EMBL COURSE EMBL-ATC May 2-4, 2019 Coordinated by D Swinkels and I Cabantchik

Techniques for Studying Iron in Health and Disease EMBL COURSE EMBL-ATC May 2-4, 2019 coordinated by D Swinkels and I Cabantchik

INTRODUCTION TO PRACTICALS-

Generation and interpretation of assay test results for biomarkers of iron homeostasis and dyshomeostasis. Rian Roelofs, Rachel van Swelm Dorine Swinkels. Radboud University. Nijmegen NL. 2-16

Biochemical and functional characterization of mitochondrial ISC assembly factors. Oliver Stehling. Institut für Zytobiologie, Philipps-Universität Marburg 17-39

Labile 2Fe-2S Clusters. Rachel Nechushtai. Institute of Life Sciences. Hebrew University, Jerusalem, Israel 40-69

Real-time monitoring of intracellular labile iron with fluorescent metal- sensors. Maya Shvartsman. EMBL HD. Rome. IT 71-88

Labile Iron IN BIOLOGICAL FLUIDS. Breno Pannia Espósito, Institute of Chemistry University of São Paulo, Brazil 89-98

Mitochondrial Iron probing. Charareh Pourzand. Department of Pharmacy & Pharmacology. Centre for Therapeutic Innovation University of Bath, UK 99-123

Generation and interpretation of assay test results for biomarkers of iron homeostasis and dyshomeostasis

Rian Roelofs, Rachel van Swelm, Dorine Swinkels Goal By the end of the day you will be able to adequately generate and interpret results of an assay for biomarkers of iron (dys)homeostasis within both a research and clinical setting.

More specifically you will: ØClinically interpret test result of a broad range of conventional and more novel iron biomarkers Ø Apply fundamentals of implementing an assay and generating a reliable test results for individual iron biomarkers in both the research and clinical setting

During the day in small groups you will:

1. Interpret test results generated (by a fully validated assay) for the diagnosis of iron disorders 2. Design of a validation plan for an assay of an analyte 3. Apply knowledge of design of a validation plan by performing a validation of a commercial hepcidin ELISA kit in the lab 4. Present analytical findings of the lab validation findings to the whole group for discussion

Day schedule 1. Interpretation test results generated (by a fully validated assay) for the diagnosis of iron disorder a. 10.20-10.35 Presentation b. 10.35-11.05 Case puzzles c. 11.05-11.25 Group discussion on case puzzels 2. Test validation a. 11.25-11.55 Presentation b. 11.55-12.25 Design practical plan c. 12.25-12.30 Provision of a practical plan d. 13.15-18.00 Practical e. 18.00-19.00 Calculation results and prepare presentation f. 20.00-21.00 Presentation and discussion

Diagnosis of iron dyshomeostasis

Clinical interpretation of test results

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RG;D^)&(&%0)'$(#)<'#*'#>)3%+%3'&.) Standardization and harmonization

True value Standardization Harmonization Equivalent results Standardization/ Harmonization Standardized harmonisation (equivalence) (true values) Ferritin Yes, but not fully Yes, but not fully

Transferrin yes yes Transferrin saturation yes yes Hepcidin now possible* now possible sTfR Efforts ongoing Efforts ongoing ZnPP no no Hb in retis no no

* Diepeveen LE et al. Clin Chem Lab Med. 2018 Epub ahead of print Take home ØSeveral iron biomarkers - derived from stores, circulation and bone marrow

Ø Diagnostic value - individual iron biomarkers: low - combination of iron biomarkers: high

ØHigh ferritin + elevated TSAT à relatively toxic High ferritin + normal TSAT à relatively safe

Ø Assay results - easily affected by pre-analytical aspects - often method depended since assays are not harmonized/ standardized

Mini-exam:

Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Reference values

F, 30yr M, 60yr F, 65yr M, 45yr F, 4yr M, 18yr (red = not standard.) Hb 9.1 10.5 14.2 13.5 7.9 8.5 Men > 13 g/dl; Wom. > 12g/dl CRP < 5 45 <5 8 < 5 < 5 < 5 mg/L Men: 30-300 µg/L; Wom.: Ferritin 15 103 1700 950 25 2700 20-200 µg/L TSAT 10 10 94 21 3 101 15-45 % Hepc. <0.5 13.2 4.2 9.4 3.1 4.3 < 0.5 -14.7 nmol/L sTfR 2.60 0.90 0.90 0.90 2.80 6.90 0.76-1.76 mg/L

ZnPP 89 81 39 75 103 95 < 40-80 mmol/mol heme

MCH 1.65 2.00 2.15 2.00 1.20 np 1.67-2.11 fmol RetHb 1.55 2.12 2.25 2.12 1.29 np 1.87-2.23 fmol DYNAMICS OF IRON-SULFUR PROTEINS - FROM SOLUTION TO CELLS - Part 2: Biochemical and functional characterization of mitochondrial ISC assembly factors

Oliver Stehling Institut für Zytobiologie und Zytopathologie Philipps-Universität Marburg, Germany

Techniques for Studying Iron in Health and Disease EMBL COURSE Fe-S proteins are associated with multiple eukaryotic compartments

Cytosol CISD2 (Miner1) Nucleotide metabolism (GPAT, DPYD) Iron homeostasis Endoplasm. (IRP1) Reticulum

Respiration (Complexes I, II, III) Citric acid cycle DNA (Aconitase, LIAS) Polymerases (POLD1) Gycosylase (NTHL1) ion CISD1 ndr (MitoNEET) cho Nucleus Mito

Eukaryotic cell

Sheftel et al. (2010) Trends Endocrinol Metab Three ‚machineries‘ cooperate to synthesize cytosolic & nuclear Fe-S proteins

Cytosol

Apo Holo CIA machinery

ISC export

ISC assembly

ion ndr cho Nucleus Mito

Eukaryotic cell The basic concept of Fe-S protein assembly

1. Scaffold 2. Sulfur supply 3. Iron supply 4. Electron supply Cytosol 5. Transfer proteins 6. Targeting factors for [4Fe-4S] assembly Mitochondrion Aconitase [4Fe-4S] ISC assembly ACP ISC machinery targeting factors LIAS ISCU1 ISCU1 ISD11 -SSH IBA57 SDH Ala NFS1 Chape- SG ISCA1 rones GS ISCA2 NFU Cys FXN 1 (B OL GLRX5 A3? FDX2 IND1 )

[2Fe-2S] Complex I DYDYDY FDX reductase proteins MFRN NAD(P)H

Iron (Fe2+)

