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1 Supplementary Materials for Localized TRPA1 channel Ca 2+ signals stimulated by reactive oxygen species promote cerebral artery dilation Michelle N. Sullivan, Albert L. Gonzales, Paulo W. Pires, Allison Bruhl, M. Dennis Leo, Wencheng Li, Agathe Oulidi, Frederick A. Boop, Yumei Feng, Jonathan H. Jaggar, Donald G. Welsh, Scott Earley* This PDF file includes: *Corresponding author. Published 6 January 2015, Sci. Signal. 8, ra2 (2015) DOI: /scisignal Fig. S1. NOX antibody specificity. Fig. S2. NADPH oxidase proteins are present in myoendothelial projections. Fig. S3. TRPA1 and NOX2 colocalize near IEL fenestrations. Fig. S4. TRPA1 does not physically interact with NOX2 but is modified by 4-HNE. Fig. S5. AITC stimulates Ca 2+ influx in endothelial cells. Fig. S6. TRPA1 sparklets in TRPA1-GFP transfected HEK 293 cells. Fig. S7. TRPA1 protein in mouse arteries. Table S1. TRPA1 sparklet properties. Table S2. Vascular reactivity of cerebral arteries from control and etrpa1 / mice. Legend for movie S1 Other Supplementary Material for this manuscript includes the following: (available at Movie S1 (.mpg format). TRPA1 sparklets in endothelial cells.
2 Figure S1. NOX antibody specificity. Top: Intact endothelium immunolabeled for NOX1, NOX2, or NOX4 (red). Autofluorescence of the internal elastic lamina is green and DAPI-stained endothelial cell nuclei are shown in blue. Bottom: Antibodies were pre-incubated with their respective antigenic blocking peptides before use in immunohistochemistry (n = 3 animals/group). Scale bar = 10 µm. 1
3 Figure S2. NADPH oxidase proteins are present in myoendothelial projections. Summary graph indication the percentage of myoendothelial projections (MEP) that contain NADPH oxidase (NOX) isoforms 1, 2, or 4. NOX1 immunolabeling was present in 16.4 ± 3.5% of internal elastic lamina fenestrations. NOX2 and NOX4 were detected within 35.4 ± 5.7% and 37.8 ± 2.6% of projections, respectively (n = 6-18 vessels/group, 3 rats). 2
4 Figure S3. TRPA1 and NOX2 colocalize near IEL fenestrations. A: There are a greater number of PLA colocalization puncta for TRPA1:NOX2 than TRPA1:NOX4 that are within or near IEL fenestrations (holes) (n = 8-14 vessels/group, 3 rats); *P 0.05 compared to TRPA1:NOX4. B: The total number of IEL fenestrations per each image analyzed did not differ between PLA experiments for TRPA1:NOX4 and TRPA1:NOX2 (n = 8-14 vessels/group, 3 rats). 3
5 Figure S4. TRPA1 does not physically interact with NOX2 but is modified by 4-HNE. A: Co-immunoprecipitation for NOX2 and TRPA1. The pull-down product and supernatant are indicated. The input sample was used as a positive control (n = 3 mice, 3 independent experiments). B: Immunoprecipitation for TRPA1 immunoblotted for 4-HNE-modified proteins. The pull-down product and supernatant are indicated. The input sample was used as a positive control (n = 3 mice, 3 independent experiments). 4
6 Figure S5. AITC stimulates Ca 2+ influx in endothelial cells. A: Depletion of intracellular stores with cyclopiazonic acid (CPA, 10 µm) treatment has no effect on basal TRPA1 sparklets in primary cerebral artery endothelial cells (n = cells/group, 3 independent cell isolations). B: Summary data indicating abolished basal and AITC (100 µm)-induced endothelial cell TRPA1 sparklets in the absence of extracellular Ca 2+ (n = 19 cells/group, 4 independent cell isolations); *P 0.05 compared with control. 5
7 Figure S6. TRPA1 sparklets in TRPA1-GFP transfected HEK 293 cells. A: Summary data indicating an increase in HEK + TRPA1-GFP + RFP cell TRPA1 sparklet frequency in the presence of AITC (100 µm) that is inhibited by HC (10 µm) (n = cells/group, 4 independent experiments); *P 0.05 compared with control, baseline. B: Summary data demonstrating that AITC (100 µm) has no effect on basal Ca 2+ channel activity of HEK cells transfected with only RFP (n = cells/group, 4 independent experiments). C: Histograms of TRPA1 sparklet amplitude (left), duration (middle), and spatial spread (right) recorded from HEK TRPA1-GFP + RFP cells. The amplitude histogram displays one prominent peak (F/F 0 = 0.13). D: Representative recordings of the fluorescence (F/F 0 ) within a region of interest on HEK 293 cells overexpressing TRPA1- GFP, stimulated with 100 µm AITC. Dotted lines indicate the opening of 1 or 2 channels at each level of fluorescence. 6
8 Figure S7. TRPA1 protein in mouse arteries. A-D: TRPA1 immunolabeling (red) in intact endothelial cells of en face mouse coronary septal (A), renal interlobar (B), 3 rd order mesenteric (C), and cerebral (D) arteries. Internal elastic lamina autofluorescence (green) and endothelial cell nuclei (blue) are shown; scale bar = 10 µm (n = 3 mice). 7
9 HEK + TRPA1-GFP Endothelial Cells Mode Mean ± SE Range Mode Mean ± SE Range Amplitude (F/F 0 ) ± ± Duration (s) ± ± Attack Time (ms) ± ± Decay Time (ms) ± ± Spatial Spread (µm 2 ) ± ± Table S1. TRPA1 sparklet properties. Mode, mean, and range for amplitude, duration, attack time, decay time, and spatial spread of TRPA1 sparklets recorded from HEK 293 cells overexpressing TRPA1-GFP (n = 792 events) or rat primary cerebral artery endothelial cells (n = 762 events). Peak Constriction Myogenic Tone Passive Diameter Control 59.3 ± 7.0 % 18.3 ± 2.2 % ± 5.4 µm etrpa1 -/ ± 3.5 % 17.2± 1.4 % ± 3.3 µm Table S2. Vascular reactivity of cerebral arteries from control and etrpa1 / mice Maximum constriction to superfusion of 60 mm KCl physiological saline solution, % myogenic tone at 60 mmhg intraluminal pressure, and the passive diameter at 60 mmhg intraluminal pressure for control (n = 6) and etrpa1 / (n = 5) mouse cerebral arteries. There were no significant differences. Supplemental Video S1. TRPA1 sparklets in endothelial cells. Representative pseudocolor TIRF microscopy recording of TRPA1 sparklets (green) induced by AITC (30 µm) in a primary cerebral artery endothelial cell. Frame rate = fps. 8
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