Cell culture and cell seeding
A549, Hep3B, and PANC-1 cells were grown in Roswell Park Memorial Institute 1640 medium (RPMI) supplemented with 10% fetal bovine serum (FBS), 1% GlutaMAX (all Thermo Fisher), and 1% Penicillin-streptomycin antibiotic cocktail (Sigma Aldrich). HMECs were grown in MCDB 131 medium supplemented with 10% FBS, 1% GlutaMAX (all Thermo Fisher), and 1% Penicillin-streptomycin antibiotic cocktail (Sigma Aldrich), 10 ng/ml hEGF (Thermo Fisher), and 1 µg/ml hydrocortisone (Sigma Aldrich). All cells were cultivated in a humidified 5% CO2 atmosphere at 37 °C until approx. 80% confluency was reached. Cells were sub-cultured twice a week by washing the confluent cells with 5 mL phosphate buffer saline (PBS) without Ca2+/Mg2+ (Thermo Fisher), detached with 1 mL trypsin-EDTA (Thermo Fischer). Cells were seeded into flat-bottom 96-well plates (Corning) before BAPO incubation and irradiation.
General synthetic techniques and analytical methods
All air- or moisture sensitive reactions were carried out under dry argon using either standard Schlenk techniques or working inside a glovebox (M-Braun Lab-Master MB 150 B-G). The glassware was stored in an oven at 130 °C for at least 16 h and cooled under vacuum prior to use. Solvents are purified using an Innovative Technology PureSolv MD 7 solvent purification system and stocked over activated molecular sieves. When degassed solvents were used, nitrogen is bubbled for a minimum of 15 minutes. Deuterated solvents were purchased from Cambridge Isotope Laboratories. All reagents were used as received from commercial suppliers unless otherwise stated. Air sensitive compounds were handled and stored in a M-Braun Lab-Master MB 150 B-G glovebox. Light sensitive compounds were handled with exclusion of light. Dialysis was performed using Spectrum Spectra/Por 6 pre-wetted standard RC Dialysis tubing, 1-50kD MWCO. Lyophilization was performed using a Christ Epsilon 2-4 -85 °C LSC plus Pilot Freeze dryer. NHS-BAPO was synthesized as previously reported.17,22 NMR spectra were recorded on Bruker Avance 200, 250, 300, 400 and 500 spectrometers operating at room temperature if not otherwise specified. Chemical shifts δ were measured according to IUPAC and are given in parts per million (ppm) relative to TMS (1H-NMR and 13C-NMR) and 85% H3PO4 in D2O (31P-NMR). The multiplicity of the signals is indicated as s (singlet), d (doublet), t (triplet), q (quartet) or m (multiplet). The abbreviation “br” describes broad signals. Absolute values of coupling constants J are given in Hertz (Hz). Mass spectrometry experiments were carried out by the MS Service (MoBiAS, Laboratory of Organic Chemistry, D-CHAB, ETH Zürich). Elemental analyses were carried out by the Micro-Laboratory (Laboratory of Organic Chemistry, D-CHAB, ETH Zürich).
For details on synthesis and characterization, see SI Fig. 2-6.
In vitro screens
When cell lines approached confluency, BAPOs were added at the respective concentrations. Untreated control wells were incubated with the respective amount of the solvent used for the corresponding BAPO preparation (media for BSA-BAPO and DMSO for the other BAPOs, maximum DMSO concentration below 1%). The plates were incubated for 1 d to allow for cellular uptake. Thereafter, the cells were washed with cell culture medium to ensure that the observed effect on cells was a result of the internalized BAPO rather than the excess in the media. BIOS was induced via irradiation with 365 nm light at 15 mW/cm2 for 30 minutes.
Irradiation
Irradiation was usually performed using the custom irradiation device described in SI Fig. 7. For fiber-based irradiation experiments, a custom setup described in SI Fig. 8 was used. For local irradiation (Fig. 1a and Fig. 2b), the emission of a mercury lamp passed through a 365/10 nm bandpass filter (Chroma) was focused onto the target area (approx. 240 μm in diameter, intensity of 9 mW/cm2) using a C-Apochromat 40x/1.2 W Korr UV-VIS-IR objective (Zeiss) installed on a commercial confocal microscopy setup (LSM980, Zeiss).
