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Supplementary figure 1: Electron microscope images of isolated mitochondria Representative pictures of mitochondrial pellets showing successful mitochondrial isolation following the protocol described in section 2.3.1.
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Supplementary figure 1: Electron microscope images of isolated mitochondria
Representative pictures of mitochondrial pellets showing successful mitochondrial isolation following the protocol described in section 2.3.1.
A
B
Supplementary figure 2: Gating strategy used to characterise isolated mitochondria from cellular debris and background.
A. Cellular debris and background were excluded from isolated mitochondria by adjusting forward (FSC) and side scatter (SSC) voltages and thresholds. B. The gating strategy was validated by staining mitochondria with MitoTracker Green. Graphs were obtained using FlowJo (v10.1). Data shown are representative of 10 placentae.
Supplementary figure 3: Results of preliminary investigation of the effect of 25, 50, and 100μg/mL ID2 or IIC5 on explant ROS production
A. Total ROS and rate of ROS production of explants during three hours of incubation with ID2 or control antibodies (n=5). B. Total ROS and rate of ROS production of explants during three hours of incubation with IIC5 or control antibodies (n=5).
Supplementary figure 4: Rate of ROS production by placental explants measured by fluorescence during incubation with ID2 or control IgG antibodies, with and without treatment compounds.
A. Rate of ROS production of explants during three hours of incubation with ID2 or control antibodies (n=16). B. Effect of 10 or 20µM melatonin (M) on the rate of ROS production of explants during three hours of incubation with ID2 (n=7). C. Effect of 0.1, 1, or 10µM MitoQ (MQ) on the rate of ROS production of explants during three hours of incubation with ID2 (n=4). D. Effect of 50 or 100nM chloroquine (CLQN) on the rate of ROS production of explants during three hours of incubation with ID2 (n=4). P-value ≤ 0.05 (*).
ID2 JO2
Concentration (μg/mL)
Mean control value
EMM
LCL
UCL
% change
% difference
CI leak
25
0.55
-0.20
-0.55
0.16
63.76
-36.24
50
0.60
-0.10
-0.47
0.28
83.96
-16.04
100
0.38
-0.10
-0.45
0.26
75.02
-24.98
CI OXPHOS
25
1.55
-0.28
-0.80
0.25
82.03
-17.97
50
1.35
0.35
-0.17
0.88
126.13
26.13
100
1.23
-0.03
-0.52
0.47
97.80
-2.20
CI + CII OXPHOS
25
6.64
-0.15
-1.00
0.71
97.82
-2.18
50
6.37
1.54
0.64
2.44
124.12
24.12
100
6.33
0.25
-0.60
1.10
103.90
3.90
CI + CII + mGPDH OXPHOS
25
8.21
-0.09
-1.09
0.92
98.92
-1.08
50
7.96
1.93
0.86
2.99
124.22
24.22
100
8.03
0.56
-0.44
1.57
107.03
7.03
CI + CII + mGPDH uncoupled
25
8.95
-0.30
-1.39
0.78
96.60
-3.40
50
8.59
1.79
0.64
2.94
120.85
20.85
100
8.95
-0.21
-1.30
0.87
97.62
-2.38
CI + CII + mGPDH uncoupled + rotenone
25
6.68
0.15
-0.73
1.02
102.17
2.17
50
6.41
1.53
0.60
2.45
123.83
23.83
100
6.51
0.31
-0.57
1.18
104.69
4.69
Complex IV
25
31.95
-1.22
-3.60
1.16
96.19
-3.81
50
26.07
3.42
0.90
5.94
113.11
13.11
100
29.70
-0.33
-2.71
2.05
98.90
-1.10
Δ CII OXPHOS
25
5.09
0.10
-0.45
0.66
102.04
2.04
50
5.02
1.18
0.60
1.77
123.58
23.58
100
5.10
0.27
-0.28
0.83
105.37
5.37
Δ mGPDH OXPHOS
25
1.57
0.06
-0.25
0.36
103.49
3.49
50
1.59
0.39
0.07
0.71
124.60
24.60
100
1.70
0.32
0.02
0.62
118.65
18.65
Δ CCCP
25
1.52
-0.07
-0.49
0.34
95.27
-4.73
50
1.49
0.51
0.07
0.95
134.06
34.06
100
1.68
-0.45
-0.86
-0.03
73.35
-26.65
Rotenone sensitive
25
2.27
-0.45
-0.99
0.09
80.29
-19.71
50
2.18
0.26
-0.31
0.84
112.10
12.10
100
2.44
-0.52
-1.06
0.02
78.78
-21.22
Supplementary table 1: Summary of comparison of ID2- to control-treated explant mitochondrial respiration rates.
Control-treated explant values (mean control value) were subtracted from ID2-treated explant values to calculate estimated marginal mean (EMM), lower confidence level (LCL), upper confidence level (UCL), mean ID2-treated explant respiration as a percentage of control respiration (% change), and the change in respiration as a percentage of control respiration (% difference). Statistically significant (p-value ≤ 0.05) changes are indicated in bold.
