (B) After stimulation, action potential duration (APD) of iPSC-derived cardiomyocytes was determined at 10%, 50% and 90% levels. (C) Lucifer Yellow dye transfer test (green) performed in H9c2 cells (bright field) stimulated with pro-inflammatory cytokines IL-1β, TNF, and IFN-γ for 48 hours. Predominance of dye observed in cells at the margin where scalpel incision was made, while absence of stimulation resulted in dye reaching more distant cells; IL-1β = Interleukin 1 beta; TNF = Tumor necrosis factor; IFN-γ = Interferon gamma; CTRL = Control group; ***P< 0.001 compared to unstimulated cells. Scale bars = 100 μm.

    Article Snippet: Heart sections were incubated overnight with anti-total Cx43 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA; SC-9059), anti-Cx43 S368 (1:100; Thermo Fisher Scientific; 48-3000), anti-Cx43 S325/328/330 (1:2000) ( ) and N-cadherin (1:100; Thermo Fisher Scientific; 33-3900) antibodies, at 4°C.

    Techniques: Functional Assay, In Vitro, Derivative Assay, Control

    Ethanol increases the activity of Cx43 hemichannels and Panx1 channels in cultured astrocytes. ( A ) Averaged Etd uptake rate (AU/min) normalized with the control condition (dashed line) by astrocytes treated for 24 h with different concentrations of ethanol (red circles). * p < 0.05, ** p < 0.005, *** p < 0.0001, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( B - E ) Representative immunofluorescence images depicting Etd and GFAP staining from dye uptake measurements (10 min exposure to Etd) in astrocytes under control conditions ( B-C ) or treated for 24 h with 100 mM ethanol ( D-E ). ( F ) Averaged Etd uptake rate (AU/min) normalized with the control condition (dashed line) by astrocytes treated for several time periods with ethanol at two concentrations: 25 mM (blue circles) or 100 mM (red circles). * p < 0.05, ** p < 0.005, *** p < 0.0001, 100 mM ethanol treatment compared to control conditions; # p < 0.05, ## p < 0.005, ### p < 0.0001, 25 mM ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( G ) Averaged Etd uptake rate (AU/min) normalized with control condition (dashed line) by astrocytes treated for 24 h with 100 mM ethanol alone or in combination with the following blockers: 200 µM La 3+ , 5 µM carbenoxolone (CBX), 500 µM Probenecid (Prob), 50 µM gap19, 50 µM gap19 I130A , 50 µM 10 panx1, siRNA Cx43 , siRNA Panx1 ; siRNA scrb and 50 µM gap19 + 50 µM 10 panx1. *** p < 0.0001, ethanol compared to control; # p < 0.05, ## p < 0.005; ### p < 0.001; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). ( H ) Time-lapse measurements of Etd uptake by astrocytes under control conditions (white circles) or treated for 24 h with 100 mM ethanol alone (red circles) or in combination with 50 µM gap19 + 50 µM 10 panx1 (black circles). Data were obtained from at least three independent experiments with three or more repeats each one (≥ 30 cells analyzed for each repeat). Calibration bar = 30 μm

    Journal: Biological Research

    Article Title: Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage

    doi: 10.1186/s40659-024-00493-2

    Figure Lengend Snippet: Ethanol increases the activity of Cx43 hemichannels and Panx1 channels in cultured astrocytes. ( A ) Averaged Etd uptake rate (AU/min) normalized with the control condition (dashed line) by astrocytes treated for 24 h with different concentrations of ethanol (red circles). * p < 0.05, ** p < 0.005, *** p < 0.0001, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( B - E ) Representative immunofluorescence images depicting Etd and GFAP staining from dye uptake measurements (10 min exposure to Etd) in astrocytes under control conditions ( B-C ) or treated for 24 h with 100 mM ethanol ( D-E ). ( F ) Averaged Etd uptake rate (AU/min) normalized with the control condition (dashed line) by astrocytes treated for several time periods with ethanol at two concentrations: 25 mM (blue circles) or 100 mM (red circles). * p < 0.05, ** p < 0.005, *** p < 0.0001, 100 mM ethanol treatment compared to control conditions; # p < 0.05, ## p < 0.005, ### p < 0.0001, 25 mM ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( G ) Averaged Etd uptake rate (AU/min) normalized with control condition (dashed line) by astrocytes treated for 24 h with 100 mM ethanol alone or in combination with the following blockers: 200 µM La 3+ , 5 µM carbenoxolone (CBX), 500 µM Probenecid (Prob), 50 µM gap19, 50 µM gap19 I130A , 50 µM 10 panx1, siRNA Cx43 , siRNA Panx1 ; siRNA scrb and 50 µM gap19 + 50 µM 10 panx1. *** p < 0.0001, ethanol compared to control; # p < 0.05, ## p < 0.005; ### p < 0.001; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). ( H ) Time-lapse measurements of Etd uptake by astrocytes under control conditions (white circles) or treated for 24 h with 100 mM ethanol alone (red circles) or in combination with 50 µM gap19 + 50 µM 10 panx1 (black circles). Data were obtained from at least three independent experiments with three or more repeats each one (≥ 30 cells analyzed for each repeat). Calibration bar = 30 μm

