Journal: Molecular Pharmacology

Article Title: A Renal-Like Organic Anion Transport System in the Ciliary Epithelium of the Bovine and Human Eye

doi: 10.1124/mol.114.096578

Figure Lengend Snippet: Immunolocalization of Na,K-ATPase, Cx43, MRP4, OAT1, and OAT3 in paraffin-embedded human eye. Na,K-ATPase was used as a marker of basolateral membranes of pigmented and nonpigmented cells. Cx43 was used as a marker for the apical membrane of both cell types. Proteins of interest are in green and nuclei are stained red with propidium iodide. The arrowheads point to pigmented cells whereas the arrows point to nonpigmented cells. The insets are magnifications of the areas near the arrow and arrowhead. Limited immunoreactivity was detected when the primary antibody was omitted (negative control). AH, aqueous humor.

Article Snippet: The mouse anti-Cx43 monoclonal antibody (clone CX-1B1) was obtained from Life Technologies (Burlington, Ontario, Canada).

Techniques: Marker, Staining, Negative Control

Journal: Prenatal Diagnosis

Article Title: Role of Myofibroblasts in the Repair of Iatrogenic Preterm Membranes Subjected to Mechanical Stimulation

doi: 10.1002/pd.6722

Figure Lengend Snippet: Myofibroblast morphology in human iatrogenic preterm AM. Fetal membranes were taken from a 31 year old patient who underwent open fetal surgery for correction of spina bifida neural tube defect that took place at 24 + 0 weeks gestational age leading to late trimester preterm delivery at 34 + 6 weeks (Case #1). Myofibroblast morphology was examined in αSMA expressing cells (green arrow) by IMF confocal microscopy in control AM specimens (A) and near the edge of the defect site (B, C). Cx43 was detected by immunostaining (pink arrow, C) and collagen by SHG imaging (red arrow). Control AM specimens were taken 5 cm away from the defect site. Signals for blue (DAPI), green (αSMA), pink (Cx43) and red (collagen) were detected by IMF confocal microscopy and SHG imaging. The dotted white lines show the length of the wound edge (WE) in the iatrogenic preterm AM specimen. Sale bar = 100 μM (A, B) and 300 μM (C). The white dotted box in (B) shows an enlarged image to show Cx43 localized in cell bodies.

Article Snippet: AM specimens were incubated with primary antibodies for mouse Cx43 (1:100, ThermoFisher Scientific, CX‐1B1) and rabbit αSMA (1:100, Abcam, ab5694) at 4°C overnight as described 44 Specimens were washed in PBS and incubated with secondary antibodies for Alexa Fluor 568 anti‐mouse or 488 anti‐rabbit at room temperature for 2 h (both 1:1000, ThermoFisher Scientific) and counterstained with 1 μg/mL DAPI for 20 min (1:1000) to detect nuclei.

Techniques: Expressing, Confocal Microscopy, Control, Immunostaining, Imaging

Journal: Prenatal Diagnosis

Article Title: Role of Myofibroblasts in the Repair of Iatrogenic Preterm Membranes Subjected to Mechanical Stimulation

doi: 10.1002/pd.6722

Figure Lengend Snippet: Cx43 plaque formation in human iatrogenic preterm ruptured amniotic membrane. Representative images obtained by confocal microscopy are shown in the epithelial (A) and fibroblast layer (B) of the preterm AM defect from one late third trimester iatrogenic preterm donor (Case #1). The distribution of Cx43 was analyzed per tissue area for comparisons between the epithelial and fibroblast layers (C) or per cell nucleus (D). Error bars represent the mean and SEM values of 6 replicates from three late trimester iatrogenic preterm donors (34 + 0 weeks to 35 + 0 weeks, Case #1–3). Significant differences are indicated by *** p < 0.001. Statistical comparisons are indicated for control AM specimens and AM defect ( p < 0.001***) in the epithelial layer ( p < 0.001 +++ ) and fibroblast layer ( p < 0.001 $$$ ). The dotted white and black lines show the length of the wound edge (WE) in the preterm AM specimen. Sale bar = 100 μM (A, B).

