T8787

For biomarker evaluation, at day 7, spheroids in each chip were washed in PBS for 5′, fixed in 4% paraformaldehyde solution (J19943.K2, Thermo Scientific) for 30′, permeabilized (0.3% Triton™ X-100 in PBS, T8787, Sigma-Aldrich) for 30′ and blocked (0.3% BSA in PBS) for 30′, using the OB1 MK3 controller to generate a flow rate of 80 µL/min. Then, triple labelling was performed overnight, in static conditions, using primary conjugated antibodies for γH2Ax (gamma H2AX [p Ser139] Antibody [Alexa Fluor 488], NB100-384AF488, Novus Bio/BioTechne), caspase-3 (CC3, Caspase-3 Antibody (31A1067) [Alexa Fluor 594], NB100-56708AF594, and KI67 (Anti-Ki67 antibody [EPR3610] (Alexa Fluor 647), ab196907, Abcam) at 1:500 dilution each. Nuclear counterstaining was performed by Hoechst 33342 at 1:5000 dilution (H3570, ThermoFisher).

After incubation with MTS, substrate scaffolds with cells were rinsed with PBS and transferred to a new Eppendorf tube with 600 µL of lysis buffer. The lysis buffer consisted of 10 mM Tris (T1503, Sigma Aldrich, Darmstadt, Germany), 1 mM EDTA (EDS, Sigma Aldrich, Darmstadt, Germany) and 4 × 10⁻⁴ % Triton X-100 (T8787, Sigma Aldrich, Darmstadt, Germany). Samples were frozen in the tubes. Samples were further vortexed three times and frozen for two more cycles. Finally, 200 µL of working solution Quant-iT™ PicoGreen dsDNA Assay Reagent (Q33120, Invitrogen, Bleijswijk, Netherlands) was transferred to a black 96-well plate with a transparent bottom. There is a fluorescently labeled probe in the working solution which starts to emit a signal after binding to dsDNA. The fluorescence was measured at an excitation wavelength of 485 nm and emission of 528 nm. The experiment was carried out in six biological repeats per group.

RPE1 cells cultured on coverslip were washed with PBS and fixed in 4% PFA at RT for 30 min, permeabilized in 0.1% Triton X-100 (T8787, Sigma, Saint Louis, MO, USA)/PBS for 15 min, washed with PBS, and then blocked in 5% FBS/PBS for 1 h at RT. Samples were then incubated with a primary antibody (diluted in blocking solution) at 4 °C overnight, washed several times with the blocking solution, and then incubated with a secondary antibody for 2 h at RT. After several washes, DNA was counterstained with DAPI (D9564, Sigma, Saint Louis, MO, USA). Samples were then mounted with mounting solution (P36934, Life Technologies, Carlsbad, CA, USA) and imaged using the Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). Primary and secondary antibodies used in this study were listed in Supplementary Table S1.

STHdh cells were exposed to 125 μM Mn and incubated at 37 °C (5% CO₂) in HBSS for 3 h. This generated “vehicle” values when Mn was co-incubated with representative amounts of DMSO (Sigma-Aldrich; D8418; St. Louis, MO, USA). Other small molecules were co-incubated with the 125 μM Mn in the HBSS to assess their impact on Mn levels. After incubation, the cells were washed three times with PBS (without Ca²⁺ and Mg²⁺) to remove the extracellular Mn. Extraction buffer (PBS containing 0.1% TritonX100 (Sigma-Aldrich; T8787; St. Louis, MO, USA) and 500 nM Fura-2) was added to lyse the cells. The fluorescence of the Fura-2 in the extraction buffer was then measured at 360_Ex/535_Em so that Mn could be quantified.

RCTEC-DBA cells were processed for immunofluorescence staining 48 h after H₂O₂ exposure with two primary antibodies for primary cilia [ARL 13B antibody (1711-1-AP, Proteintech, USA) and anti-acetylated α tubulin antibody (T7451-100ML, Sigma-Aldrich, USA)] and secondary antibodies [Alexa Fluor 488 (1711-1-AP, Proteintech) and Alexa Fluor 555 (ab150082, Abcam, UK)], and nuclei were counterstained with DAPI (KS042, Dojindo, Japan). The immunofluorescence procedure was performed by inducing the following solutions into inlet tip 3 sequentially by aspirating from outlet tip 2 at 2 μl min⁻¹ using a 1-ml syringe: 20 μl of 4% paraformaldehyde (163-20145, Wako, Japan) for 10 min, 40 μl of phosphate-buffered saline (PBS; 11482-15, Nacalai Tesque, Inc., Japan) for 20 min, 30 μl of 0.3% Triton X (T8787, Sigma-Aldrich) for 15 min, 100 μl of 3% bovine serum albumin (BSA; A9418-100G, Sigma-Aldrich) for 50 min, 120 μl of PBS containing ARL-13B antibody and anti-acetylated α tubulin antibody for 60 min, 120 μl of PBS containing Alexa Fluor 488, Alexa Fluor 555, and DAPI for 60 min, and 240 μl of PBS for 120 min. Images of primary cilia were recorded as three-dimensional data using structured illumination microscopy (BZ-X710, Keyence Corporation, Japan), and length of primary cilia was measured after projecting two dimensions using ImageJ/FIJI (1.52h).²⁰ (link)

For BrdU staining, antigen retrieval (DNA denaturation) was performed with 10mM sodium citrate (pH6) at 95 °C for 20 min, permeabilized in 0.1% Triton X-100 (T8787; Sigma) in PBS (PBST) for 3 × 10 min, then blocked with 20% goat serum (G6767; Sigma) in PBST for 30 to 60 min at room temperature. For P-cadherin staining only, 20% donkey serum (D9663; Sigma) was used instead of goat. Specimens were incubated with primary antibodies at 4 °C overnight. Primary antibodies were as follows: anti-RFP (1:500, 600-401-379; Rockland Immunochemical), goat anti-P-cadherin (1:200, AF761; R&D Systems), rabbit anti-non-muscle myosin IIB (1:200, 909901; BioLegend), and rat anti-BrdU (1:200, ab6326; Abcam). After six 1- to 2-h PBST washes, specimens were incubated at 4 °C overnight with Alexa Fluor–conjugated secondaries (Life Technology). Nuclei and F-actin were counterstained with DAPI (4′,6-diamidino-2-phenylindole; 1:5000, 62247; Thermo Fisher Scientific) and Alexa Fluor 488/635 Phalloidin (1:500, A12379, A34054; Invitrogen), respectively. Specimens were washed 6 times with PBST (1 to 2 h per wash) and then mounted on glass slides with 50% glycerol (356352; Calbiochem) in PBS. Z-stacks were acquired by a confocal microscope (TCS SP5; Leica) with oil immersion 40× and 63× objectives.