Braymer and Lill (2017) JBC IRP1 [4Fe-4S] [4Fe-4S]

)

SDH ? 3

A L

O LIAS

2B 2A B ( CIAO CIAO 1 Lill et al. (2015) Eur (2015) al. et Lill Cell J Biol

U F

N 6. Targeting factors Targeting 6. MMS19 IND1 Aconitase [4Fe-4S] Complex Complex I ISC factors IBA57 ISCA2 ISCA1 targeting ISC ISC CIAO3 export SG 2. Sulfur supply Sulfur 2. GS X-S 5. Transfer 5. eins ABCB7 GLRX5 t e-2S] F CFD1 pro [2 rones

Chape-

n o

35 i

NBP

1. Scaffold 1. r FDX2

NAD(P)H d ISCU1

n o Core GLRX

Fe? h

G c machinery machinery

) o

FDXreductase t assembly ISC ISC assembly ISC

2+ 3 i

GLRX M 3. Iron supply? Iron 3. FXN ISCU1 − e CIA- PIN1 MFRN DY DY DYDY -SSH Iron (Fe Iron − supply ACP e NFS1 ‒ NDOR1 ISD11 FAD FMN The basic concept of Fe-S protein assembly protein Fe-S of concept basic The 4. e 4. Ala − Cys 2e CIA Cytosol machinery NADPH 1 IRP [4Fe-4S] [4Fe-4S]

)

SDH ? 3

A L

O LIAS

2B 2A B ( CIAO CIAO 1

U F

N MMS19 IND1 Aconitase [4Fe-4S] Complex Complex I ISC ISCA2 factors IBA57 ISCA2 ISCA1 targeting ISC ISC CIAO3 export SG GS X-S eins ABCB7 GLRX5 t e-2S] F 2 CFD1 pro [ rones

Chape-

n o

35 i

NBP r FDX2

NAD(P)H d Vinzent Schulz ISCU1

3 n

FDX2 o Core GLRX

Fe? h

G c machinery

) o

FDXreductase t assembly ISC

2+ 3 i

GLRX M FXN ISCU1 − e CIA- PIN1 MFRN DY DY DYDY -SSH Iron (Fe Iron − ACP e NFS1 NDOR1 ISD11 FAD FMN (Bio)chemical characterisationISCfactors of Ala − Cys 2e CIA ytosol C machinery NADPH 1 V VV V IRP Ó ÓÓ Ó [4Fe-4S] [4Fe-4S] LAS) mt Fe )

SDH ? 3 LIAS LIAS A

(HGC L Mitochondrial [Fe-S] defects [Fe-S] LIAS O

2B 2A B ( [4Fe-4S] defects [4Fe-4S] CIAO CIAO 1 Cytosolic-nuclear U F

N (Multiple mitochondrial mitochondrial (Multiple dysfunction syndrome) dysfunction MMS19 4) Ó ÓÓ Ó IND1 Aconitase [4Fe-4S] Complex Complex I ISC ISCA2 factors ISCA2 ISCA2 IBA57 (MMDS ISCA2 ISCA1 targeting ISC ISC CIAO3 export SG GS X-S ABCB7 GLRX5 teins o CFD1 [2Fe-2S] pr rones

Chape- n

L) o

Ó ÓÓ Ó

35 i

NBP r FDX2

NAD(P)H d ISCU1

3 n

FDX2

(Hyperglycinemia, lacticacidosis, (Hyperglycinemia, o Core GLRX FDX2 FDX2

and seizures / PDH-LIAS deficiency) PDH-LIAS / seizures and Fe? h (MEOA

G c machinery

) o

FDXreductase t assembly ISC

2+ 3 i

GLRX M FXN ISCU1 − e CIA- PIN1 MFRN DY DY DYDY -SSH Iron (Fe Iron − ACP e NFS1 NDOR1 ISD11 FAD FMN Ala − Cys 2e ÌÌ CIA ytosol C machinery General cellular General defects [Fe-S] mt Fe myopathy mitochondrial (Episodic and atrophy optic without or with leukoencephalopathy) reversible NADPH FDX2 and ISCA2 defects: prototypes for Fe-S assembly diseases assembly Fe-S for prototypes defects: ISCA2 and FDX2 Characterisation of Fe-S assembly defects

[4Fe-4S] Fe-S protein CIA machinery stability

IRP1 Lactate TFR expression Cytosolic formation (Tf binding) aconitase Cytosol X-S activity Cytosolic-nuclear ISC [Fe-S] defects Mitochondrion export AconitaseMitochondrial [4Fe-4S]aconitase LIAS activity activity ACP LIAS ÓÓÓ LIAS(HGCLAS) ISCU1 ISCU1 ISD11 -SSH SDH Ala Chape- SG ISCA2 ÓÓÓ SDH NFS1 activity rones GS (MMDS4) Cys FXN FDX2 ÓÓÓ GLRX5 FDX2 (MEOAL) Subunit DY [2Fe-2S] stabilityComplex I DYDY Ferrochelataseproteins Core ISC machinery: MFRN stability ISC targeting factors: General cellular Mitochondrial [Fe-S] defect (Fe2+) [4Fe-4S] defect

Stehling et al. (2014) Biochimie Reporter approaches for the characterisation of Fe-S assembly defects

Mock Cytosol LDH Scramble activity FDX2 Cytosolic ISCA2 aconitase activity LIAS Total Viable lysate cell CS activity HeLa mt mt mt Digitonin mt Complex IV treatment activity Mitochondrial Lactate LIAS aconitase formation activity mt activity mt TFR expression SDH (Tf binding) Fe-S protein stability activity Tf-FITC Membranes/ Activity fluorescence Immunoblotting Organelles measurements Determination of aconitase (mt and cytAco) activity

96-Well-Plate

Citrate cis-Aconitate Isocitrate NADP+ NADPH

a -Ketoglutarate Isocitrate Dehydrogenase (IDH)

Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002. Determination of lactate dehydrogenase (LDH) activity

Mitochondria Lactate Cytosol Dehydrogenase Pyruvate (LDH) Dehydrogenase Pyruvate Lactate

NADH NAD+

96-Well-Plate

Principles of Biochemistry, 4th edition Lehninger A, Nelson D (ed), Cox M (ed) Freeman; 2004 Determination of succinate dehydrogenase (SDH) activity

SDH A Substrate

FAD

[2Fe-2S]

Fe-S [4Fe-4S] DCPIPred DCPIPox B (colorless) (blue) Matrix SDH [3Fe-4S]