    Article Snippet: siRNA duplexes against mouse Cx43 or Panx1 were predesigned and obtained from Origene (Rockville, MD). siRNA (10 nM) was transfected with Oligofectamine (Invitrogen) according to the Origene application guide for Trilencer-27 siRNA.

    Techniques: Activity Assay, Cell Culture, Control, Immunofluorescence, Staining

    Ethanol increases the activity of large-pore channels in HeLa cells transfected with Cx43 or Panx1. ( A - D ) Representative images depicting Cx43 EGFP (green), Panx1 EGFP (green) and DAPI (blue, 5 µM and 10 min of exposure) labeling by HeLa-Cx43 EGFP ( A-B ) and HeLa-Panx1 EGFP ( C-D ) cells under control conditions or treated for 24 h with 100 mM ethanol. Insets at the right of each panel depict the respective DAPI labeling alone (top) or plus the phase view merged with EGFP (bottom). ( E-F ) Time-lapse measurements of DAPI uptake by HeLa-Cx43 EGFP ( E ) and HeLa-Panx1 EGFP ( F ) cells under control conditions (white circles) or treated for 24 h with 100 mM ethanol (red circles). ( G ) Averaged DAPI uptake rate (AU/min) normalized with the control condition (dashed line) by parental HeLa, HeLa-Cx43 EGFP and HeLa-Panx1 EGFP cells treated for 24 h with 100 mM ethanol. In some experiments, HeLa-Cx43 EGFP or HeLa-Panx1 EGFP cells were treated for 24 h with 100 mM ethanol plus 50 µM gap19 or 50 µM 10 panx1, respectively. * p < 0.05, ** p < 0.01, ethanol treatment compared to control conditions (two-way ANOVA followed by Tukey’s post-hoc test). Data were obtained from at least three independent experiments with three or more repeats each one (≥ 20 cells analyzed for each repeat). Calibration bar = 15 μm

    Journal: Biological Research

    Article Title: Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage

    doi: 10.1186/s40659-024-00493-2

    Figure Lengend Snippet: Ethanol increases the activity of large-pore channels in HeLa cells transfected with Cx43 or Panx1. ( A - D ) Representative images depicting Cx43 EGFP (green), Panx1 EGFP (green) and DAPI (blue, 5 µM and 10 min of exposure) labeling by HeLa-Cx43 EGFP ( A-B ) and HeLa-Panx1 EGFP ( C-D ) cells under control conditions or treated for 24 h with 100 mM ethanol. Insets at the right of each panel depict the respective DAPI labeling alone (top) or plus the phase view merged with EGFP (bottom). ( E-F ) Time-lapse measurements of DAPI uptake by HeLa-Cx43 EGFP ( E ) and HeLa-Panx1 EGFP ( F ) cells under control conditions (white circles) or treated for 24 h with 100 mM ethanol (red circles). ( G ) Averaged DAPI uptake rate (AU/min) normalized with the control condition (dashed line) by parental HeLa, HeLa-Cx43 EGFP and HeLa-Panx1 EGFP cells treated for 24 h with 100 mM ethanol. In some experiments, HeLa-Cx43 EGFP or HeLa-Panx1 EGFP cells were treated for 24 h with 100 mM ethanol plus 50 µM gap19 or 50 µM 10 panx1, respectively. * p < 0.05, ** p < 0.01, ethanol treatment compared to control conditions (two-way ANOVA followed by Tukey’s post-hoc test). Data were obtained from at least three independent experiments with three or more repeats each one (≥ 20 cells analyzed for each repeat). Calibration bar = 15 μm

    Article Snippet: siRNA duplexes against mouse Cx43 or Panx1 were predesigned and obtained from Origene (Rockville, MD). siRNA (10 nM) was transfected with Oligofectamine (Invitrogen) according to the Origene application guide for Trilencer-27 siRNA.