Article Snippet: AM specimens were incubated with primary antibodies for mouse Cx43 (1:100, ThermoFisher Scientific, CX‐1B1) and rabbit αSMA (1:100, Abcam, ab5694) at 4°C overnight as described 44 Specimens were washed in PBS and incubated with secondary antibodies for Alexa Fluor 568 anti‐mouse or 488 anti‐rabbit at room temperature for 2 h (both 1:1000, ThermoFisher Scientific) and counterstained with 1 μg/mL DAPI for 20 min (1:1000) to detect nuclei.

Techniques: Membrane, Confocal Microscopy, Control

Journal: Prenatal Diagnosis

Article Title: Role of Myofibroblasts in the Repair of Iatrogenic Preterm Membranes Subjected to Mechanical Stimulation

doi: 10.1002/pd.6722

Figure Lengend Snippet: Myofibroblast nuclei deformation in human iatrogenic preterm AM. AM specimens were immunostained for αSMA to detect myofibroblast (pink arrow) nuclei (blue arrow, A). Circularity values for the nuclei shape of myofibroblasts expressing αSMA in the epithelial layer were compared to the fibroblast layer in control specimens and AM defect (B). Nuclei values close to 1 represent a perfect circle in contrast to zero, which represents a more elongated shape. The total number of nuclei counted ranged from 1202 to 1582 from 6 specimens (Case #1–3). Signals for blue (DAPI), green (αSMA) and pink (Cx43) were detected by immunofluorescence confocal microscopy. The dotted white lines show the length of the wound edge (WE) in the iatrogenic preterm AM specimen. Scale bar = 100 μM. *** p < 0.001.

Article Snippet: AM specimens were incubated with primary antibodies for mouse Cx43 (1:100, ThermoFisher Scientific, CX‐1B1) and rabbit αSMA (1:100, Abcam, ab5694) at 4°C overnight as described 44 Specimens were washed in PBS and incubated with secondary antibodies for Alexa Fluor 568 anti‐mouse or 488 anti‐rabbit at room temperature for 2 h (both 1:1000, ThermoFisher Scientific) and counterstained with 1 μg/mL DAPI for 20 min (1:1000) to detect nuclei.

Techniques: Expressing, Control, Immunofluorescence, Confocal Microscopy

Journal: Prenatal Diagnosis

Article Title: Role of Myofibroblasts in the Repair of Iatrogenic Preterm Membranes Subjected to Mechanical Stimulation

doi: 10.1002/pd.6722

Figure Lengend Snippet: Effects of mechanical stimulation in human iatrogenic preterm AM. Preterm AM specimens were traumatized with a needed to create a 0.8‐mm defect and subjected to cyclic tensile strain (CTS) for 24 h. Mechanical stimulation was applied intermittently at 2% strain and 1 Hz frequency in the presence and absence of 50 μM Cx43 antisense (Cx43as). Absolute values for GAG (A), collagen (B) and elastin content (C) were normalized to dry tissue weight. PGE 2 release was quantified in media samples (D). Explants cultured without cyclic tensile strain (−CTS) were compared to +CTS specimens. Error bars represent the mean and SEM values of 24 replicates from three late third trimester iatrogenic preterm donors (34 + 0 weeks to 35 + 0 weeks, Case #1–3). Significant comparisons are indicated for −CTS and +CTS conditions where *** p < 0.001. All other comparisons (not indicated) were not significantly different.

Article Snippet: AM specimens were incubated with primary antibodies for mouse Cx43 (1:100, ThermoFisher Scientific, CX‐1B1) and rabbit αSMA (1:100, Abcam, ab5694) at 4°C overnight as described 44 Specimens were washed in PBS and incubated with secondary antibodies for Alexa Fluor 568 anti‐mouse or 488 anti‐rabbit at room temperature for 2 h (both 1:1000, ThermoFisher Scientific) and counterstained with 1 μg/mL DAPI for 20 min (1:1000) to detect nuclei.

Techniques: Cell Culture

Journal: Prenatal Diagnosis

Article Title: Role of Myofibroblasts in the Repair of Iatrogenic Preterm Membranes Subjected to Mechanical Stimulation

doi: 10.1002/pd.6722

Figure Lengend Snippet: Effects of mechanical stimulation on gene expression in human iatrogenic preterm AM. Preterm AM specimens were traumatized with a needed to create a 0.8‐mm defect and subjected to cyclic tensile strain (+CTS) for 4 and 24 h. Mechanical stimulation was applied intermittently at 2% strain and 1 Hz frequency in the presence and absence of 50 μM Cx43 antisense (Cx43as). Gene expression of Cx43 (A) and TGFβ (B) are presented as ratio values and normalized to control values. In all cases, error bars represent the mean and SEM values of 12 replicates from three late third trimester iatrogenic preterm donors (34 + 0 weeks to 35 + 0 weeks, Case #1–3). Significant comparisons are indicated for −CTS and +CTS conditions where *** p < 0.001. All other comparisons (not indicated) were not significantly different.