Ubiquinone

Ubiquinole Heme b Decyl- Decyl- ubiquinone ubiquinole

Succinate Fumarate Succinate D Malonate Dehydrogenase IMS SDHC SDH (SDH)

Principles of Biochemistry, 4th edition Succinate-coenzyme Q reductase (SQR), Lehninger A, Nelson D (ed), Cox M (ed) Freeman; 2004 Respiratory Complex II Determination of cytochrome-c oxidase (COX) activity

O2 + 4H+ 2 H2O

Cyt-c (red) Cyt-c (ox) Cytochrome- oxidase (COX)

Principles of Biochemistry, 4th edition Lehninger A, Nelson D (ed), Cox M (ed) Freeman; 2004 Determination of citrate synthase (CS) activity

96-Well-Plate Citrate synthase (CS)

TNB DTNB (yellow) (colorless)

Acetyl-CoA Coenzyme-A

Oxaloacetate Citrate Citrate synthase (CS)

Principles of Biochemistry, 4th edition Lehninger A, Nelson D (ed), Cox M (ed) Freeman; 2004 The pyruvate dehydrogenase complex requires lipoate for the oxidative decarboxylation of pyruvate to acetyl-CoA

The lipoyl cofactor contains a [4Fe-4S] cluster–derived thiol (SH)SH moiety

a -KGDH

Å Decarboxylation Ç Substrate oxidation, S-S reduction É Transesterification Ñ + Ö Lipoate regeneration

Principles of Biochemistry, 4th edition TPP, thiamine pyrophosphate Lehninger A, Nelson D (ed), Cox M (ed) Freeman; 2004 Lipoyl synthase (LIAS) converts the protein-bound octanoyl lipid moiety into the lipoyl cofactor

e- S-Adensosine Methionine (SAM) Octanoyl chain

AdoMet radical cluster LIAS

5’-deoxyadenosyl Lipoyl chain Radical (DOA·)

Auxiliary cluster

a -Lipoate antibody (Immunoblotting)

Lanz ND, et al. LCP lipoate carrier protein (lysine residue) Biochemistry. 2014; 53(28):4557-72. Cofactor maturation determines Fe-S protein stability and can be assessed by immunoblotting

Cytosol Proteolysis

CIA machinery Apo Holo DPYD

instable stable

instable ISC export

Apo Holo FECH stable

Proteolysis Holo NDUFA13 Holo NTHL1 ISC assembly stable stable

ion ndr cho Nucleus Mito

Eukaryotic cell Optional: Determination of TFR1 expression

FITC

holo- Fe TFR1 Plasma Transferrin Fe membrane

Cytosol

tratransferrinnsferrin receptor (TFR1) mRNA stabilized 5’ 3’

holo-IRP1 IRE (cytAco) apo-IRP1

ISCCore ISCExp CIA

IRE, Iron Responsive Element [Fe-S] assembly deficiency elevates lactate formation

Lactate Dehydrogenase Pyruvate (LDH) Ó LIAS Dehydrogenase Pyruvate Lactate (Lipoate)

NADH NAD+

Aconitase Ó

=> => RCC-I

RCC-I

=> RCC-I

RCC-II Ó (SDH) a -KGDH (Lipoate)

Principles of Biochemistry, 4th edition Lehninger A, Nelson D (ed), Cox M (ed) Freeman; 2004 Optional: Determination of lactate formation

Lactate Dehydrogenase (LDH) Pyruvate Lactate

NADH NAD+

Diaphorase

DCPIPox DCPIPred (blue) (colorless) Characterisation of Fe-S assembly defects

[4Fe-4S] Fe-S protein CIA machinery stability

LIASLIAS ÓÓÓ SDH Core ISC machinery: SDH FDX2 ÓÓÓ ÓÓÓ General cellular [Fe-S] defects ISCA2 activity

Subunit stabilityComplex I Ferrochelatase stability ISC targeting factors: Mitochondrial [4Fe-4S] defects Acknowledgements

Roland Lill

Vinzent Schulz Ulrich Mühlenhoff

Debarati Bandyopadhyay Joseph Braymer Brigitte Niggemeyer Marie Brück Nadine Richter Sven Freibert Magdalena Rakwalska-Bange Ralf Rößer Stafanie Thelen Martin Stümpfig Arunkumar Upadhyay

GIF Leibniz & Koselleck programs SFB 593, 987, TR1 GRK1216, LI 415 SPP 1710, 1927 Marie Curie The 2Fe-2S NEET proteins’ Cluster

May 2nd 2019 – LAB Proteins with Fe-S Clusters

• Identified ~50 years ago as acid-labile prosthetic groups contained within a class of electron carriers proteins called ferredoxins

• In recent years, over 100 proteins that contain [Fe-S] clusters, called [Fe-S] proteins, were described

• Fe-S clusters were shown to control protein structure and to act as environmental sensors; modulators of gene regulation and participate in radical generation

• Three major processes required to sustain life on earth – nitrogen fixation; photosynthesis and respiration – involve the obligate participation of [Fe-S] proteins.

• Today we will learn about:2Fe-2S The Nitrogenase proteins

Howard J. B., Rees D. C. PNAS 2006;103:17088-17093

Electron transfer

Psa D PsaE

The Ferredoxin structure is from Aphanothece sacrum (Tsukihara, Fukuyama et al. 1990)

Photosystem I structure is from Synechococcus elongatus (Jordan, Fromme et al. 2001). Respiration Structure of Complex I - Thermus thermophilus NEET proteins

• mitoNEET was discovered in 2004 as the cell target of the Insulin sensitizing drug pioglitazone (a TZD= thiazolidinedione) (Colca et al (2004) Am J Physiol Endocrinol Metab 286, E252-60)

• The name mitoNEET was given because of a a special sequence Asn- Glu-Glu-Thr (NEET) found at the C-terminus

• The protein has a CDGSH domain (usually in zinc finger proteins)

• The human mitoNEET gene (CISD1) was cloned and identified as one of the 3 members of the CISD family

• The protein was localized at the outer mitochondrial & ER membrane (Willey et al (2007) PNAS 104, 5318-5323)

“Reddish” Protein Structure of NEET proteins ‘NEET fold’

β-Cap

Cluster binding domain

Homodimers

Karmi et al. JBIC 2018 9 Structure of the NEET proteins Unique “NEET-fold” & 3Cys:1His Cluster Ligation

CISD1 CISD2

(A) Ribbon diagrams of the soluble parts of mNT (B) The cluster-binding site highlighting the CISD1 (cyan) and NAF-1 CISD2 (blue) His and three relevant Cys residues.