    Techniques: Activity Assay, Transfection, Labeling, Control

    Ethanol does not modulate levels and plasma membrane distribution of Cx43 and Panx1 in cultured astrocytes. ( A-L ) Representative confocal images depicting Cx43 (cyan, left panel) or Panx1 (cyan, right panel) immunostaining in combination with DAPI (magenta) and WGA (red) labeling by astrocytes under control conditions ( A-C and G-I ) or treated for 24 h with 100 mM ethanol ( D-F and J-L ). The size of the z-step acquisition for all images was 0.3 μm. Calibration Bar: 10 μm. Insets: 2X magnification of the indicated area of panels C, F, I and L. ( M - N ) Quantification of membrane, intracellular and total staining of Cx43 (M) and Panx1 (N) normalized to control conditions (dashed line) by astrocytes treated for 24 h with 100 mM ethanol. ( O ) Quantification of Manders’ overlap coefficient for Cx43 or Panx1 with WGA by astrocytes under control conditions (white bars) or treated for 24 h with 100 mM ethanol (red bars). ( P ) Total Cx43 (upper panel) and Panx1 (bottom panel) levels by astrocytes under control conditions or treated for 1, 24, 48–72 h with 100 mM ethanol. Total levels of each analyzed band were normalized according to the levels of GADPH detected in each lane. ( Q-R ) Quantification of total levels of Cx43 ( Q ) and Panx1 ( R ) normalized to control (dashed line) in astrocytes treated for 1, 24, 48–72 h with 100 mM ethanol. Averaged data were obtained from three independent experiments

    Journal: Biological Research

    Article Title: Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage

    doi: 10.1186/s40659-024-00493-2

    Figure Lengend Snippet: Ethanol does not modulate levels and plasma membrane distribution of Cx43 and Panx1 in cultured astrocytes. ( A-L ) Representative confocal images depicting Cx43 (cyan, left panel) or Panx1 (cyan, right panel) immunostaining in combination with DAPI (magenta) and WGA (red) labeling by astrocytes under control conditions ( A-C and G-I ) or treated for 24 h with 100 mM ethanol ( D-F and J-L ). The size of the z-step acquisition for all images was 0.3 μm. Calibration Bar: 10 μm. Insets: 2X magnification of the indicated area of panels C, F, I and L. ( M - N ) Quantification of membrane, intracellular and total staining of Cx43 (M) and Panx1 (N) normalized to control conditions (dashed line) by astrocytes treated for 24 h with 100 mM ethanol. ( O ) Quantification of Manders’ overlap coefficient for Cx43 or Panx1 with WGA by astrocytes under control conditions (white bars) or treated for 24 h with 100 mM ethanol (red bars). ( P ) Total Cx43 (upper panel) and Panx1 (bottom panel) levels by astrocytes under control conditions or treated for 1, 24, 48–72 h with 100 mM ethanol. Total levels of each analyzed band were normalized according to the levels of GADPH detected in each lane. ( Q-R ) Quantification of total levels of Cx43 ( Q ) and Panx1 ( R ) normalized to control (dashed line) in astrocytes treated for 1, 24, 48–72 h with 100 mM ethanol. Averaged data were obtained from three independent experiments

    Article Snippet: siRNA duplexes against mouse Cx43 or Panx1 were predesigned and obtained from Origene (Rockville, MD). siRNA (10 nM) was transfected with Oligofectamine (Invitrogen) according to the Origene application guide for Trilencer-27 siRNA.

