Tweaking the NRF2 signaling cascade in human myelogenous leukemia cells by artificial nano-organelles

Significance Elevated levels of reactive oxygen species (ROS) are at the core of several chronic inflammatory pathologies, including fibrosis and neurodegenerative disorders. The NRF2 signaling axis is a predominant player in restoring redox homeostasis inside cells. Exploiting NRF2 activation for reboosting cellular ROS detoxification has been suggested as promising therapeutic strategy in age-related inflammatory diseases. Here, we undertake an alternative approach to expand on the ROS defense by introducing artificial nano-organelles (AnOs) with an advanced design into leukemic K562 cells and dissect how they engage in endogenous redox signaling. The demonstrated modulation of signaling pathways by these AnOs paves the way for the development of adjuvant therapies in situations where NRF2 activation is not sufficiently effective.


B C
Table S1.Quantification of dyes per fluo-AnOs by FCS.
Table S2.Table S3.Particle concentration of DL633-AnOs and A647-AnOs by nanoparticle tracking analysis (NTA).Reporter cells were seeded at 25,000 100 µl -1 per well and AnOs were added after 24 h.Nonfluorescent AnOs or A647-labelled AnOs with CPPs were supplemented as indicated.Medium with potassium iodide as electron donating co-substrate was administered 7 h after AnO supplementation, and H2O2 was added after an additional hour.24 h after the oxidative insult with H2O2, cells were analyzed by flow cytometry, microscopy and/or RT-qPCR to check for NRF2 activation state and cell viabilities.

Technical details for "Preparation of CPP-functionalized fluo-AnOs"
The dried polymer concentration was 10 mg mL -1 with both dyes, DyLight 633 NHS ester or Atto647N DOPE, at a concentration of 2 µM.After rehydration in PBS, resulting fluo-AnOs were extruded with an Avanti mini-extruder (Avanti Polar Lipids, Alabama, USA) through a polycarbonate (PC) membrane with a 100 nm diameter pore size for 11 times.Non-embedded fluorescent dye was separated from polymersomes by size exclusion chromatography (SEC, Sepharose 2B column; 37 cm length).Then, 1 µL of the DBCO-coupled CPP (7 mM in PBS) was added to 1 mL of the LPO-AnO suspension and stirred at 37 °C for 16 h in the dark.

Technical details for "AnO characterization by static (SLS) and dynamic light scattering (DLS)"
Multi-angle light scattering data were recorded on a spectrometer (LS Instruments, Switzerland) equipped with a 21 mW 633 nm He-Ne laser.All experiments were measured at scattering angles between 40 and 135° at 25 °C in round-bottom cuvettes (10 x 0.9-1.0mm, Boro 3.3, LS Instruments (Fribourg, Switzerland).For both static (SLS) and dynamic (DLS) light scattering measurements, selfassembly dispersions diluted to 0.05 mg mL -1 were used without filtration.Hydrodynamic radii (Rh) were obtained from DLS measurements from the mean of three independent measurements over the whole range of angles using second order cumulant analysis.Polydispersity indices (PDI) were calculated from the 90° DLS measurements.For SLS analyses, mean intensities (N = 3) were plotted against the respective angle and fitted with a Mie scattering model (MiePlot, UK) for ɳ = 1.35 and 5 % polydispersity.The radius R obtained from the best fit was transformed into the radius of gyration (Rg) using the formula for spherical structures Rg 2 = (3/5)R 2 .

Technical details for "Transmission electron microscopy (TEM)"
For TEM, aliquots of AnOs in PBS at 0.25 mg mL -1 were adsorbed to a freshly glow-discharged, carbon-coated, parlodion-(2 % in n-butyl acetate) copper grid (Quantifoil, Germany).Then, the grid was washed four times with 50 µL water.After blotting excess liquid, the specimen was negatively stained with a 2% uranyl acetate solution (5 µL) for 10 s, washed three times with water and dried by blotting.TEM images were recorded on a CM100 transmission electron microscope (Philips, Eindhoven, The Netherlands) at an acceleration voltage of 80 kV.

Technical details for "Fluorescence correlation spectroscopy"
FCS experiments were conducted at 20 °C using a 40x water immersion objective (C-Apochromat 40x, NA 1.2; Carl Zeiss, Jena, Germany).For each measurement, 10 µL of sample were placed on a 22 × 50 mm glass slide, and a 633 helium/neon laser was used to excite DyLight 633.
The laser was passed through the MBS488/561/633 filter and the signal was detected in the range of 657-690 nm with a pinhole aperture of 39 µm.Fluorescent fluctuations over time were recorded for 30 x 5 s.The raw data was processed and analyzed using ZEN software.