Paddock et al. PNAS. 2007 & Conlan et al. JMB, 2009 Possible Function of NEET Proteins

Cluster Donor protein?

Apo-acceptor protein

Ola Karmi Henri-Baptiste Marjault

Dr. Yang-Sung Sohn Paddock et al., PNAS, 2007 DAY 2-part I (May 3rd)

Ola Karmi, PhD. Student Henri-Baptiste Marjault, PhD. Student 4Cys 2Cys:2His 3Cys:1His Specific absorption peak due to the [2Fe-2S] cluster presence for the different coordination types:

Ferredoxin (4Cys::) 423 nm

NEET proteins (3Cys::1His)458 nm Apo-ferredoxin Holo-NAF-1 Holo-ferredoxin

423458 Absorption Peak – 458nm The ‘NEET – fold’ Labile Cluster

Stable Cluster

Tamir, Sagi, et al. PLoS One 8.5 (2013). Tamir, Sagi, et al. Biological 16 Crystallography 70.6 (2014).

NAF-1 Ferredoxin

"Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS.

In SDS-PAGE the electrophoretic mobility of proteins depends primarily on their molecular mass, in native PAGE the mobility depends on both the protein's charge and its hydrodynamic size. 1. Reduction of the apo-ferredoxin 2. Mix reduced apo-Fdx+ Holo NAF-1/ Holo H114C

200µl Fdx Distribute 25µl/ tube Reduced 4. Run the NATIVE Gel

3. Load the NATIVE Gel

Reduced Apo-Fdx+ Holo-NAF-1NAF 1 1 2 3 4 5 6 7 8 9 10 Time Time Time Time Time Time Time Holo- Time 0’ Holo-Fdx 60’ 0’ 5’ 10’ 15’ 30’ 60’ NAF-1 H114C H114C

2. Transfer into 96 wells plate 1. Reduction of the apo-ferredoxin

3. Add holo-NEET proteins (equi-concentratrion)

4. Follow the UV-Vis spectrum using the plate reader with a specific attention for the absorbance at 458 nm (NEET protein) and 423 nm (Ferredoxin protein). DAY 2-part II (May 3rd)

Dr. Yang-Sung Sohn

1. To observe the involvement of NEET proteins in iron homeostasis of cancer cells. How does the suppression or the over-expression of NAF-1 protein affect on iron homeostasis in cancer cells?

2. To learn how to measure mitochondrial iron level changes such as iron overload caused by NAF-1 protein suppression or iron full utilization caused by NAF-1 protein over-expression by confocal microscopy

3. To learn how to analyze the data of such experiment. v v

1. Cell incubation with RPA (0.5µM) in DMEM-Hepes for 15 minutes at 37°C.

2. Washing cells and replenishing cells with the flesh DMEM- Hepes medium.

3. Taking images randomly at least 5 different areas in each cell lines with confocal microscopy.

---

To prove that indeed the rpa quenching was caused by iron we use an iron chelator Deperiprone(DFP) (100µM) which in princple to reverse the penotype in the NAF-1(-) cell lines for 30 minutes and 1- 3 processes is repeated for the measurements. WT NAF-1(+) NAF-1(-)

-DFP

+DFP *** 1.1 *** 1

0.9

0.8

0.7

RPA Flourescence (r. u.) (r. Flourescence RPA 0.6

0.5 Control NAF-1 (+) NAF-1 (-) (-DFP) (+DFP) Real-time monitoring of intracellular labile iron with fluorescent metal-sensors.

Dr. Maya Shvartsman EMBL HD May 04, 2019 What is labile cell iron and why is it important?

• Labile cell iron (LCI) is the small fraction of intracellular iron, which is readily exchangeable, redox-active and chelatable (0.1- 3uM). • Potentially dangerous, due to its ability to generate reactive oxygen species. • Labile iron levels in a cell or an organelle change with cell state and in disease.

Cabantchik, Frontiers Pharmacol 2014 Prus and Fibach, Br. J. Haematol 2008 Why do we need real time monitoring of labile iron?

• To study iron transport into cells and organelles - kinetics. (Which Fe forms and how fast can cells acquire?)

• To screen for molecules which could facilitate iron transport into cells (compensating for Fe deficiency, therapeutic use).

• To study the action of iron chelators (therapeutic use).

 Using “turn-off” probes for real time measurements: fast, direct, and flexible (sec-min). How do we measure labile cell iron? In this workshop, we will use two turn-off metal-sensors: calcein green (CALG) for cytosol, RPA for mitochondria. Controls: untreated (-Fe) control, chelators for specific recovery of fluorescence. Cell permeant and impermeant chelators. chelator Chelator - Fe Cyto Mito Fe Fe Fe CALG CALG-Fe CALG

Fe chelator FI - Fe +Fe

CALG – cytosol (lexc 488nm, lem 520nm) RPA – mitochondria (lexc 560nm, lem 610nm) Time (min) The assay for labile Fe measurement is flexible.

• Flow cytometry – for suspension cells. • Reads signals from cells, can distinguish cell types. • Can yield data from thousands of cells in each sample. • If working in tubes, only one sample at a time can be run. • Taking samples out for measurements may be necessary.

• Plate reader – adherent cells. • Reads cellular and non-cellular signals (background from medium or dye attached to plate). • Plate reader – 96 kinetics in one run. • No need to take samples out.

• Microscope – adherent cells. • Precise localization of processes. • Relatively low sample size, complex image processing. • Photobleaching! Different techniques of assay give similar results.

Cyto Mito

None (t=0’) +FeHQ 5uM (t=20’) +SIH 50uM Labile cell iron measurement – experiment stages

• Think and plan the experiment: aim, hypothesis, cell model, controls, instruments, conditions. • *Controls: experimental groups or spectral controls (unstained cells, compensation, etc).

• Stain the cells.

Measure iron ingress kinetics. • Cyto Mito • *Adding cell impermeant and permeant chelators.

• Analyze the data. The experiment of today – 2 groups.

Group 1: Endocytosis – “E” Group 2: The “HK” compound How would an endocytosis inhibitor What would the hinokitiol molecule affect Fe ingress? do to Fe transport into cells?