Viability assays
Cells were incubated with 4 μM calcein AM (CalA, Corning) and 2 μM ethidium homodimer-1 (EHD, Sigma Aldrich) in media. The assay relies on detecting the loss of membrane integrity and esterase activity as a measure of cell viability. In alive, metabolizing cells, non-fluorescent CalA is rapidly converted to fluorescent calcein by cytosolic esterases. Upon excitation, emission indicates live cells. On the other hand, EHD cannot penetrate the membrane of alive cells. If the plasma and nuclear membranes are compromised during cell death, EHD enters the cell and intercalates into DNA. Thus, nuclear fluorescence from EHD indicates dead/dying cells. After approx. 10 min, images of the center of the wells (approx. 1.7x1.7 mm2) were acquired utilizing a plate reader (ImageXpress Pico, Molecular Devices). The detected fluorescent signal was analyzed using Fiji. CalA and EHD fluorescence was analyzed utilizing the same approach. An appropriate threshold was applied to all images in the stack. To account for uneven fluorescence signals, heterogeneous cell size, and confluency of cells, binary transformation, appropriate particle size threshold, and Watershed algorithm were applied to the analysis, respectively. Parameters were kept identical for identical cell lines and channels within one experiment. Viability was calculated as the ratio of live cells to the total number of cells. Cells that showed both CalA and EHD fluorescence, which was occasionally observed, were counted as dead.
Morphological analysis
Bright field images were acquired directly after BIOS induction as well as two, six, and eight hours after. Stress granules were quantified using appropriate intensity and particle size thresholds. Granule count was then normalized by the number of cells in the field of view.
Rescue experiments
Cells were co-treated with 30 μM BSA BAPO and a respective concentration of sodium ascorbate. Irradiation, cell viability assay, imaging and image analysis were performed as described above.
Tissue phantoms and fiber-based irradiation
To investigate the optical effects tissue exhibits on BIOS activation (e.g., scattering and absorption), tissue-mimicking phantoms were prepared as described before.23 Briefly, 2 g bacteriological agar (VWR) and 0.05 g of aluminum oxide (Thermo Fisher) were dissolved in 55 mL of water supplemented with 25 μL of ink (Pelikan). The mixture was then heated to 94 °C, poured into rectangular aluminum molds (3 mm depth), and left to cool at room temperature. The mold was then cooled at 4 °C for 24 h in order to solidify. The next day, cells were treated using 80 μM alanine BAPO and were either covered by phantoms or not. To mimic fiber-based irradiation as required for activation in tissue, a custom setup was used (see SI Fig. 8 for details). The power at the sample was the same as for the previous experiments. The cell viability assay, imaging, and image analysis were done as described above.
In vivo zebrafish larvae screens
Two days post-fertilization (dpf) zebrafish larvae (wild type AB strain) were either treated with 10 μM BSA-BAPO or 10 μM BSA directly added to the water. After 1 d, the larvae were washed thoroughly, anesthetized in E3 medium containing 0.02% MS-222 (PharmaQ, Tricaine PharmaQ), and irradiated using the irradiation device (15 mW/cm2 for 30 min). For local irradiation experiments, we used Tg(mpx:GFP)uvm1 transgenic zebrafish expressing GFP under regulatory elements of the mpx gene to label neutrophils.24 BSA/BSA-BAPO incubation was performed as described above. For irradiation, larvae were anesthetized in 0.02% MS-222 and mounted in a lateral position in 1% low melting point agarose (Ultra-PureTM Low Melting Point, Invitrogen 16520) between two microscope cover glasses. During irradiation, larvae were covered with 0.01% MS-222-containing E3 medium to keep preparations from drying out. Irradiation was performed by passing filtered light from a mercury lamp through a C-Apochromat 40x/1.2 W Korr UV-VIS-IR40x objective (Zeiss) installed on a commercial confocal microscopy setup (LSM980, Zeiss). Fluorescent images were acquired using a Plan-Apochromat 10x/0.45 M27 objective installed on a laser scanning confocal microscope (LSM980, Zeiss). Brightfield overviews were acquired using a Leica M205 FCA stereo microscope equipped with a Leica DMC6200 C color camera.
Statistics
Due to the multitude of conditions to be compared, we do not show significance levels via symbols in the respective plots. All statistical analysis is summarized in a comprehensive excel sheet, available as additional information. Graphs show mean +/-SD. As three or more groups were compared in every experiment, a one-way ANOVA followed by Bonferroni correction was performed.
Software
OriginPro 8G was used for data visualization, analysis, and statistics. Images were analyzed using ImageJ. Figures were compiled in Adobe Illustrator.
References
22 Widera, A. et al. ACTIVE-BAPO – A Versatile Transfer Agent for Photoactive Bis(acyl)phosphane Oxide Units. Chemistry – A European Journal 29, e202203842, doi:https://doi.org/10.1002/chem.202203842 (2023).
23 Ntombela, L., Adeleye, B. & Chetty, N. Low-cost fabrication of optical tissue phantoms for use in biomedical imaging. Heliyon 6, e03602, doi:https://doi.org/10.1016/j.heliyon.2020.e03602 (2020).
24 Mathias, J. R. et al. Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish. J Leukocyte Biol 80, 1281-1288, doi:10.1189/jlb.0506346 (2006).