Enzyme quantification by bicinchoninic acid assay
The LPO encapsulation efficiency in AnOs was calculated using the enhanced Pierce bicinchoninic acid (BCA) assay according to the supplier's protocol (#23227, Thermo Scientific).AnOs were first ruptured by sonication and then incubated with ethanol at a ratio of 3:1 (v/v; final volume: 200 µL) at 37 °C for 1 h.The solution was passed through a 0.2 μm nylon membrane filter device (Whatman) and mixed with the BCA assay reagent at a 1:2 ratio (v/v).LPO standards and samples were incubated for 2 h at 37 °C, then the absorbance was measured at 562 nm using a SpectraMax id3 plate reader.

Technical details for "Isolation of PBMCs from buffy coats"
For isolating immune cells from blood, buffy coats were diluted ~ 1:4 in PBS and 15 mL lymphoprep™ density gradient medium (#07851, STEMCELL Technologies) was filled per SepMate tube (#85450, STEMCELL Technologies) before 35 mL of diluted buffy coat was added without disturbing the density gradient medium.Blood cells were separated by centrifugation at 1,200 g for 10 min and the PBMC containing top layer was harvested.The PBMC fraction from several SepMate tubes were pooled and pelleted by centrifugation at 120 g for 10 min (no break) and then washed by resuspension in 50 mL PBS followed by centrifugation at 300 g for 8 min.For freezing, PBMCs were normalized to 100 x 10 6 mL -1 after centrifugation at 300 g for 5 min and resuspended in FBS containing 10 % DMSO.
Cells were frozen at -80 °C and transferred the day after to liquid nitrogen for long term storage.For thawing PBMCs, PBMC aliquots were thawed at 37 °C in the presence of 250 µL 1 mg mL -1 DNAse (#DN25-100mg, Sigma-Aldrich) to avoid precipitates.

Technical details for "NRF2 activity assay for K562 reporter cells using flow cytometry"
Cell seeding: Generally, reporter cells were seeded in 100 µL RPMI medium supplemented with 50 µg mL -1 gentamycin at 2.5 × 10 4 cells per well.If cells were pre-treated with BSO, cells were alternatively seeded in 50 µL RPMI medium and treated 6 h later with 50 µL 2x BSO in RPMI resulting in 200 µM BSO per well (BSO: #B2640-500MG, Sigma-Aldrich).After 24 h of culture, 20 µL of AnOs in PBS were added to the cells for a final AnO concentration in the well of 10 % (v/v).After 7 h incubation, 50 µL RPMI culture medium containing a 4x KI concentration (#207969-100G, Sigma-Aldrich) were added to each well resulting in a final KI concentration of 6 µM.After 1 h of KI exposure, cells were treated with 50 µL RPMI culture medium containing H2O2 (H2O2 solution purum p.a., ≥35%, Sigma-Aldrich, #95299-500ML) at 4x the concentration of the final concentration per well (total medium volume 200 µL).After 24 h incubation with H2O2, cells were examined by flow cytometry or microscopy, or RNA was extracted for performing RT-qPCR.

Processing of cells for flow cytometry:
In preparation for flow cytometry, non-internalized AnOs were washed away by repeated centrifugation and resuspension steps, and dead cells were labelled with violet live/dead™ stain (#L34963, Thermo Scientific) according to the supplier's protocol.After AnO and/or H2O2 treatment, cells were harvested by centrifugation (160 g, 3 min) in U-bottom 96 well plates and washed in 1x 200 µL PBS by resuspension and centrifugation (160 g, 3 min).For labelling dead cells, cell pellets were resuspended in 100 µL violet live/dead™ dye in DMSO, diluted 1:1000 in PBS, and incubated for 30 min at RT in the dark.The labelling reaction was stopped by adding 100 µL FACS buffer (PBS with 1% bovine serum albumin from PAN Biotech, #P06-1391100, 2 mM EDTA from Sigma, #E6511) and cells were washed 2x with 200 µL FACS buffer by centrifugation (160 g, 3 min) and resuspension.

Acquisition details for flow cytometry:
Dead cells were quantified with the live/dead™ stain.K562 reporter cells expressed GFP under control of a constitutive CMV promotor to report global protein expression and mCherry in response to NRF2 activation.Uptake of AnOs in K562 reporter cells was investigated by either quantifying DL633 or A647 fluorescence.Per data point a total of 5,000 events was recorded.
Cells were analyzed at a flow speed of 1 µL s For NRF2 activity assessment, mCherry intensity medians were divided by GFP intensity medians for each sample and fold changes were calculated by normalizing to the respective control condition.