    Techniques: Membrane, Cell Culture, Immunostaining, Labeling, Control, Staining

    Ethanol-induced Cx43 hemichannel activity depends on TLR4 and IL-1β/TNF-α/p38 MAPK/iNOS-dependent signaling. ( A ) Averaged Etd uptake rate (AU/min) normalized with control condition (dashed line) by astrocytes treated for 24 h with 100 mM ethanol alone or in combination with the following agents: 0.5 µM TAK-242, 100 ng/mL of IL-1ra + 100 ng/mL of sTNF-αR1, 1 µM SB203580, 1 µM L-N6, 20 nM 0.5 µM TAK-242 + 50 µM gap19 or 0.5 µM TAK-242 + 50 µM 10 panx1. * p < 0.005, ethanol treatment compared to control conditions; # p < 0.01; ## p < 0.005; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). Data were obtained from at least three independent experiments with three or more repeats each one (≥ 30 cells analyzed for each repeat). ( B-C ) Averaged data of IL-1β ( B ) and TNF-α ( C ) released by astrocytes under control conditions (white circles) or treated for several time periods with 100 mM ethanol (red circles). * p < 0.05, ** p < 0.01, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( D-E ) Representative fluorescence micrographs of basal NO production (DAF-FM, green) by astrocytes under control conditions ( D ) or treated for 24 h with 100 mM ethanol ( E ). Calibration Bar: 20 μm. ( F ) Average DAF-FM fluorescence normalized to control conditions (dashed line) by astrocytes treated with 100 mM ethanol for several time periods. * p < 0.05, ** p < 0.005, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( G ) Average DAF-FM fluorescence normalized to control conditions (dashed line) by astrocytes treated for 72 h with 100 mM ethanol alone or in combination with the following agents: 0.5 µM TAK-242, 50 µM gap19, 50 µM 10 panx1 or 0.5 µM TAK-242 + 50 µM gap19. *** p < 0.005, ** p < 0.001, effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test)

    Journal: Biological Research

    Article Title: Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage

    doi: 10.1186/s40659-024-00493-2

    Figure Lengend Snippet: Ethanol-induced Cx43 hemichannel activity depends on TLR4 and IL-1β/TNF-α/p38 MAPK/iNOS-dependent signaling. ( A ) Averaged Etd uptake rate (AU/min) normalized with control condition (dashed line) by astrocytes treated for 24 h with 100 mM ethanol alone or in combination with the following agents: 0.5 µM TAK-242, 100 ng/mL of IL-1ra + 100 ng/mL of sTNF-αR1, 1 µM SB203580, 1 µM L-N6, 20 nM 0.5 µM TAK-242 + 50 µM gap19 or 0.5 µM TAK-242 + 50 µM 10 panx1. * p < 0.005, ethanol treatment compared to control conditions; # p < 0.01; ## p < 0.005; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). Data were obtained from at least three independent experiments with three or more repeats each one (≥ 30 cells analyzed for each repeat). ( B-C ) Averaged data of IL-1β ( B ) and TNF-α ( C ) released by astrocytes under control conditions (white circles) or treated for several time periods with 100 mM ethanol (red circles). * p < 0.05, ** p < 0.01, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( D-E ) Representative fluorescence micrographs of basal NO production (DAF-FM, green) by astrocytes under control conditions ( D ) or treated for 24 h with 100 mM ethanol ( E ). Calibration Bar: 20 μm. ( F ) Average DAF-FM fluorescence normalized to control conditions (dashed line) by astrocytes treated with 100 mM ethanol for several time periods. * p < 0.05, ** p < 0.005, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( G ) Average DAF-FM fluorescence normalized to control conditions (dashed line) by astrocytes treated for 72 h with 100 mM ethanol alone or in combination with the following agents: 0.5 µM TAK-242, 50 µM gap19, 50 µM 10 panx1 or 0.5 µM TAK-242 + 50 µM gap19. *** p < 0.005, ** p < 0.001, effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test)

    Article Snippet: siRNA duplexes against mouse Cx43 or Panx1 were predesigned and obtained from Origene (Rockville, MD). siRNA (10 nM) was transfected with Oligofectamine (Invitrogen) according to the Origene application guide for Trilencer-27 siRNA.

