Supplementary Video 1. Loss of MitoTracker in WT BMDM. WT BMDM were LPS primed
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1 Supplementary Video 1. Loss of MitoTracker in WT BMDM. WT BMDM were LPS primed for 3-4 hrs and treated with 20 μm nigericin two minutes into imagining. Cells were labeled with MitoTracker (red) and psiva (green) prior to imaging. Cells were imaged via confocal live cell microscopy for one hour. Video clip begins two frames prior to nigericin treatment. Supplementary Video 2. No Loss of MitoTracker in NLRP3 -/- BMDM. NLRP3 -/- BMDM were LPS primed for 3-4 hrs and treated with 20 μm nigericin two minutes into imagining. Cells were labeled with MitoTracker (red) and psiva (green) prior to imaging. Cells were imaged via confocal live cell microscopy for one hour. Video clip begins two frames prior to nigericin treatment. Supplementary Video 3. Nigericin induces loss of mtdna in WT BMDM. LPS primed WT BMDM were labeled with PicoGreen (green) and MitoTracker Red (red) and then treated with nigericin and imaged for one hour using wide field live cell imaging. Video clip begins at nigericin treatment. Supplementary Video 4. Nigericin does not induce loss of mtdna in NLRP3 -/- BMDM. NLRP3 -/- BMDM were labeled with PicoGreen (green) and MitoTracker Red (red) and then treated with nigericin and imaged for one hour using wide field live cell imaging. Video clip begins at nigericin treatment. Figure S1. MMP and LMP are NLRP3 inflammasome dependent in BMDM (A) WT or NLRP3 KO BMDM were LPS primed and treated with either 3 mm ATP or 20 μm nigericin with or without 50 mm KCl. Percent of cells/field retaining MitoTracker red signal after 0, 5, 10, 15, 30, 45, and 60 min of treatment during live cell wide field imaging were recorded. All lines not easily seen are at 100% behind the green line. (n 5, ***, P< for WT LPS Nig vs WT
2 LPS KCl Nig and WT LPS ATP vs WT LPS KCl ATP). (B) WT or NLRP3 -/- BMDM labeled with LysoTracker Green were treated with 100 ng/ml tetanolysin O (TLO) following LPS priming. Live cell microscopy was used to detect loss of LysoTracker Green signal following treatment for 30 minutes. Data are displayed as five representative fields/cell type. Scale bar= 50 μm. (C) WT BMDM were LPS primed and treated with 20 μm nigericin with or without 50 mm KCl. Cells were labeled with LysoTracker Green and imaged via wide field live cell microscopy. Nigericin was added after 2 min. LysoTracker MFI was normalized to initial signal after background subtraction. Data from five fields (LPS Nig) or 10 fields (LPS KCl Nig) are shown. (5 min, ***, P<0.0001). Figure S2. Processed IL-1 is detected after 15 min nigericin stimulation WT BMDM were LPS primed or left unprimed and then treated with 20 μm nigericin for 2-30 minutes. Lysates and precipitated supernatants were blotted for mature IL-1 (17kD) or pro IL-1 (33kD). Figure S3. Mitochondrial ROS, but not CA-074 Me influences LMP (A) LPS primed and nigericin exposed WT BMDM were either treated with CA-074 Me or left untreated. Cells were labeled with MitoTracker Red and LysoTracker Green. Data were recorded as percent of cells/field that lost signal after one hour of live cell imaging. At least 10 cells per field were counted. Error bars represent ± s.d. (n=10 fields for LPS Nig; n=15 fields for LPS Nig CA-074 Me). (B) WT BMDM were LPS primed and treated with 3mM ATP with or without 500 μm MitoTEMPO. LysoTracker green signal was recorded as percent of cells/field retaining signal at 0, 5, 10, 15, 30, 45, and 60 min after addition of ATP in live cell wide field imaging (n 5, ***, P=0.0008).
3 Table S1. Values of MitoTacker MFI in cells that lost or retained signal WT BMDM were LPS primed and treated with nigericin with or without 500 μm MitoTEMPO and labeled with MitoTracker Red. MFI with background subtraction was recorded after 60 minutes of live cell wide field imaging. Cells with MFI values <300 were considered to have lost MitoTracker signal. Each MFI value recorded is from a single cell from one of five representative fields. (mean MFI in No loss of MitoTracker column = , s.d. ± : mean MFI for Loss of MitoTracker column = , s.d. ± ).
4 Heid Table S1 MitoTracker MFI No Loss of MitoTracker Loss of MitoTracker LPS Nigericin LPS Nigericin MitoTEMPO 500 μm
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Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures
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