Analysis details for calculating cell viabilities:
Debris was excluded by polygonal gating in FSC-A:SSC-A plots while conserving dead cells and two populations were detected in violet live/dead™ histograms: live and dead cells with low or high violet live/dead™ intensity, respectively.Histogram gating was used to demarcate dead cells (= high violet live/dead™ intensity) from live cells to determine cell viabilities.Data were exported and analyzed in Microsoft Excel to calculate the percentage of living cells per condition and data were plotted in Prism (V9.2.0).

Technical details for: "Killing and rescue assay with PBMCs using flow cytometry"
Cell seeding: PBMCs were seeded in 100 µL PBMC medium at 10 5 cells per wells.AnO supplementation, as well as KI and H2O2 were identical as for K562 reporter cells, but in PBMC medium.After 24 h incubation with H2O2, PBMCs were processed for flow cytometry and/or microscopy.PBMCs were prepared for flow cytometry as K562 reporter cells.Uptake of AnOs in T-lymphocytes was quantified by measuring A647 fluorescence.

PBMC viability assessment and gating for T-lymphocytes:
To gate the live and dead T-lymphocyte populations for later analyses, PBMCs were stained for CD3, CD14 and CD19 using conjugated antibodies.Conjugated antibodies were purchased from BioLegend (PE-CD3: #981004; BV750-CD14: #367135, AF488-CD19: #363037).PBMCs were washed in 1x 200 µL FACS buffer by centrifugation and resuspension (300 g, 3 min).Fc receptors on PBMCs were blocked with 100 µL Human TruStain FcX™ (#422301, BioLegend) diluted 1:20 in FACS buffer for 10 min at RT. PBMCs were washed by centrifugation (300 g, 3 min) and resuspension in 1x 200 µL FACS buffer.PBMCs were then stained for CD3, CD14 and CD19 by antibodies diluted 1:100 in FACS buffer for 30 min at 4°C in the dark.PBMCs were washed by adding 100 µL PBS followed by centrifugation (300 g, 3 min) and resuspension in 1x 200 µL PBS followed by another centrifugation (300 g, 3 min).Dead cells were labelled with violet live/dead™ stain (diluted 1:1000 in PBS) for 30 min at RT in the dark and the reaction was stopped by adding 100 µL FACS buffer followed by centrifugation (300 g, 3 min).PBMCs were washed again with FACS buffer as before.Cells were then fixed in 100 µL 4 % formaldehyde (#P087.5,Carl Roth) for 10 min at RT in the dark and the reaction was stopped by addition of 100 µL FACS buffer followed by centrifugation (300 g, 3 min).PBMCs were washed again with FACS buffer as before.Cells were resuspended in 200 µL FACS buffer and analyzed by flow cytometry.
PBMCs were analyzed on a BD LSRFortessa and fluorochrome spillover was compensated using single-stained controls (for antibodies: CompBead Plus #560497, Becton Dickinson; for live/dead stain: Arc Amine Reactive Compensation Bead Kit, #A10628, Thermo Fisher) prepared according to the instructions of the manufacturer.

Acquisition details for flow cytometry:
Cells were analyzed at a flow speed of 1.5 µL s

Technical details for RT-qPCR
For gene expression analysis in reporter cells, RNA was extracted using the RNeasy Plus Mini Kit (#74136, Qiagen) according to the instructions of the manufacturer.500 ng of RNA were reverse transcribed to cDNA using the iScript™ Advanced cDNA Synthesis Kit (#1725038, BioRad) according to supplier's protocols.Then, gene expression was analyzed by RT-qPCR in technical triplicates on a CFX Connect TM Optics Module (BioRad) light cycler using SYBR master mix (#1725271, BioRad) according to the manufacturer's instructions.Primers were purchased from Microsynth.
Per well of a Hard-Shell® 96-Well PCRHSP9655 Plate (#HSP9655, Biorad), we mixed 10 µL of 2 x SYBR master mix (#1725271, BioRad), 4 µL of diluted cDNA, 2 µL each of forward and reverse primer, and 2 µL RNAse/DNAse free H2O.The plate was sealed with Microseal ® B Adhesive Sealers (#MSB-1001, BioRad) and the qPCR reaction was observed on a CFX Connect TM Optics Module (BioRad) light cycler over a total of 40 cycles after 3 min denaturation at 95 °C: a) 10 s at 95 °C, b) 30 s at 62 °C, melting curve: increase of 0.5 °C every 5 s until 95 °C.Primer dimers were excluded by melting curve analysis.

