human adult dermal lymphatic microvascular ecs (hdly-mvecs)  (Lonza)

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: Juxtamembrane 2 (JM2) is not cytotoxic. Human microvascular endothelial cells (HMVECs) were treated with JM2 at 12.5, 25, 50, 100, and 200 μM concentrations for 2 h at 37°C, 5% CO2. The media were then sampled for lactate dehydrogenase (LDH) as a marker for cell death. No significant differences were observed between control (No Peptide) and any concentration of JM2. n = 3, with each n representing the average of 3 replicates; error bars represent SE.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Marker, Concentration Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: JM2 specifically enhances connexin43 (Cx43)-β-tubulin interaction. A and B: Cx43-HeLa cell lysates were incubated with β-tubulin-glutathione S-transferase (GST) bound to glutathione-Sepharose beads plus vehicle (H2O; Veh) or 25 μM JM2. Eluted proteins were analyzed by immunoblotting for Cx43 (A) or β-tubulin (B). β-Tubulin pulled down Cx43 in control conditions (vehicle), and this was increased in the presence of JM2 (A, “β-tubulin Pull-Down”), indicating that JM2 specifically enhances the interaction between Cx43 and β-tubulin. This was supported by the observation that the input amounts of Cx43 and β-tubulin subjected to pull-down were equivalent between Veh and JM2 treatments (A and B “Input” blots, respectively) and that JM2 did not affect the amount of β-tubulin pulled down by a β-tubulin antibody (B, “β-tubulin Pull-Down”). C: HMVECs were treated for 2 h with either vehicle (H2O; “Veh”), 50 μM JM2, or 50 μM control peptide (CP). Cells were fixed and labeled for Cx43-β-tubulin interaction (green), and the nucleus (blue). Differential interference contrast (DIC; gray scale) was used to approximate cell-cell borders (dashed lines). Boxed regions indicate the location of the enlarged insets. Scale bar = 10 μm. D: Cx43-β-tubulin interaction was quantified as the number of Duolink signals detected per cell. The number of signals detected in JM2-treated cells was significantly greater than in vehicle (Veh) or control peptide (CP). n = 3, with each n representing the average of 5 replicates; error bars represent SD. Nonlinear level adjustments were applied to Western blot images to enhance visibility. ****P < 0.001.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Incubation, Western Blot, Labeling

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: JM2 decreases gap junction (GJ) size while increasing labeling for microtubules. HMVECs were treated for 2 h with either vehicle (H2O; “Veh”) or 50 μM JM2. A: cells were fixed and labeled for Cx43 (cyan), α-tubulin (yellow), and the nucleus (magenta); Scale bar = 10 μm. B: total Cx43 fluorescence was unaffected by JM2. C and D: similarly, GJ number was measured and found to be unaffected by JM2 (C), while there was a significant decrease in the level of cell border-associated Cx43 fluorescence (D). E: consistent with the reduction in cell border Cx43 fluorescence, GJ size was reduced. F: concomitantly, the level of intracellular Cx43 fluorescence was significantly increased in JM2-treated cells. G: microtubule fluorescence was also significantly increased by JM2 treatment. For all graphs, n = 3, with each n representing the average of 5 replicates; error bars represent SD. *P < 0.05, **P < 0.01.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Labeling, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: JM2 increases the accumulation of Cx43-containing secretory vesicles. HMVECs were treated for 2 h with either vehicle (H2O; “Veh”) or 50 μM JM2. A: cells were fixed and labeled for Cx43 (red), the trans-Golgi network (TGN) protein TGN38 (green), and the nucleus (blue). Arrows indicate a number of puncta with Cx43 TGN38 colocalization that are likely secretory vesicles; Scale bar = 10 μm. B–D: colocalization of Cx43 and TGN38 was significantly increased by JM2 as assessed by Pearson’s coefficient (B), the amount of colocalized pixels as a % of total Cx43 pixels (C), and the amount of colocalized pixels as a % of total TGN38 pixels (D); n = 3, with each n representing the average of 3 replicates; error bars represent SD. *P < 0.05, **P < 0.01.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Labeling

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: JM2 treatment inhibits both gap junction intercellular communication and hemichannel function. A: HMVECs were treated with either vehicle (H2O and EtOH), 50 μM JM2, or 50 μM flufenamic acid (FFA) for 2 h, and calcein-AM was loaded during the final 30 min. Selected cells (arrows) were then photobleached by high-intensity laser light and recovery was monitored. Scale bar = 20 μm. B: average fluorescence readings were plotted over time. n = 3, with each n representing the average of 3 replicates; error bars represent SE. C and D: nonlinear regression to an exponential decay function was used to determine the maximum predicted recovery (F∞; C) and the recovery rate constant (k; D). Error bars represent SD. E: connexin43 (Cx43) and and wild-type (WT) HeLa cells were treated with either vehicle (H2O and EtOH; “Veh”), 50 μM JM2, 50 μM FFA, or 5 μM mefloquine (MFQ) for 2 h, then exposed to ethidium bromide (EtBr) for 15 min. Cells were fixed and imaged, and the quantified relative fluorescence is displayed. Dashed line indicates the level of autofluorescence in Cx43-HeLa cells. F: HMVECs were treated, imaged, and quantified as in E. For graphs in E and F, n = 4, with each n representing the average of 5 replicates; error bars represent SD. *P < 0.05, **P < 0.01, ***P< 0.001, ****P < 0.001.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: A control peptide does not affect hemichannel function. HMVECs were treated with either vehicle (H2O and EtOH; “Veh”), 50 μM control peptide (CP), or 50 μM FFA for 2 h, then exposed to EtBr for 15 min. Cells were fixed and imaged, and the quantified relative fluorescence is displayed. No significant difference was observed between vehicle control and control peptide treatment conditions; n = 3, with each n representing the average of 5 replicates; error bars represent SD. *P < 0.05.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Fluorescence