Article Snippet: AM specimens were incubated with primary antibodies for mouse Cx43 (1:100, ThermoFisher Scientific, CX‐1B1) and rabbit αSMA (1:100, Abcam, ab5694) at 4°C overnight as described 44 Specimens were washed in PBS and incubated with secondary antibodies for Alexa Fluor 568 anti‐mouse or 488 anti‐rabbit at room temperature for 2 h (both 1:1000, ThermoFisher Scientific) and counterstained with 1 μg/mL DAPI for 20 min (1:1000) to detect nuclei.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Cx43 mediates changes in myofibroblast contraction and collagen release in human amniotic membrane defects after trauma

doi: 10.1038/s41598-021-94767-4

Figure Lengend Snippet: Cx43 expression by myofibroblasts in wounded AM defects. After trauma, we observed increased cellularity around the wound edge (WE) and the presence of αSMA expressing myofibroblasts that had migrated into the defect site and released collagen in CAM and PAM specimens (Top view). In the epithelial layer, AECs formed a thick layer around the WE and the cells did no not migrate into the defect site (cross-sectional view). In the fibroblast layer, the dense region of collagen fibres in the defect site were highly polarized and begin to contract the edges of the wound (Cross-sectional view). We observed punctate staining of Cx43 expression (pink) by myofibroblasts with cytoplasmic localization after four days of trauma (high magnification) in CAM and PAM defects. Blue (nuclear), green (αSMA), pink (Cx43) and red signals (collagen) were detected by confocal microscopy and SHG imaging, respectively. Scale bars = 20 μΜ and 50 μM.

Article Snippet: Whole-mount control and wounded specimens were fixed in 4% PFA for 2 h and incubated with primary antibodies for mouse Cx43 (diluted 1:100, ThermoFisher Scientific, CX-1B1), rabbit vimentin (1:500, Abcam, ab137321) or rabbit αSMA (1:100, Abcam, ab5694) at 4 °C overnight, as previously described , .

Techniques: Expressing, Staining, Confocal Microscopy, Imaging

Journal: Scientific Reports

Article Title: Cx43 mediates changes in myofibroblast contraction and collagen release in human amniotic membrane defects after trauma

doi: 10.1038/s41598-021-94767-4

Figure Lengend Snippet: Cx43 protein expression in AM defects after trauma. The distribution of Cx43 was analysed per unit tissue area for comparisons between epithelial and fibroblast layer ( A ) and per AEC or myofibroblast cell nuclei ( B ) in control and wound edge (WE) specimens. In all cases, error bars represent the mean and SEM values for n = 9 replicates, where membranes were taken from three donors. Significant differences were found as indicated by ***p < 0.001. Comparisons between CAM or PAM specimens in the epithelial and fibroblast layer are indicated by +++ p < 0.001. All other comparisons were not statistically significant (not indicated).

Article Snippet: Whole-mount control and wounded specimens were fixed in 4% PFA for 2 h and incubated with primary antibodies for mouse Cx43 (diluted 1:100, ThermoFisher Scientific, CX-1B1), rabbit vimentin (1:500, Abcam, ab137321) or rabbit αSMA (1:100, Abcam, ab5694) at 4 °C overnight, as previously described , .

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Cx43 mediates changes in myofibroblast contraction and collagen release in human amniotic membrane defects after trauma

doi: 10.1038/s41598-021-94767-4

Figure Lengend Snippet: The effect of trauma on Cx43 and TGFβ 1 gene expression. Term human CAM and PAM specimens were cultured for up to four days after trauma. Gene expression of Cx43 ( A ) and TGFβ 1 ( B ) were presented as ratio values and normalised to Day 1 controls. In all cases, error bars represent the mean and SEM values of 9 replicates from three separate donors, where (*, ** or ***) indicates significant comparisons for CAM or PAM specimens. All other comparisons (not indicated) were not significantly different.