Technical details for: "Quantification of glutathione"
For determining cellular glutathione levels, K562 reporter cells were seeded in 1.2 mL PRMI medium at a concentration of 0.5 x 10 6 cells mL -1 in transparent, flat-bottom 6-well microtiter plates (#Z707759, TPP).After 6 h, cells were treated without or with 200 µM BSO by adding 1.2 mL RPMI medium at a 2x BSO concentration.KI was added 24 h after seeding by supplementing 1.2 mL RPMI medium at a 4x concentration resulting in 6 µM per well and 1 h later cells were treated without or with 40 µM H2O2 in 1.2 mL RPMI medium also at 4x of the final concentration of the well.After 24 h, cells were harvested by centrifugation (160 g, 3 min) and washed 1x in 1 mL PBS.Cells were then further processed according to the instructions of the kit's manufacturer.The amount of chromogenic product was measured after 30 min in technical triplicates on a TECAN Spark 10M UV/VIS plate photometer at 405 nm.Images were exported as tagged image file (TIF) and processed in Image J Version 2.9.0.Z-stacks were merged as maximum intensity projections.For improved visibility, channel look-up-tables (LUT) were changed and brightness/contrast was adjusted accordingly for all imaged ROIs (DL633: LUT "fire" min.15 max.140; WGA488: LUT "green" min.5 max.255; Hoechst: LUT "grays" min.0 max.
Acquisition details for imaging with airyscan superresolution microscopy (Figure 6) AnO uptake in PBMCs was examined on a Zeiss LSM 880 AxioObserver.16-bit images were acquired as z-stacks (3 planes, range 2 µm, distance 1 µm) using a plan-apochromat 63x NA 1.40 DIC M27 objective with oil immersion with 1888x1888 pixels which scales to 67x67 µm in size.Fluorophores were excited in the listed order using a gain of 800 and a BP 495-550 + LP 570 filter for all channels: A647: HeNe633 laser at 633 nm with 80 % intensity WGA488: Argon laser at 458 nm with 10 % intensity Hoechst: Diode 405-30 at 405 nm with 9 % intensity Superresolution images were calculated in ZEN 2012 software using the 2D airyscan processing algorithm with constant processing strength of 7.3 to allow for comparable intensities between conditions.Images were exported as CZI files for further processing in Image J Version 2.9.0.Zstacks were merged as maximum intensity projections and for improved visibility, central regions with cells of interest were cropped (from pixels xy 185, 185 to xy 1450, 1450).LUTs were changed and brightness/contrast was adjusted accordingly for all imaged ROIs (A647: LUT "fire" min.50 max.1500; WGA488: LUT "green" min.20 max.1500; Hoechst: LUT "grays" min.0 max.7000).

Figure S3 .
Figure S3.LPO functionality of active and inactive A647-AnOs with or without CPP.

Figure S6 .
Figure S6.Experimental timeline of treating reporter cells with AnOs and required substrates.Figure S7.Dampened NRF2 transcriptional activation by scavenging intracellular H2O2 with AnOs (RT-qPCR of mCherry).
Figure S6.Experimental timeline of treating reporter cells with AnOs and required substrates.Figure S7.Dampened NRF2 transcriptional activation by scavenging intracellular H2O2 with AnOs (RT-qPCR of mCherry).

Figure
Figure S8.H2O2 scavenging by AnOs reduces NRF2 driven mCherry expression in a dose

Figure S1 .
Figure S1.AnOs with surface coupled CPP.(A) Chemical composition of the DBCOfunctionalized cell penetrating peptide (CPP) used for coupling to the surface of AnOs.Oneletter code identifies amino acids constituting the dendrimer.(B) TEM micrographs of DL633-melAnOs-CPP (active), harboring fluorescent dyes in their membrane.(C) TEM micrographs of A647-melAnOs-CPP (active) showing the typical morphology of collapsed polymersomes.Scale bar: 1000 nm.

Figure S4 .
Figure S4.Uptake of inactive and active CPP-functionalized DL633-AnOs and A647-AnOs in MCF-7 cells.Images of MCF-7 cells incubated with different samples of DL633-AnOs or A647-AnOs for 24 h obtained by confocal laser scanning microscopy.Cells were fixed with 4 % paraformaldehyde and cell nuclei were stained with Hoechst 33342.Images present transmission light channel merged with blue fluorescence of cell nuclei (Hoechst 33342) and red fluorescence of DL633-AnOs (top panels) and A647-AnOs (bottom panels).Scale bar = 20 µm.