No Fe +TfFe 5uM +FeHQ 5uM No Fe +TfFe 5uM +FAS 5uM (E-1) (E-2) (E-3) (HK-1) (HK-2) (HK-3)

No Fe +TfFe 5uM FeHQ 5uM No Fe +TfFe 5uM FAS 5uM +CQ 20uM +CQ 20uM +CQ 20uM +HK 2.5uM +HK 2.5uM +HK 2.5uM (E-4) (E-5) (E-6) (HK-4) (HK-5) (HK-6)

TfFe = Transferrin-iron TfFe = Transferrin-iron FeHQ = FeCl3: 8’-HQ 1:1 (Fe(III) complex) FAS = Fe(II) ammonium sulfate CQ = chloroquine (endocytosis inhibitor) CQ = chloroquine (endocytosis inhibitor)

• Take samples out of your source plates at 0’, 10’, 20’, 30’, 40’, 50’, 60’, 70’. Put them on ice! • Add Fe to source plates after 0’ time. DFO after 40’. SIH after 50’. 1. Stain your cells

• Mitochondrial staining – RPA. • Accumulates according to the voltage across mitochondrial inner membrane.

RPA

• Cytosolic staining - calcein green CALG (AM): an uncharged acethomethoxyl ester is hydrolyzed to carboxylic acids in the cell.

Staining: usually at 37°C RPA: 0.1-1uM, 15min, 3:1 RPA:Fe CALG: 0.125 – 0.25uM, 10 min, 1:1 CALG-Fe CALG Calibrate for cell type & instrument! (AM) Use microscopy for localization! What are the factors that affect your staining?

• Concentration of metal sensor. • Temperature (37C by default). • Time • Cell density (concentration of cells in the tube). • Metabolic state and viability of cells. • Number of mitochondria/cell (mitochondrial dyes). • Quenching of metal sensor by contaminant Fe in medium (RPA) – add DFO during staining. 2. Measure iron ingress kinetics - I

1. Have a “no Fe” control in each experiment. 2. You can measure baseline signal in all samples before adding Fe (10-20mins).

No Fe control

3. Metal-sensors can be leaking out of cells (export by multidrug resistance pumps, or cells damaged). Adding an inhibitor like probenecid could be necessary. Baseline/”no Fe” fluorescence stability

Good Leakage Dye accumulation FI FI + probenecid FI

Time Time Time (min) (min) (min) 2. Measure iron ingress kinetics - II

1. Add Fe (TfFe, Fe(II) or Fe(III) salts). Measure signal. 2. Kinetics to run at 37C (your source plates) ! 3. In flow cytometry experiments, samples have to be taken out, put on ice to stop reaction, and preferrably also diluted. 4. Take care to account for the volume you take out of your source plate.

Samples on ice 2. Measure iron ingress kinetics - III

1. Add a cell-impermeant iron chelator to stop Fe ingress into cells (DTPA or DFO 50uM). Measure signal. 2. This is also your internal control, to prove your reaction to Fe is intracellular and your cells are intact. 2. Measure iron ingress kinetics - IV

1. Add a high affinity cell-permeant iron chelator which will get into the cells and take iron from the metal-sensor (SIH or DFP or DFR 50uM). NOTE: Works for calcein. But NOT for all metal-sensors.

This is the control to prove that fluorescence decreased due to Fe. 3. Analyze the data.

• Extract mean fluorescence values from each timepoint and sample. • Plot the fluorescence values as a function of time.

• Normalize your fluorescence (F/F0 or F/Fmax). • Calculate change in labile cell Fe (Delta of normalized fluorescence) = “relative” quantification. Fe chelator FI

Δ(F/Fmax) Δ(F/F0)

Time (min) For absolute quantification (in uM Fe or no. of atoms/cell), additional experiments and calculations are needed: Measure, how much metal sensor is inside a cell. Measure packed volume of cells/organelles. Correct for metal sensor : Fe stoichiometry. Relative vs Absolute quantification

“Relative”: compare treatments. “Absolute”: get kinetic parameters (Vmax, K1/2), Fe concentrations. Summary

• Fluorescent “off” metal sensors are a valuable tool to measure labile cell iron in real time (sec – min).

• This experimental system is flexible: can be adapted to any cell type and instrument.

• Powerful tool: Can be used to compare treatments or calculate kinetic parameters and Fe concentartions in cells and compartments.

• Many factors can affect the outcome of experiment: staining conditions, metabolic state of cells, type of instrument used, etc.

• Calibration of experimental conditions is highly recommended for each new user. 5/4/2019

TECHNIQUES FOR STUDYING IRON IN HEALTH AND DISEASE DAY 3 (May 4th 2019) FLUORESCENCE MONITORING OF LABILE IRON TRAFFICKING IN CELLS AND IRON SPECIATION IN ANIMAL FLUIDS AND ORGANS

Station 2: LABILE IRON IN BIOLOGICAL FLUIDS Breno Pannia Espósito University of São Paulo Institute of Chemistry

Non transferrin-bound iron NTBI (Potentially hazardous)

LPI Labile plasma iron (Almost certainly dangerous) Redox-active Should be chelated

• LPI is generated by forms of iron which can catalyze the oxidation of ascorbate

• LPI is an indicator of the extent of iron-mediated oxidative damage in vivo

• LPI correlates with several IO conditions and is a valuable parameter to follow up chelation regimens

1 5/4/2019

Give/don’t give antipyretic

Give/don’t give iron chelator

LPI

Who’s LPI?

LPI standard

Advantages of a non-natural standard for LPI: LPI is defined operationally by 1. Stable (non-aggregating) comparing a sample with the 2. Redox-active behavior of a Fe(nta) standard 3. Non-biodegradable

2 5/4/2019

Chemical principle of the fluorimetric detection of LPI by means of the autoxidation of ascorbic acid (Hasc) catalysed by iron. Reactive oxygen species are generated which convert dihydrorhodamine (DHR) into fluorescent rhodamine (Rhod).

Treatment with a strong iron chelator (deferiprone; brown square) halts the iron reduction step and hence the whole cascade of reactions, preventing DHR to be oxidized.

Effect of SOD and catalase

SOD )

-1 80

SOD+L1 60

CAT

40 CAT+L1

DHR oxidation rate (F.U. DHRoxidation min rate 0.1 1 10 100 activity (units/ml)

Ascorbil radicals were not identified (EPR) in preliminary tests.

3 5/4/2019

• Part a: LPI and its quantification

Calibration for the LPI experiment. Kinetic curves of DHR oxidation are obtained for known concentrations of Fe(nta), the LPI standard, in the absence (a) or presence (b) of deferiprone, a strong iron chelator that allows for the unequivocal assessment of iron-dependent-only oxidation of DHR. DHR oxidation rates are obtained as slopes (m) for each kinetic curve; in the presence of deferiprone m tends to zero.