Article Snippet: Whole-mount control and wounded specimens were fixed in 4% PFA for 2 h and incubated with primary antibodies for mouse Cx43 (diluted 1:100, ThermoFisher Scientific, CX-1B1), rabbit vimentin (1:500, Abcam, ab137321) or rabbit αSMA (1:100, Abcam, ab5694) at 4 °C overnight, as previously described , .

Techniques: Gene Expression, Cell Culture

Journal: Experimental mechanics

Article Title: Probing Endothelial Cell Mechanics Through Connexin 43 Disruption

doi: 10.1007/s11340-018-00445-4

Figure Lengend Snippet: Effect of chalcone treatment on Cx43 structure. Immunostaining was performed in HUVEC monolayers after 6 hours of chalcone treatment. Green color represents Cx43 and Blue represents DAPI. Figure labels are as follows- control (a, b, and c), 0.2 μg/mL chalcone treated cells (d, e, and f) and 2μg/mL chalcone treated cells (g, h, and i). (obj: 20x; scale bar = 200 μm).

Article Snippet: After permeabilization, HUVEC monolayers were incubated with a 2% BSA blocking solution at 37°C and subsequently incubated with the following primary antibodies; mouse monoclonal Cx43 antibody (CX-1B1, Thermo-fisher), mouse monoclonal Cx40 antibody (2F9A11, Thermo-fisher), rabbit polyclonal Cx37 antibody (42–4400, Thermo-fisher), mouse monoclonal ZO-1 antibody (ZO-1 1A12,Thermofisher), rabbit polyclonal VE-Cadherin antibody (PA5–19612, Thermo-fisher) overnight at 4°C.

Techniques: Immunostaining

Journal: Experimental mechanics

Article Title: Probing Endothelial Cell Mechanics Through Connexin 43 Disruption

doi: 10.1007/s11340-018-00445-4

Figure Lengend Snippet: Phase contrast images of HUVEC monolayers after Cx43 disruption. Control phase contrast images of HUVECs at 30 mins (a), 2 hours (b) and 6 hours (c). Phase contrast images of HUVECs treated with 0.2μg/mL chalcone at 30 mins (d), 2 hours (e) and 6 hours (f). Phase contrast images of HUVECs treated with 2μg/mL chalcone at 30 mins (g), 2 hours (h) and 6 hours (i). Scale bar 500 × 500 μm.

Article Snippet: After permeabilization, HUVEC monolayers were incubated with a 2% BSA blocking solution at 37°C and subsequently incubated with the following primary antibodies; mouse monoclonal Cx43 antibody (CX-1B1, Thermo-fisher), mouse monoclonal Cx40 antibody (2F9A11, Thermo-fisher), rabbit polyclonal Cx37 antibody (42–4400, Thermo-fisher), mouse monoclonal ZO-1 antibody (ZO-1 1A12,Thermofisher), rabbit polyclonal VE-Cadherin antibody (PA5–19612, Thermo-fisher) overnight at 4°C.

Techniques:

Journal: Experimental mechanics

Article Title: Probing Endothelial Cell Mechanics Through Connexin 43 Disruption

doi: 10.1007/s11340-018-00445-4

Figure Lengend Snippet: Average normal intercellular stresses (Pa) of HUVEC monolayers during Cx43 inhibition. Figure labels show average normal intercellular stresses of HUVECs at 30 mins (a), 2 hours (b) and 6 hours (c) of control HUVECs and at 30 mins (d), 2 hours (e) and 6 hours (f) of HUVECs treated with 0.2μg/mL chalcone and at 30 mins (g), 2 hours (h) and 6 hours (i) of HUVECs treated with 2μg/mL chalcone. Scale bar 500 × 500 μm.

Article Snippet: After permeabilization, HUVEC monolayers were incubated with a 2% BSA blocking solution at 37°C and subsequently incubated with the following primary antibodies; mouse monoclonal Cx43 antibody (CX-1B1, Thermo-fisher), mouse monoclonal Cx40 antibody (2F9A11, Thermo-fisher), rabbit polyclonal Cx37 antibody (42–4400, Thermo-fisher), mouse monoclonal ZO-1 antibody (ZO-1 1A12,Thermofisher), rabbit polyclonal VE-Cadherin antibody (PA5–19612, Thermo-fisher) overnight at 4°C.

