Non activated macrophages
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Non activated macrophages -60 mV +80 mV 300 pA 200 ms J774. G8 cells 2 mM LaCl3 10 µM CBX 1 mM Prob 1 µM Mefl Activated Peritoneal Macrophages 100 µM LaCl3 10 µM CBX 5 pA 200 ms 1 mM Prob 1 µM Mef 100 pA 200 ms A B
description
+80 mV. -60 mV. B. J774. G8 cells. A. Non activated macrophages. 2 mM LaCl3. 5 pA. 200 ms. Activated Peritoneal Macrophages. 10 µM CBX. 100 µM LaCl3. 1 mM Prob. 10 µM CBX. 1 mM Prob. 1 µM Mefl. 1 µM Mef. 300 pA. 100 pA. 200 ms. 200 ms. C. D. E. - PowerPoint PPT Presentation
Transcript of Non activated macrophages
Non activated macrophages
-60 mV
+80 mV
300 pA
200 ms
J774. G8 cells
2 mM LaCl3
10 µM CBX
1 mM Prob
1 µM Mefl
Activated Peritoneal Macrophages
100 µM LaCl3
10 µM CBX
5 pA
200 ms
1 mM Prob
1 µM Mef
100 pA200 ms
A B
-80 -40 40 80 120
-120-80-40
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Control
FFA
LaCl3+
CBX
Probenecid
J774.G8 cells
Mefloquine
Voltage (mV)
Cu
rren
t (p
A)
-80 -40 40 80 120
-120
-80
-40
40
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160
200 Control
FFA
LaCl3+
CBX
Probenecid
Activated Peritoneal Macrophages
Mefloquine
Voltage (mV)
Cu
rren
t (p
A)
-80 -40 40 80 120
-120
-80
-40
40
80
120
160
200
Non activated Peritoneal Macrophages
Control
Voltage (mV)
Cu
rren
t (p
A)
C D
E
Supplementary Fig. 1 Functional mouse Pannexin- 1 ionic currents in non- activated peritoneal macrophages, LPS activated peritoneal macrophages and J774 cells. A: Ionic currents in response to voltage steps from -120 to 80 mV recorded from LPS activated peritoneal macrophages. Prob (1 mM), CBX (10 M) and Mef (1 M) block the outward current. B: Ionic currents in response to voltage steps from -120 to 80 mV recorded from J774 cells. Prob (1 mM), CBX (10 M) and Mef (1 M) block the outward current. In non-activated peritoneal macrophages, we observed only tiny ionic currents. C- E: I-V relationship of LPS activated peritoneal macrophages, J774 cells or non-activated peritoneal macrophages, respectively. In C and D, the J774 cells and LPS activated macrophages were treated or not with 2 mM LaCl3, 10 M CBX, 1 mM Prob, 100 M FFA or 1 M Mef. In E, we plotted the I-V relationship of non activated macrophages. The values represent the means SD of 2-4 experiments performed on different days in the case of the LPS activated peritoneal macrophages and J774 cells. Non-activated peritoneal macrophages values represent the means SD of 25 records performed on five days.
Activated Peritoneal Macrophages
100 µM LaCl3
10 µM CBX
1 mM Prob
1 µM Mef
Control
Washout
LaCl3
Control
Washout
Prob
Control
Washout
CBX
Control
Washout
Mef
A B
C D
Supplementary Fig. 2. Functional mouse Pannexin- 1 ionic currents in LPS activated peritoneal macrophages. Representative traces of 2 s voltage ramps from the holding potential of -120 mV to +80 mV applied in activated peritoneal macrophages before, during and after drug administration in whole-cell patchclamp configuration. In A: 100 μM LaCl3, B: 1 mM Prob, C: 10 μM CBX or D: 1 μM Mef.
J774. G8 cells
v
v
100 µM LaCl3
10 µM CBX
1 mM Prob
1 µM Mef
Control
Washout
LaCl3
Control
Washout
Prob
Control
Washout
Mef
Control
Washout
CBX
A B
C D
Supplementary Fig. 3. Functional mouse Panx1 ionic currents in J774 cells. Representative traces of 2 s voltage ramps from the holding potential of -120 mV to +80 mV applied in J774 cells before, during and after drug administration in whole-cell patch-clamp configuration. In A: 100 μM LaCl3, B: 1 mM Prob, C: 10 μM CBX or D: 1 μM Mef.
Control
Non-activated Peritoneal Macrophages
Supplementary Fig. 4. Functional mouse Panx1 ionic currents in non-activated peritoneal macrophages. Representative traces of 2 s voltage ramps from the holding potential of -120 mV to +80 mV applied in non-activated peritoneal macrophages before and after to wash the solution in whole-cell patch-clamp configuration.
CTL
Acetate + 5 mM ATP
Acetate
0.0
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0.6
0.8
1.0
CTL
Acetate
ATP
ATP + Acet n.s.
permeabilized cells / total cells
***
***
5 mM ATP
Supplementary Fig. 5. Dye uptake assay with hemichannels blockage by low intracellular pH induced by acetate. A: Dye uptake assay in activated peritoneal mouse macrophages treated or not with acetate 10 mM for inhibition of hemichannels. Left panels are contrast phase, and right panels are EB uptake. B: Quantification of fluorescence within a square (mean of 100 cells per square) treated 10 mM for inhibition of hemichannels . ANOVA analyses were performed on the raw data. *p˂0.05, **p˂0.01, and ***p˂0.001 compared to the ATP group. Experiments were performed in triplicate, and the quantification repeated at least in three days.
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