(c) Oxidation rates in the absence of deferiprone (def) are linearly dependent on iron concentration. (d) The difference between oxidation rates in the absence and presence of def is also linearly dependent on iron concentration, and it is the calibration curve used to quantify LPI in unknown samples.

4 5/4/2019

Artifacts?

• EDTA • Turbid samples • Hemolysis (because of color)

a 0 60 120 180

0.015 ) 80

-1 Control ) -1 400 HH 60

300 A 0.010 (F.U. min

A 465 465 40 200 0.005 100 20 DHR rate oxidation

0 ∆ DHR oxidation 0.000 0 DHR oxidation rate (F.U. x min x rate (F.U. DHR oxidation 0 20 40 60 80 0 15 80 [Fe] (µµµM) [HbO2] in serum (mM) Relationship with Tf saturation

Starters

• DHR dihydrorhodamine 123 (di)hydrochloride Biotium – Frozen 100 mM in dmso; 12 µL aliquots; only 1× thaw • Ascorbic acid – Frozen 8 mM in water; 150 µL aliquots; use and discard • HBS: treated with Chelex® 100 sodium form 1 g/100 mL • Serum samples – Might be thawed 2×; probably more

5 5/4/2019

Starters

• Iron standard: ferric

nitrilotriacetate, Fe(nta) (NH4)2Fe(SO4)2 – Redox-active yellow complex – Prepared by the incubation of ferrous ammonium sulfate with nta3- (1:3 mol:mol) in water for ~ 1 h at 37oC

• O-donor ligands form more stable complexes with Fe(III)

• Fe(II), which reacts faster, is readily oxidized by O2 in the buffer

10 µµµL sample 10 µµµL sample 190 µµµL dhr+asc 190 µµµL dhr+asc+deferiprone

Fluorescence reading F 485/535 nm o t 40’/37 C m m Calc slope (m) sample w/o chel. sample w/ chel. from 15 a 40’

Slopes x endpoints!

6 5/4/2019

FeNTA: 1 2 3 4 5 6 0 A s1 - def B s2 - def C s3 - def D s4 - def E s1 + def C/C -def F C/C+ def s2 + def G s3 + def 20 µµµM H s4 + def

(a) LPI

s1 – s4: “surprise” samples in Plasma-Like Buffer

• Part b: Effect of iron source and chelator characteristics on the LPI assay

7 5/4/2019

chelators: 0 60 µ M

7 8 9 10 11 12 edta FeCl3 10 µµµM dfo FeCIT 10 µµµM edta FeNTA 10 µµµM dfo FeNP 10 µµµM edta dfo edta dfo

(b) properties of Fe and chel

EDTA DFO

Results: LPI quantification (PLM)

Calibration curve (PLM) • Effect on pseudo-first order 60000 conditions (DHR = 50 µM) and 50000 on auto-oxidation of HAsc (40 40000 mM) 30000

20000

10000

DHR oxidation rate (a.u./min) rate oxidationDHR 0 Fe(nta) = 20 µM 0 5 10 15 20 25 Fe (µµµM) Fe(nta) = 5 µM

Calibration curve (linear) y = 5065.6x + 11794 R² = 0.897 70000 • Source of protein for PLM 60000 (here: BSA; no human material 50000 40000 allowed, either HSA or plasma) 30000 20000 10000

DHR oxidation rate (a.u./min) rate oxidationDHR 0 0 2 4 6 8 10 12 Fe (µµµM)

8 5/4/2019

Results: LPI quantification (HBS)

Calibration curve 180000 160000 140000 120000 Different 100000 DHR 80000 60000 source!! 40000 20000

DHR oxidation rate (a.u./min) rate oxidationDHR 0 0 5 10 15 20 25 Fe (µµµM)

Calibration curve y = 14365x + 25353 R² = 0.9453 180000 160000 140000 120000 100000 80000 60000 40000 20000

DHR oxidation rate (a.u./min) rate oxidationDHR 0 0 2 4 6 8 10 12 Fe (µµµM)

Results: Effect of chelator and iron source

EDTA preserved samples “Wrong” chelators

9 5/4/2019

Clinical significance of LPI Increased LPI has been related to: • Neurological damage after ischemia (1), • Endothelial dysfunction in thalassemic children (2), • Cognitive defects in Alzheimer’s patients (3), • Complications of myelodysplastic syndrome (4), especially infection and tissue damage (5), • Increased morbidity in: – Transfused patients (6) – Diabetes (7)

(1) Curr Med Chem 2007,14: 857-874 (2) Pediatric Cardiol 2008,29: 130-135 (3) J Alzheimers Dis 2008,13: 225-232 (4) Leuk Lymph 2008,49: 427-438 (5) Blood 2009,114:5251-5255 (6) Biochim Biophys Acta 2009,1790:694-701 (7) Diabetes Care 2004,27: 2730-2732

Clinical significance of LPI LPI detection has been applied to: • Assess the efficiency of transferrin therapy on thalassemic mice (1), • Assess chelator (deferasirox) efficiency on MDS (2,3a), aplastic anemia, or acute myeloid leukemia (3b-c) • Compare different chelation protocols in patients with thalassemia major: dfo/deferiprone (4) or deferasirox (5), • Compare the efficiency of antioxidants (vitamin C, tocopherol) on the blocking of Fe-induced HDL oxidation in models of CVD (6), • Asses chelation therapy compliance (7) and tailored chelation regimens (8)

(1) Nature Med 2010,16:177-180 (2) Acta Haematol 2009,122:165-173 (3) (a) Haematologica 2008,93:91-92 (b) Transfusion 2015,55:1613- 1620 (c) Eur J Hematol 2016,96:19-26 (4) Br J Haematol 2009,147:744-751 (5) Eur J Haematol 2009,82:454-457 (6) Antiox Redox Signal 2010,12:209-217 (7) Blood Cells Mol Dis 2018,71:1-4 (8) Hematology 2017,22:183-191

10 Mitochondrial Iron probing

Charareh Pourzand Department of Pharmacy & Pharmacology Centre for Therapeutic Innovation University of Bath, UK

4th May 2019 Fluorescence Monitoring of Labile Iron trafficking in Cells and Iron Speciation in Animal Fluids and Organs Intracellular Labile Iron Pools

Fe Tf Tf TfR Ft H+ Ft Proton pump

(Cytosolic) LIP Lysosome

Nucleus

Cytosol Tf: transferrin Ft: ferritin

LIP: labile iron pool Aroun et al. Photochem. Photobiol. Sci. (2012) LIP and UVA-induced necrosis in skin fibroblasts

UVA

Ft degradation Cytosol Lysosome

(Cytosolic) LIP

Nucleus

Pourzand et al. PNAS (1999) Zhong et al. J. Invest. Dermatol. (2004) Reelfs et al. J. Invest. Dermatol. (2004) Reelfs et al. Curr. Drug Metab. (2010) Aroun et al. Photochem. Photobiol. Sci. (2012) Research Focus from 2012-to date (Pourzand-Hider)

 Design and evaluation of mitochondrial iron sensors to:

 Monitor the mitochondrial labile iron pool under normal,

oxidative stress and pathological conditions.