Techniques: Inhibition

Journal: Experimental mechanics

Article Title: Probing Endothelial Cell Mechanics Through Connexin 43 Disruption

doi: 10.1007/s11340-018-00445-4

Figure Lengend Snippet: Maximum shear intercellular stresses (Pa) of HUVEC monolayers during Cx43 inhibition. Figure labels show maximum shear intercellular stresses of HUVECs at 30 mins (a), 2 hours (b) and 6 hours (c) of control HUVECs and at 30 mins (d), 2 hours (e) and 6 hours (f) of HUVECs treated with 0.2μg/mL chalcone and at 30 mins (g), 2 hours (h) and 6 hours (i) of HUVECs treated with 2μg/mL chalcone. Scale bar 500 × 500 μm.

Article Snippet: After permeabilization, HUVEC monolayers were incubated with a 2% BSA blocking solution at 37°C and subsequently incubated with the following primary antibodies; mouse monoclonal Cx43 antibody (CX-1B1, Thermo-fisher), mouse monoclonal Cx40 antibody (2F9A11, Thermo-fisher), rabbit polyclonal Cx37 antibody (42–4400, Thermo-fisher), mouse monoclonal ZO-1 antibody (ZO-1 1A12,Thermofisher), rabbit polyclonal VE-Cadherin antibody (PA5–19612, Thermo-fisher) overnight at 4°C.

Techniques: Inhibition

Journal: Experimental mechanics

Article Title: Probing Endothelial Cell Mechanics Through Connexin 43 Disruption

doi: 10.1007/s11340-018-00445-4

Figure Lengend Snippet: rms traction (Pa) distributions of HUVEC monolayers during Cx43 disruption. Figure label shows control HUVECs (a, b and c), 0.2 μg/mL chalcone treated HUVECs (d, e and f) and 2 μg/mL chalcone treated HUVECS (g, h and i) at before any chalcone treatment (labels a, d and g), after 2 hours of experiment onset (labels b, e and h) and after 6 hours of experiment onset (labels c, f and i). Scale bar 500 × 500 μm.

Article Snippet: After permeabilization, HUVEC monolayers were incubated with a 2% BSA blocking solution at 37°C and subsequently incubated with the following primary antibodies; mouse monoclonal Cx43 antibody (CX-1B1, Thermo-fisher), mouse monoclonal Cx40 antibody (2F9A11, Thermo-fisher), rabbit polyclonal Cx37 antibody (42–4400, Thermo-fisher), mouse monoclonal ZO-1 antibody (ZO-1 1A12,Thermofisher), rabbit polyclonal VE-Cadherin antibody (PA5–19612, Thermo-fisher) overnight at 4°C.

Techniques:

Journal: Experimental mechanics

Article Title: Probing Endothelial Cell Mechanics Through Connexin 43 Disruption

doi: 10.1007/s11340-018-00445-4

Figure Lengend Snippet: Velocity in HUVEC monolayers during Cx43 inhibition. Figure labels are as follows—control (a, b and c), 0.2 μg/mL chalcone treated HUVECs (d, e and f) and 2μg/mL chalcone treated HUVECs (g, h and i) are showing velocity distributions at before chalcone treatment (labels a, d and g), after an hour of chalcone treatment (labels b, e and h) and at the end of experiment (labels c, f and i).

Article Snippet: After permeabilization, HUVEC monolayers were incubated with a 2% BSA blocking solution at 37°C and subsequently incubated with the following primary antibodies; mouse monoclonal Cx43 antibody (CX-1B1, Thermo-fisher), mouse monoclonal Cx40 antibody (2F9A11, Thermo-fisher), rabbit polyclonal Cx37 antibody (42–4400, Thermo-fisher), mouse monoclonal ZO-1 antibody (ZO-1 1A12,Thermofisher), rabbit polyclonal VE-Cadherin antibody (PA5–19612, Thermo-fisher) overnight at 4°C.