Concentration (μg/mL)
Mean control value
EMM
LCL
UCL
% change
% difference
CI leak
25
-2.58
1.50
-1.31
4.30
42.08
-57.92
50
-2.02
1.23
-1.77
4.23
39.22
-60.78
100
0.55
-0.53
-3.34
2.27
3.03
-96.97
CI OXPHOS
25
-0.82
0.94
-1.61
3.49
-14.96
-114.96
50
-0.31
-0.07
-2.80
2.66
122.79
22.79
100
1.37
-0.11
-2.66
2.44
92.12
-7.88
CI + CII OXPHOS
25
0.15
2.43
0.42
4.44
1720.40
1620.40
50
1.73
-0.15
-2.30
1.99
91.20
-8.80
100
2.21
-0.41
-2.42
1.60
81.31
-18.69
CI + CII + mGPDH OXPHOS
25
15.21
3.56
-2.51
9.64
123.44
23.44
50
16.03
0.22
-6.27
6.71
101.37
1.37
100
15.63
5.66
-0.42
11.74
136.22
36.22
CI + CII + mGPDH uncoupled
25
19.76
3.39
-2.52
9.31
117.17
17.17
50
20.04
-1.83
-8.16
4.49
90.86
-9.14
100
19.70
4.40
-1.52
10.32
122.33
22.33
CI + CII + mGPDH uncoupled + rotenone
25
21.16
2.56
-4.76
9.88
112.10
12.10
50
21.19
-1.56
-9.38
6.26
92.63
-7.37
100
19.57
6.86
-0.46
14.18
135.03
35.03
Antimycin A
25
33.83
5.11
-3.63
13.85
115.12
15.12
50
31.94
2.02
-7.32
11.37
106.34
6.34
100
33.93
7.46
-1.28
16.21
122.00
22.00
Δ CII OXPHOS
25
0.97
1.49
-0.16
3.14
253.94
153.94
50
2.04
-0.08
-1.84
1.68
95.97
-4.03
100
0.83
-0.31
-1.95
1.34
63.24
-36.76
Δ mGPDH OXPHOS
25
15.06
1.13
-4.14
6.40
107.53
7.53
50
14.30
0.37
-5.26
6.00
102.60
2.60
100
13.42
6.07
0.81
11.34
145.26
45.26
Δ CCCP
25
4.55
-0.17
-2.54
2.21
96.33
-3.67
50
4.01
-2.05
-4.59
0.49
48.82
-51.18
100
4.07
-1.26
-3.64
1.12
69.02
-30.98
Rotenone sensitive
25
1.40
-0.83
-3.72
2.05
40.50
-59.50
50
1.14
0.27
-2.81
3.36
123.83
23.83
100
-0.13
2.45
-0.43
5.34
-1829.73
-1929.73
Supplementary table 2: Summary of comparison of ID2- to control-treated explant mitochondrial ROS production rates.
Control-treated explant values (mean control value) were subtracted from ID2-treated explant values to calculate estimated marginal mean (EMM), lower confidence level (LCL), upper confidence level (UCL), mean ID2-treated explant ROS production rates as a percentage of control explant ROS production rates (% change), and the change in explant ROS production rates as a percentage of control explant ROS production rates (% difference). Statistically significant (p-value ≤ 0.05) changes are indicated in bold.
Concentration (μg/mL)
Mean control value
EMM
LCL
UCL
% change
% difference
CI leak
25
0.55
-0.15
-0.59
0.30
73.48
-26.52
50
0.60
-0.09
-0.54
0.36
85.16
-14.84
100
0.38
0.27
-0.15
0.69
171.55
71.55
CI OXPHOS
25
1.55
-0.35
-0.88
0.17
77.08
-22.92
50
1.35
-0.12
-0.65
0.40
90.81
-9.19
100
1.23
0.23
-0.26
0.73
119.14
19.14
CI + CII OXPHOS
25
6.64
-0.77
-1.78
0.23
88.35
-11.65
50
6.37
0.61
-0.40
1.61
109.51
9.51
100
6.33
0.65
-0.30
1.60
110.24
10.24
CI + CII + mGPDH OXPHOS
25
8.21
-0.79
-1.95
0.38
90.42
-9.58
50
7.96
0.95
-0.22
2.11
111.88
11.88
100
8.03
0.73
-0.37
1.84
109.13
9.13
CI + CII + mGPDH uncoupled
25
8.95
-1.21
-2.42
0.00
86.51
-13.49
50
8.59
0.74
-0.47
1.94
108.56
8.56
100
8.95
0.11
-1.03
1.25
101.25
1.25
CI + CII + mGPDH uncoupled + rotenone
25
6.68
-0.73
-1.62
0.15
89.03
-10.97
50
6.41
0.67
-0.22
1.55
110.39
10.39
100
6.51
0.30
-0.54
1.13
104.55
4.55
Complex IV
25
31.95
-1.32
-4.30
1.66
95.87
-4.13
50
26.07
2.20
-0.78
5.18
108.44
8.44
100
29.70
-1.06
-3.87
1.75
96.44
-3.56
Δ CII OXPHOS
25
5.09
-0.43
-1.17
0.32
91.64
-8.36
50
5.02
0.73
-0.01
1.47
114.57
14.57
100
5.10
0.40
-0.30
1.11
107.91
7.91
Δ mGPDH OXPHOS
25
1.57
-0.01
-0.29
0.27
99.26
-0.74
50
1.59
0.34
0.06
0.61
121.22
21.22
100
1.70
0.08
-0.18
0.35
104.87
4.87
Δ CCCP
25
1.52
-0.44
-0.98
0.09
70.79
-29.21
50
1.49
-0.28
-0.81
0.26
81.43
-18.57
100
1.68
-0.38
-0.88
0.13
77.44
-22.56
Rotenone sensitive
25
2.27
-0.48
-1.05
0.09
78.99
-21.01
50
2.18
0.07
-0.50
0.64
103.36
3.36
100
2.44
-0.17
-0.71
0.37
93.07
-6.93
Supplementary table 3: Summary of comparison of IIC5- to control-treated explant mitochondrial respiration rates.