 Evaluate the susceptibility of cells to iron-mediatedmediated oxidative damage.

Potential applications:

 Mitochondrial iron sensors as diagnostic and prognostic tools for

 Pathologies with particular sensitivity to oxidative stress

 Mitochondrial iron-overload diseases (e.g. Friedreich’s ataxia, Parkinson’s disease) Abbate et al, Amer. J. Haematol, 2013  Mitochondrial iron sensors as probes to evaluate the chelation Reelfs et al, JWMS, 2015 Abbate et al, Biochem J. 2015 Abbate et al, Chem Comun, 2016 potency of clinically approved iron chelators for treatment of iron Reelfs et al, Amer. J. Haematol 2016 Reelfs et al, J. Invest Dermatol, 2016 overload diseases. Reelfs et al, Metallomics, 2019 Research Focus from 2012-to date (Pourzand-Hider)

 Design and evaluation of mitochondrial iron chelators to:

 Adjust the excess harmful mitochondrial labile iron under oxidative stress and pathological conditions.

 To protect the cells from excess mitochondrial labile iron contributing to iron- mediated oxidative damage in the organelles.

Potential applications:

Pathologies with particular sensitivity to oxidative stress

 Skin Photoprotection ( Integration to sunscreen formulations)

 Therapy of mitochondrial iron overload diseases Abbate et al, Amer. J. Haematol, 2013 Reelfs et al, JWMS, 2015 Abbate et al, Biochem J. 2015 (e.g. Friedreich’s ataxia, Parkinson’s disease) Abbate et al, Chem Comun, 2016 Reelfs et al, Amer. J. Haematol 2016 Reelfs et al, J. Invest Dermatol, 2016 Reelfs et al, Metallomics, 2019 Research Focus from 2012-to date (Pourzand-Hider)

Challenges

1 Membrane penetration 2 Metal selectivity

Zn 3 Cu Subcellular specificity Fe

Mg Research Focus from 2012-to date (Pourzand-Hider)

1st generation of mitochondrial iron sensors

FLUOROPHORE SELECTIVE IRON CHELATOR F

TARGETING MOIETY

F Fe

Abbate et al, Amer. J. Haematol, 2013 Reelfs et al, JWMS, 2015 Abbate et al, Biochem J. 2015 Targeting mitochondria: the “SS tetrapeptides”

H-DMT-D-Arg-Phe-Lys-NH2 H-D-Arg-DMT-Lys-Phe-NH2 H-Phe-D-Arg-Phe-Lys-NH2

The “SS tetrapeptides” as “druggable” compounds

 Alternating aromatic and basic residues

 Basic residues provide for localization in the inner mitochondrial membrane

Small & Water Soluble

Freely penetrate cells in an energy-independent manner despite a net +3 charge

Resistant to peptidase degradation Zhao, G.M., Qian, X., Schiller, P.W., Szeto, H.H., 2003. J. Pharmacol. Exp. Ther. 307, 947-954. Able to pass the BBB Zhao, K., Luo, G.X., Zhao, G.M., Schiller, P.W., Szeto, H.H., 2003. J. Pharmacol. Exp. Ther. 304, 425-432. Zhao, K., Zhao, G.M., Wu, D., Soong, Y., Birk, A.V., Schiller, P.W., Szeto, H.H., 2004. J. Biol. Chem. 279, 34682-34690. Long elimination half-life Zhao, K., Luo, G.X., Giannelli, S., Szeto, H.H., 2005. Biochem. Pharmacol. 70, 1796-1806. Szeto HH, Ann N Y Acad Sci. 2008;1147:112-21. Dansylated mitochondria-targeted iron sensors

Abbate et al, Amer. J. Haematol, 2013 Reelfs et al, JWMS, 2015 Abbate et al, Biochem J. 2015 Chimeric iron sensor peptides: 1st generation iron sensors

Fl R + + Fl F chimeric catechol compound HBl (bidentate iron chelator)

mitochondria HPO Non-chelator - Iron Iron - - chelator chelator SS-peptide Fe SS-peptide Fe - - -

mitochondrion Fl Fl

Abbate V*, Hider R, Pourzand C, Reelfs O*. Amer. J. Haematol. (2013) Abbate V*, Reelfs O*,, Hider R, Pourzand C. Biochem. J. (2015) Evaluation of Iron responsiveness

Fluorescence profiles In solution In cells

a aChelator compound 750 Fe(HQ) Fe addition 600 3 m Fe removal 180 +100 M deferiprone 450 + Fe (III) .]

300 140

Fluorescence Intensity Fluorescence 150 fluorescence

0 450 475 500 525 550 575 600 100 Control wavelengthnm [nm] 10BP17G 13BP19 b Non-chelator compound 60 14BP22 400 Fluorescence [a.u.] Fluorescence

350 [a.u Fluorescence 300 + Fe (III) 20 250 -10 0 10 20 30 40 50 60 70 80 200 Time [min] 150 Time [min] Fluorescence intensity Fluorescence fluorescence 100

450 475 500 525 550 575 600 625 650 wavelengthnm [nm] Subcellular distribution of lead compound BP19 Fluorescence microscopy Compound Chimeric iron sensor peptides: 2nd generation: “intrinsically fluorescent” iron chelator Merge

BH22 = chelator AND fluorophore

F K

R +Fe(HQ)3

• More efficient chemical synthesis. 90 + DFP • Improved physico-chemical properties. Fluorescence [a.u.] Fluorescence

40 -10 0 10 20 30 40 50 60 Time [min]

Abbate V*, Reelfs O*,, Kong X, Pourzand C, Hider RC. Chem Com (2016) Current Research Design and evaluation of mitochondrial iron sensors/chelators to  monitor and adjust the mitochondrial labile iron pool  to protect cells from oxidative stress-induced damage Potential applications:  Skin Photoprotection ( Integration to sunscreen formulations):  Occupational and recreational exposure  Pathologies with particular sensitivity to oxidative stress (e.g. mitochondria iron-overload diseases including Friedreich’s ataxia)