Techniques: Inhibition

Journal: Oncotarget

Article Title: Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

doi:

Figure Lengend Snippet: A. Endothelial cell localization of Cx32 in CRC cell-conditioned media. HMEC were stimulated with SW480-CM or SW620-CM for 6 h and double-stained for ZO-1 and Cx32. Representative micrographs showing the strong labelling of Cx32 induced by SW620-CM and the combined image of co-localization with ZO-1 (yellow); DAPI staining of nuclei ( n = 3, bar 20 μm). B. SW620-CM increase the Cx32 expression in HMEC. A higher Cx32 protein level was detected in response to SW620-CM compared with SW480-CM by immune-blot analysis (no cell expression in unstimulated HMEC). Representative of 5 experiments (Hsc70 as loading control; 150 μg/lane). C-D. In vitro tubulogenesis assay of HMEC pretreated or not (control IgG) with inhibitory monoclonal antibody against Cx32 (anti-Cx32 mAb). HMEC were plated on Matrigel-coated 24-well plates, incubated with SW620-CM for 6 h, and photographed. C. Representative photos of tube formation in HMEC intracellularly delivered with 0.2 μg anti-Cx32 mAb or control IgG (Bar 80 μm). The dotted areas are enlarged in the inserts on the right. Arrows indicated branch points. D. Number of branch points per field of view was quantified (at least 80 single cells were scored; mean ± SD, n = 4; * P < 0.01 vs control).

Article Snippet: Rabbit polyclonal anti-Cx43 (710700), mouse monoclonal anti-Cx43 (CX-1B1), anti-Cx32 (CX-2C2) and ZO-1 (ZO1-1A12) antibodies were from Invitrogen.

Techniques: Staining, Expressing, In Vitro, Incubation

Journal: European Journal of Histochemistry : EJH

Article Title: Diagnosis of sudden cardiac death due to early myocardial ischemia: An ultrastructural and immunohistochemical study

doi: 10.4081/ejh.2018.2866

Figure Lengend Snippet: Scoring method chosen for the evaluation of immunohistochemistry.

Article Snippet: Immunostaining was performed with the following primary antibodies: antifibronectin (rabbit monoclonal antibody, ab32419, diluted 1:200; Abcam, Cambridge, UK), anti-Cx43 (Abcam; rabbit monoclonal antibody, ab11370, diluted 1:2000), anti-npCx43 (Invitrogen, mouse monoclonal antibody, CX-1B1, diluted 1:50), anti-ZO-1 (Invitrogen, Carlsbad, CA, USA; mouse monoclonal antibody, ZO1-1A12, diluted 1:50).

Techniques: Immunohistochemistry

Journal: European Journal of Histochemistry : EJH

Article Title: Diagnosis of sudden cardiac death due to early myocardial ischemia: An ultrastructural and immunohistochemical study

doi: 10.4081/ejh.2018.2866

Figure Lengend Snippet: Immunohistochemistry performed against several potential clinical markers on control and AMI. A) Subject n. 27; fibronectin staining was graded as 1. B) Subject n. 13; fibronectin staining was graded as 3. C) Subject n. 22 (control); Cx43 resulted positive (grade 2) at ID, whereas it was negative in the cytoplasm. D) Subject n. 12; Cx43 resulted weakly positive (grade 1) at ID, whereas it was appreciably positive in the cytoplasm (grade 1). E) Subject n. 26 (control); npCx43 staining resulted weakly positive in the ID (grade 1), negative in the cytoplasm. F) Subject n. 11; npCx43 staining resulted strongly positive in the cytoplasm (grade 3).

Article Snippet: Immunostaining was performed with the following primary antibodies: antifibronectin (rabbit monoclonal antibody, ab32419, diluted 1:200; Abcam, Cambridge, UK), anti-Cx43 (Abcam; rabbit monoclonal antibody, ab11370, diluted 1:2000), anti-npCx43 (Invitrogen, mouse monoclonal antibody, CX-1B1, diluted 1:50), anti-ZO-1 (Invitrogen, Carlsbad, CA, USA; mouse monoclonal antibody, ZO1-1A12, diluted 1:50).

Techniques: Immunohistochemistry, Staining

Journal: European Journal of Histochemistry : EJH

Article Title: Diagnosis of sudden cardiac death due to early myocardial ischemia: An ultrastructural and immunohistochemical study

doi: 10.4081/ejh.2018.2866

Figure Lengend Snippet: P-value estimation from the Mann-Whitney U test.