Control-treated explant values (mean control value) were subtracted from IIC5-treated explant values to calculate estimated marginal mean (EMM), lower confidence level (LCL), upper confidence level (UCL), mean IIC5-treated explant respiration as a percentage of control respiration (% change), and the change in respiration as a percentage of control respiration (% difference). Statistically significant (p-value ≤ 0.05) changes are indicated in bold.
Concentration (μg/mL)
Mean control value
EMM
LCL
UCL
% change
% difference
CI leak
25
0.65
-2.23
-4.71
0.25
-243.57
-343.57
50
0.12
-0.66
-3.32
1.99
-440.65
-540.65
100
-0.06
-0.90
-3.39
1.58
1540.60
1440.60
CI OXPHOS
25
2.20
-2.99
-5.93
-0.04
-35.90
-135.90
50
1.30
-0.98
-4.13
2.17
24.52
-75.48
100
0.34
0.05
-2.89
3.00
116.16
16.16
CI + CII OXPHOS
25
3.33
-1.75
-4.29
0.79
47.39
-52.61
50
2.76
-0.82
-3.54
1.89
70.08
-29.92
100
1.44
0.35
-2.19
2.89
124.53
24.53
CI + CII + mGPDH OXPHOS
25
20.02
-0.58
-3.57
2.41
97.10
-2.90
50
16.24
2.41
-0.79
5.61
114.87
14.87
100
19.85
-1.55
-4.55
1.44
92.17
-7.83
CI + CII + mGPDH uncoupled
25
21.70
-1.46
-4.97
2.04
93.25
-6.75
50
18.99
1.83
-1.92
5.58
109.62
9.62
100
21.27
-1.17
-4.68
2.34
94.48
-5.52
CI + CII + mGPDH uncoupled + rotenone
25
23.42
-1.02
-5.31
3.27
95.63
-4.37
50
20.37
2.60
-1.99
7.18
112.74
12.74
100
22.60
-0.09
-4.38
4.21
99.62
-0.38
Antimycin A
25
39.20
-2.62
-7.07
1.83
93.31
-6.69
50
33.00
5.09
0.33
9.86
115.44
15.44
100
36.54
-0.93
-5.38
3.53
97.47
-2.53
Δ CII OXPHOS
25
1.13
1.25
-0.12
2.62
210.78
110.78
50
1.45
0.21
-1.26
1.67
114.18
14.18
100
1.10
0.29
-1.09
1.66
126.04
26.04
Δ mGPDH OXPHOS
25
16.70
1.16
-1.30
3.62
106.94
6.94
50
13.48
3.24
0.62
5.87
124.06
24.06
100
18.41
-1.92
-4.38
0.54
89.59
-10.41
Δ CCCP
25
1.68
-0.95
-3.17
1.28
43.46
-56.54
50
2.75
-0.56
-2.94
1.82
79.73
-20.27
100
1.42
0.38
-1.85
2.61
126.48
26.48
Rotenone sensitive
25
1.72
0.51
-2.43
3.45
129.64
29.64
50
1.38
0.70
-2.44
3.85
150.87
50.87
100
1.33
1.06
-1.89
4.00
179.60
79.60
Supplementary table 4: Summary of comparison of IIC5- to control-treated explant mitochondrial ROS production rates.
Control-treated explant values (mean control value) were subtracted from IIC5-treated explant values to calculate estimated marginal mean (EMM), lower confidence level (LCL), upper confidence level (UCL), mean IIC5-treated explant ROS production rates as a percentage of control explant ROS production rates (% change), and the change in explant ROS production rates as a percentage of control explant ROS production rates (% difference). Statistically significant (p-value ≤ 0.05) changes are indicated in bold.