Figure S5 .Figure S6 .
Figure S5.CPP-dependent uptake of active A647-AnOs in MCF-7 cells.Images of MCF-7 cells incubated with different samples of A647-AnOs for 24 h as obtained by confocal laser scanning microscopy.Cells were fixed with 4 % paraformaldehyde and cell nuclei were stained with Hoechst 33342.Images present transmission light channel merged with blue fluorescence of cell nuclei (Hoechst 33342) and red fluorescence of A647-AnOs.Scale bar = 50 µm.

Figure S12 .
Figure S12.Identification of immune cell subpopulations in PBMCs under basal (= no H2O2) and stressed conditions (= 500 µM H2O2) using flow cytometry.PBMCs from donor #1 and #2 were seeded at a density of 100,000 in 100 µl per well and either oxidative stress was induced by addition of 500 µM H2O2 (A donor #1, C donor #2) or cells were kept under basal conditions (B donor #1, D donor #2) for 24 h before analysis by flow cytometry.A total of N = 10,000 events (excluding debris) was analyzed for each condition.Debris was excluded by stringent gating in FSC-A:SSC-A plots and the T-cell, monocyte and B-cell subpopulations were identified by staining for CD3, CD14 and CD19 using tagged antibodies (CD3-PE, CD14-BV750, CD19-AF488).The percentage of B-cells was less than 1 % of all cells which is why we excluded them from further analysis.For the T-cell and monocyte subpopulation single cells were gated in FSC-H:FSC-A plots and violet LIVE/DEAD stain was used to determine percentages of live and dead cells.The violet LIVE/DEAD stain could only discriminate between live and dead T-cells, as for monocytes peak separation was not achieved.Live T-cells (blue), dead T-cells (orange) and total monocytes (green) were overlayed in FSC-A:SSC-A plots (E) to determine a gate for live & dead T-lymphocytes which was used for further analyses.

H 2 O 2 concentration live cells, % Flow cytometry: reporter cell viability sgCTRL sgNFE2L2 0 50 100 150 90.5 48.1 89.4 74.8 live cells, % Rescue of NRF2 knockdown cells by A647-melAnOs-CPP
Flow cytometry standard files were exported from BD LSRFortessa DIVA software and analyzed in FlowJo using hierarchical gating: 1) stringent, polygonal gate framing the live-cell population in FSC-A:SSC-A plots while excluding dead cells and debris; 2) diagonal, rectangular gate in FSC-H:FSC-A plots to exclude doublets; 3) GFP-A intensity histogram plot: two populations were detected and GFP-negative cells were excluded from further analysis.Means of GFP intensities of GFP positive cells, means of mCherry intensities and DL633 or A647 median fluorescent intensities were exported as table from FlowJo and further analyzed in Microsoft Excel.DL633/A647 intensity histograms were plotted in FlowJo using the layout editor.

Technical details for: "Fluorescence microscopy"
Details for deposition of cells by cytospin K562 reporter cells or PBMCs were seeded and treated with labelled AnOs, KI and H2O2 as described for flow cytometry.After incubation, cells from 2-3 wells of the 96-well culture plate were pooled.Pooled cells were washed by resuspension in 200 µL PBS and centrifugation (300 g for 3 min).Cells were fixed in 200 µL 4 % formaldehyde (#P087.5,Carl Roth) for 10 min at RT in the dark and cells were harvested by centrifugation (300 g, 3 min).Cells were washed again with PBS buffer as before.Plasma membranes were stained with 200 µL CF®488A Wheat Germ Agglutinin (WGA488, #29022-11, Biotium) diluted 1:1000 in PBS for 15 min at RT in the dark and pelleted and washed as before.Finally, cells were resuspended in 50 µL PBS and deposited with cytofunnels (Fisherbrand™ Single Cytology Funnels, #10-354, Thermo Fisher Scientific) and filter cards (#5991022, Thermo Fisher Scientific) on cytoslides TM (#5991051, Thermos Andon) using a Shandon Cytospin 2 Centrifuge (Thermo Fisher Scientific) at 300 rpm for 3 min.After air drying the cell layer, cells were immersed in Prolong™ Glass Antifade Mountant with NucBlue™ Stain (#P36981, Thermo Fisher Scientific), which contained Hoechst stain for visualizing nuclei, and glass slides were sealed with nail polish.