Article Snippet: Immunostaining was performed with the following primary antibodies: antifibronectin (rabbit monoclonal antibody, ab32419, diluted 1:200; Abcam, Cambridge, UK), anti-Cx43 (Abcam; rabbit monoclonal antibody, ab11370, diluted 1:2000), anti-npCx43 (Invitrogen, mouse monoclonal antibody, CX-1B1, diluted 1:50), anti-ZO-1 (Invitrogen, Carlsbad, CA, USA; mouse monoclonal antibody, ZO1-1A12, diluted 1:50).

Techniques:

Journal: Oncotarget

Article Title: Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

doi:

Figure Lengend Snippet: A. Endothelial cell localization of Cx32 in CRC cell-conditioned media. HMEC were stimulated with SW480-CM or SW620-CM for 6 h and double-stained for ZO-1 and Cx32. Representative micrographs showing the strong labelling of Cx32 induced by SW620-CM and the combined image of co-localization with ZO-1 (yellow); DAPI staining of nuclei ( n = 3, bar 20 μm). B. SW620-CM increase the Cx32 expression in HMEC. A higher Cx32 protein level was detected in response to SW620-CM compared with SW480-CM by immune-blot analysis (no cell expression in unstimulated HMEC). Representative of 5 experiments (Hsc70 as loading control; 150 μg/lane). C-D. In vitro tubulogenesis assay of HMEC pretreated or not (control IgG) with inhibitory monoclonal antibody against Cx32 (anti-Cx32 mAb). HMEC were plated on Matrigel-coated 24-well plates, incubated with SW620-CM for 6 h, and photographed. C. Representative photos of tube formation in HMEC intracellularly delivered with 0.2 μg anti-Cx32 mAb or control IgG (Bar 80 μm). The dotted areas are enlarged in the inserts on the right. Arrows indicated branch points. D. Number of branch points per field of view was quantified (at least 80 single cells were scored; mean ± SD, n = 4; * P < 0.01 vs control).

Article Snippet: Rabbit polyclonal anti-Cx43 (710700), mouse monoclonal anti-Cx43 (CX-1B1), anti-Cx32 (CX-2C2) and ZO-1 (ZO1-1A12) antibodies were from Invitrogen.

Techniques: Staining, Expressing, In Vitro, Incubation

Journal: Oncotarget

Article Title: Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

doi:

Figure Lengend Snippet: A. Both SW480 and SW620 cell-conditioned media triggered ATP accumulation in HMEC bath medium within 6 h. Extracellular ATP was measured by Luciferase assay (means ± S.D. n = 4; * P -values < 0.01 vs control). B. Panx-1 mRNA expression in HMEC after 6 h of control or CRC cell-CM exposures. A drastic decrease in Panx-1 expression was observed with SW620-CM (means ± S.D. n = 3; * P -values < 0.05 vs control). C. Panx-1 protein expression in HMEC was unchanged by exposure to SW480- and SW-620-CM for 6 h (mean ±SD, P -value s = 0.4980 Mann-Whitney U test; n = 4). D. Cell surface localization of Panx-1 was decreased in HMEC exposed to SW620-CM for 6 h. Arrows indicated Panx-1 plaques at the plasma membrane. DAPI staining of nuclei. Optical section of 0.5 μm thickness ( n = 5, Bar 12 μm). E. SW620-CM-triggered ATP release is inhibited by gap junction blocker, carbenoxolone (carben., 100 μM, 30 min) and by neutralizing Cx32 antibody (0.2 μg anti-Cx32 mAb) in HMEC (means ± S.D. * P -values < 0.01 vs control; n = 3).

Article Snippet: Rabbit polyclonal anti-Cx43 (710700), mouse monoclonal anti-Cx43 (CX-1B1), anti-Cx32 (CX-2C2) and ZO-1 (ZO1-1A12) antibodies were from Invitrogen.