    Techniques: Activity Assay, Control, Fluorescence

    The ethanol-induced activation of hemichannels and pannexons triggers the release of ATP and glutamate by different pathways in cultured astrocytes. ( A ) Averaged Etd uptake rate (AU/min) normalized with control condition (dashed line) by astrocytes treated for 24 h with 100 mM ethanol alone or in combination with the following agents: 200 nM A740003, 200 µM oATP, 200 nM A740003 + 50 µM 10 panx1, 200 nM A740003 + 50 µM gap19 or 200 nM A740003 + 0.5 µM TAK-242. *** p < 0.001, ethanol treatment compared to control conditions; # p < 0.05; ### p < 0.005; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). ( B ) Averaged data of ATP release by astrocytes under control conditions (white circles) or treated with 100 mM ethanol for different time periods (red circles). * p < 0.05, ** p < 0.01, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( C ) Averaged data of ATP release by astrocytes under control conditions (dashed line) or treated for 24 h with 100 mM ethanol alone or in combination with the following agents: 50 µM 10 panx1, siRNA Panx1 , 50 µM gap19, siRNA Cx43 , 200 nM A740003, 200 µM oATP or 0.5 µM TAK-242. ** p < 0.01, ethanol treatment compared to control conditions; ## p < 0.01; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). ( D-E ) Averaged data of IL-1β ( D ) and TNF-α ( E ) release by astrocytes under control conditions (white bar) or treated for 72 h with 100 mM ethanol (red bars) or in combination with the following agents: 50 µM 10 panx1, siRNA Panx1 , 50 µM gap19, siRNA Cx43 , 200 nM A740003, 200 µM oATP or 0.5 µM TAK-242. *** p < 0.005, ethanol treatment compared to control conditions; ### p < 0.005; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). ( F ) Averaged data of glutamate release by astrocytes under control conditions (white circles) or treated with 100 mM ethanol for different time periods (red circles). * p < 0.05, ** p < 0.01, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( G ) Averaged data of glutamate release by astrocytes under control conditions (dashed line) or treated for 24 h with 100 mM ethanol alone or in combination with the following agents: 50 µM 10 panx1, siRNA Panx1 , 50 µM gap19, siRNA Cx43 , 200 nM A740003, 200 µM oATP or 0.5 µM TAK-242. ** p < 0.01, ethanol treatment compared to control conditions; ## p < 0.01; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). Data were obtained from at least three independent experiments with three or more repeats each one (≥ 30 cells analyzed for each repeat)

    Journal: Biological Research

    Article Title: Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage

    doi: 10.1186/s40659-024-00493-2

    Figure Lengend Snippet: The ethanol-induced activation of hemichannels and pannexons triggers the release of ATP and glutamate by different pathways in cultured astrocytes. ( A ) Averaged Etd uptake rate (AU/min) normalized with control condition (dashed line) by astrocytes treated for 24 h with 100 mM ethanol alone or in combination with the following agents: 200 nM A740003, 200 µM oATP, 200 nM A740003 + 50 µM 10 panx1, 200 nM A740003 + 50 µM gap19 or 200 nM A740003 + 0.5 µM TAK-242. *** p < 0.001, ethanol treatment compared to control conditions; # p < 0.05; ### p < 0.005; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). ( B ) Averaged data of ATP release by astrocytes under control conditions (white circles) or treated with 100 mM ethanol for different time periods (red circles). * p < 0.05, ** p < 0.01, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( C ) Averaged data of ATP release by astrocytes under control conditions (dashed line) or treated for 24 h with 100 mM ethanol alone or in combination with the following agents: 50 µM 10 panx1, siRNA Panx1 , 50 µM gap19, siRNA Cx43 , 200 nM A740003, 200 µM oATP or 0.5 µM TAK-242. ** p < 0.01, ethanol treatment compared to control conditions; ## p < 0.01; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). ( D-E ) Averaged data of IL-1β ( D ) and TNF-α ( E ) release by astrocytes under control conditions (white bar) or treated for 72 h with 100 mM ethanol (red bars) or in combination with the following agents: 50 µM 10 panx1, siRNA Panx1 , 50 µM gap19, siRNA Cx43 , 200 nM A740003, 200 µM oATP or 0.5 µM TAK-242. *** p < 0.005, ethanol treatment compared to control conditions; ### p < 0.005; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). ( F ) Averaged data of glutamate release by astrocytes under control conditions (white circles) or treated with 100 mM ethanol for different time periods (red circles). * p < 0.05, ** p < 0.01, ethanol treatment compared to control conditions (one-way ANOVA followed by Tukey’s post-hoc test). ( G ) Averaged data of glutamate release by astrocytes under control conditions (dashed line) or treated for 24 h with 100 mM ethanol alone or in combination with the following agents: 50 µM 10 panx1, siRNA Panx1 , 50 µM gap19, siRNA Cx43 , 200 nM A740003, 200 µM oATP or 0.5 µM TAK-242. ** p < 0.01, ethanol treatment compared to control conditions; ## p < 0.01; effect of pharmacological agents compared to ethanol treatment (one-way ANOVA followed by Tukey’s post-hoc test). Data were obtained from at least three independent experiments with three or more repeats each one (≥ 30 cells analyzed for each repeat)

    Article Snippet: siRNA duplexes against mouse Cx43 or Panx1 were predesigned and obtained from Origene (Rockville, MD). siRNA (10 nM) was transfected with Oligofectamine (Invitrogen) according to the Origene application guide for Trilencer-27 siRNA.

    Techniques: Activation Assay, Cell Culture, Control