ASSESSMENT OF CELLULAR OXYGEN GRADIENTS WITH A PANEL OF PHOSPHORESCENT OXYGEN-SENSITIVE PROBES Ruslan I. Dmitriev, Alexander V. Zhdanov, Greg Jasionek, Dmitri B. Papkovsky Biochemistry Department, University College Cork, Cork, Ireland SUPPLEMENTARY DATA DNA sequence insert (5 3 ): Green EGFP encoding sequence; Yellow R 9 encoding sequence. Gray restriction sites for cloning (BamHI and Hin diii). No color linker region. ggatccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacgg CCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCG GCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGtCcTTCAGCCGCTACCCCGACCAC ATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGG CAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAG AAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCA GAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACC CCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTAC AAGagatctggaggtggaAGACGTCGAAGACGTCGAAGACGTCGAtaagctt Predicted ER9Q protein, includes sequence encoded by the vector pqe: Red vector derived sequence; Green EGFP; Yellow R 9 ; 143 Da, 265 a.a., pi 8.76 MRGSHHHHHHGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYG VQSFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNS HNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAA GITLGMDELYKRSGGGRRRRRRRRR Figure S1. DNA coding and amino acid sequences of ER9Q protein. 1
A Absorbance, a.u..2.15.1.5 Excitation Em ission Phosphorescence,a.u. B 1 1 C Phosphorescence lifetime, μ s 8 15 2 3 4 5 6 Wavelength, nm.5 1 1.5 2 PtCP-TFN conc., μm 1 7 8 9 1 Time, min Average Phosphorescence F1,cps Cellviability, % 1 11 1 9 8 7 1.5 μ M/ FCCP -.5 μ M/ DMSO -.5 μ M/ baseline Figure S2: Evaluation of the TFN-PtCP probe for O2 sensing. A: Excitation and emission spectra in air-saturated solution. B: Average phosphorescence intensity counts (F1) from PC12 cells loaded with -2 μm TFN-PtCP for 2 h; effect on cell viability is also shown. C: Monitoring of oxygenation of PC12 cells loaded with TFN-PtCP (.5, 2 μm for 2 h) and exposed to metabolic with 1 μm FCCP or DMSO (solvent) and 2.5 mm, presented in phosphorescence lifetime (τ) scale. Bars indicate time of effectors addition. 2
A ER9Q ER9Q brightfield ER9Q TMRM B Average phosphorescence, cps 1 1 1 8 F1 2 4 6 8 1 12 14 16 Staining time,h Cellviability,% τ, μs C 1uM FCCP 2uM FCCP 1 7 9 11 τ, μs D 55 15 1 7 8 16 h FCCP- 6 h D MSO- 6 h FCCP- 2hD MSO- 2hFCCP- Figure S3: Evaluation of ER9Q-PtCP protein with PC12 cells. A: Fluorescent images of PC12 cells stained with ER9Q protein (green, plasma membranes) and mitochondria-specific dye TMRM (red). B: Average phosphorescence intensity counts (F1) obtained with PC12 cells incubated with 1 μm ER9Q- PtCP for different time intervals; cell viability is also shown (right axis). C, D: Monitoring of oxygenation of PC12 cells stained with ER9Q-PtCP probe at rest and upon metabolic with uncoupler (FCCP) and Ca 2+ chelator (), presented in phosphorescence lifetime scale (τ). Bars indicate the time of effector treatment. 3
TFN-Alexa 499 Chlathrin-mediated endosomes dextran 1,-Alexa 488 Macropinosomes CTX-Alexa 488 Lipid raft-mediated endosomes GFP-Rab6 Trans-Golgi network MitoCase12 Mitochondria Nano2 Hoechst 33342 Figure S4. The intracellular localization of Nano2 probe in MEF cells compared to markers of endocytosis and mitochondria. Brightfield and merged fluorescence (obtained from the same optical slices) images of the live MEF cells are shown. Cells were stained with Nano2 probe and with markers of organelles and imaged as described in Materials and methods section. Scale bar μm. 4
Phosphorescence Intensity (t1), cps 7 1 blank control Per-NP ER9Q 1 7 8 9 1 Figure S5: Respiration profiles of MEF cells exposed to mimics of PC and IC probes and measured using MitoXpress probe. Cells were incubated in conditions of routine staining with probes in presence of non-phosphorescent mimics of Nano2 (Per-NP) and ER9Q-PtCP (unlabeled ER9Q protein) and subjected to OCR analysis with the help of MitoXpress probe. 5