Techniques: Luciferase, Expressing, MANN-WHITNEY, Staining

Journal: Oncotarget

Article Title: Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

doi:

Figure Lengend Snippet: A. IL-8 secretion in conditioned media from SW480 and SW620 cells was examined through ELISA. CRC cells were exposed or not to the HMEC-CM. All cell media were collected after 6 h (mean ± SD, * P -values < 0.01 Mann-Whitney U test and Kruskal-Wallis test; n = 4). B. SW620-CM increase the endothelial expression of the CXCR2 receptor. A slight but significant increase in optical density (OD; relative to control) of bands was detected in response to SW620-CM compared with unstimulated HMEC ( P -values < 0.01 Mann-Whitney U test and Kruskal-Wallis test; n = 4). No inhibitory effect was observed by pre-treating HMEC with SB225002 (200 nM), the CXCR2 antagonist. Representative of 4 experiments (Hsc70 as loading control; 100 μg/lane). C–D. Endothelial CXCR2 conveys angiogenic effects of SW620-CM. HMEC were pretreated or not with neutralizing anti-CXCR2 antibody (anti-CXCR2 mAb; 10 μg/ml) or SB225002. Cells were exposed to SW620-CM or human recombinant rh IL-8 (1 ng/ml) for 6 h. C. Representative Images of tube formation (Bar 80 μm). The dotted areas are enlarged in the inserts on the right. Arrows indicated branch points. D. Number of branch points per field of view was quantified (mean ± SD, n = 4; * P < 0.01 vs control). E. Blocking CXCR2 significantly diminished SW620-CM-induced expression of Cx32 in HMEC ( P -values < 0.01 vs SW620-CM Mann-Whitney U test and Kruskal-Wallis test; n = 3). HMEC were exposed to cell-conditioned media for 6 h. In some cases, HMEC were pretreated with anti-CXCR2 mAb or SB225002, as indicated. This is a representative of three experiments with similar results (Hsc70 as loading control; 100 μg/lane).

Article Snippet: Rabbit polyclonal anti-Cx43 (710700), mouse monoclonal anti-Cx43 (CX-1B1), anti-Cx32 (CX-2C2) and ZO-1 (ZO1-1A12) antibodies were from Invitrogen.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Expressing, Recombinant, Blocking Assay

Journal: Oncotarget

Article Title: Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

doi:

Figure Lengend Snippet: In vitro tubulogenesis assay of HMEC pretreated or not (control IgG) with inhibitory monoclonal antibody against Cx32 (0.2 μg/ml anti-Cx32mAb). HMEC were plated on Matrigel-coated 24-well plates, incubated with SW620-CM or human recombinant rh IL-7 (1 ng/ml) for 6 h, and photographed (Bar 80 μm). The dotted areas are enlarged in the inserts on the right. Arrows indicated branch points. Histogram shows the number of branch points per field of view (at least 80 single cells were scored; mean ± SD, n = 4; * P < 0.01 vs control). Blocking Cx32 decreases rh IL-7- and SW620-CM-induced tube formation (* P < 0.05 vs control; n = 3).

Article Snippet: Rabbit polyclonal anti-Cx43 (710700), mouse monoclonal anti-Cx43 (CX-1B1), anti-Cx32 (CX-2C2) and ZO-1 (ZO1-1A12) antibodies were from Invitrogen.

Techniques: In Vitro, Incubation, Recombinant, Blocking Assay

Journal: Oncotarget

Article Title: Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

doi:

Figure Lengend Snippet: The diagram shows the endothelial cell (EC) expression of both Cx32 and Cx43 as well as their ability to form hemi-channels or gap junction channels with CRC cells at the microvascular level. Cancer cells from a primary tumor (here, SW480 cells) locally invade the surrounding tissue, enter the microvasculature of the blood system (passive intravasation), survive and translocate through the bloodstream to micro-vessels of distant tissues. SW480 cells release HSP27 that favors the establishment of GJIC, via Cx43-channels, with the underlying endothelium. This direct cell-to-cell communication contributes to their trans-endothelial migration TEM (active extravasation). In contrast, cancer cells from a metastatic site (here, SW620 cells) release larger amount of chemokines, increasing the endothelial expression of the receptor CXCR2. In turn, CXCR2 promotes both endothelial Cx32 expression and tubulogenesis. The release of ATP through Cx32 hemi-channels from ECs and the subsequent ATP-mediated activation of purinergic P2Y2 receptors could modulate crosstalk between ECs and metastatic cancer cells, favoring neo-angiogenesis in metastatic foci.

Article Snippet: Rabbit polyclonal anti-Cx43 (710700), mouse monoclonal anti-Cx43 (CX-1B1), anti-Cx32 (CX-2C2) and ZO-1 (ZO1-1A12) antibodies were from Invitrogen.

Techniques: Expressing, Migration, Activation Assay