Abbate et al, Amer. J. Haematol, 2013 Reelfs et al, JWMS, 2015 Abbate et al, Biochem J. 2015 Abbate et al, Chem Comun, 2016 Reelfs et al, Amrer. J. Haematol 2016 Reelfs et al, J. Invest Dermatol, 2016 Reelfs et al, Metallomics, 2019

) 2004 ( Dermatol. Invest. J. al. et Zhong

High Necrosis High Low Necrosis Low

High ATP depletion ATP High Low ATP depletion ATP Low

Low mitochondrial damage mitochondrial Low High mitochondrial damage mitochondrial High

UVA

High basal Cytosolic LIP Cytosolic basal High Cytosolic LIP Cytosolic basal Low

Skin fibroblasts Skin keratinocytes Skin

> Cell susceptibility to oxidative stress oxidative to susceptibility Cell > - Susceptibility SS < LIP

High mitochondrial LIP? mitochondrial High Low mitochondrial LIP? mitochondrial Low

High basal Cytosolic LIP Cytosolic basal High Cytosolic LIP Cytosolic basal Low

4/ORF7 FEK HaCaT cells HaCaT

fibroblasts fibroblasts Skin keratinocytes Skin

Mito-LIP <- Mito-LIP Susceptibility SS models Cell >

Preparations for Mito for Preparations Susceptibility SS measurement: -LIP Mitochondrial LIP fluorimetric quantification

Remove condition medium (CM) from the plates Wash the plates twice by adding 3 ml PBS and aspirating Trypsinize for about 4 min 20 s (for HaCaT cells) in incubator and add back the respective CM for each conditions Place the cell suspensions in 3 x 15 ml falcon tubes and spin them at 1000 rpm for 5 min Trypsin Aspirate supernatant and resuspend in 700 µL of buffer (HEPES pH 7.3, 150 mM NaCl) Transfer 200 µL of cells in triplicates to blackwalled 96-well plates Transfer 50 µL to eppendorf make a 1:1 volume ratio with trypan blue @ 1000 rpm for 5 min Count cells

700 µL Buffer Buffer only

Unstained cells

Cells + BP19

Cells + BP19 + DFO Procedure for the preparation of Fe:NTA complex (Calibration curve)  Mix Fe solution and NTA in a 1:3 molar ratio prepared in MilliQ water  Add 40 µL of 17.4 mM Fe solution + 120 µL of 17.4 mM NTA 120 µL of µL of  Let stand for 1 h at RT for complex to form 40 17.4 17.4 mM NTA  Add 10 µL of Fe:NTA complex solution to 395 µL of MOPS = 107.16 µM mM Fe solution  Then, add 80 µL of 107.16 µM solution to 720 µL MOPS = 10.716 mM  Make the premixes in the relevant eppendorf tubes numbered from 0 to 1.0 mM as follow: Eppendorf # Volume from 10.716 uM Premix volume of MOPS FeNTA (µL) per well 0 0 250 0.1 4.65 245.35 0.2 9.3 240.7 395 µL of µL of 0.3 13.95 236.05 10 Fe:NTA MOPS 0.4 18.65 231.35 0.5 23.3 226.7 0.6 27.95 226.7 Transfer 80 µL 0.7 32.65 217.35 0.8 37.3 212.7 0.9 41.95 208.05 720 µL MOPS 1.0 46.65 203.35  Add all premixes to a blackwalled 96 well plate Dilution of BP19 for Fe:NTA calibration curve Prepare a 20 µM solution of BP19 with two consecutive dilutions from a 50 mM stock solution of BP19. First make up a 100 µM solution by adding 2 µL of 50 mM of BP19 to 998 µL of MOPS solution Then make up a 20 µM solution by adding 800 µL of 100 µM solution to 3.2 mL of MOPS solution Add 50 µL of 20 µM BP19 solution to all the wells related to the calibration curve Mix with a multichannel pipette and incubate for 10-15 min at RT

3.2 ml MOPS 998 µL MOPS µL of µM 2 µL of 100 mM 800 100 of BP19 of BP19 Add 50 µL of 20 µM BP19 solutions to wells 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0.8 0.9 1.0

Mix with a multichannel 0.8 0.9 1.0 pipette and incubate for 10-15 min 0.8 0.9 1.0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Buffer only 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Unstained cells 0.8 0.9 1.0

0.8 0.9 1.0 Cells + BP19

0.8 0.9 1.0 Cells + BP19 + DFO 0.8 0.9 1.0 Read fluorescence on a fluorescence plate reader (37°C), Excitation wavelength 330 nm, Emission wavelength 550 nm b D ʹ BP19 ͳ ʹǣ

[a.u] ǡ ȏǤǤȐ ȏǤǤȐ BP19 BP19 Fluorescence Fluorescence

ͳǣ Cells incubated with: BP19 DFO + BP19 BP19 ͵Ϊ ͵Ϊ m 6 6 m Cell counts tot cells *10 Fluo/10 Fluo Mean SD Mean cells*106/ml F2-F1/F2 F2-F1 mLIP [mM] (haemocytometer) in 700 ml cells Healthy ȏ Ȑ ȏ Ȑ Ctrl 27890 28378 30308 28859 1279 249 241 235 242 2.42 1.69 17059 19 46155 42347 44251 2693 181 197 171 183 1.83 1.28 34544 DFO + 19 39475 37198 35140 37271 2168 124 130 125 126 1.26 0.88 42146 0.18 7602 0.18 FRDA Ctrl 28504 28241 26835 27860 897 100 111 82 98 0.98 0.68 40751 19 38413 39208 38811 562 70 65 62 66 0.66 0.46 84432 DFO + 19 47523 50022 48773 1767 69 51 56 59 0.59 0.41 118764 0.29 34332 0.80

Calibration curve Fe [mM] Fluo delta Fluo SLOPE (delta F curve) 43079 0 90424 0 0.1 84844 5580 100000 Fluo quenching 60000 Fluo change (delta F) 0.2 85599 4825 0.3 80000 0.4 70753 19671 40000 0.5 64921 25503 60000 y = 43079x 0.6 66431 23993 y = -42352x + 89898 R² = 0.9494 0.7 56603 33821 40000 R² = 0.9498 0.8 54858 35566 20000

0.9 32173 Fluo change [a.u.] 58251 [a.u.] Fluorescene 20000 1 46069 44355 0 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 Fe [mM] Fe [mM]