Dpbs buffer

The T₁ and T₂ values of MT218 and Gd(HP-DO3A) solutions were determined using a 3T MRS 3000 scanner (MR Solutions, Guildford, UK). Solutions of MT218 were prepared at concentrations of 0, 0.25, 0.5, 1, and 2 mM in DPBS buffer (Gibco, Gaithersburg, MD). T₁ values were measured using an inversion recovery-fast low angle shot (IR-FLASH) sequence (τ = 10 ms, TE = 4 ms, FA = 8°, echoes/frames = 128, FOV = 40 mm × 40 mm, slice thickness = 2 mm, number of averages = 1, time delay = 4,000 ms, sample period = 200, matrix = 256 × 128). T₂ values were obtained using a multi-echo multi-slice (MEMS) sequence (TE = 15 ms, repetition time = 15 ms, echoes = 10, FOV: 40 mm × 40 mm, slice thickness = 1 mm, number of averages = 1, matrix = 256 × 192). The r₁ and r₂ relaxivities were calculated from the slopes of 1/T₁ and 1/T₂ vs. Gd concentration plots, respectively.

Alkylation-fixation of a monolayer of HeLa Kyoto cells on 35-mm MatTek glass-bottom dishes was performed 2–3 days after transfection. Alkylation-fixation of neuronal cultures plated on 35-mm MatTek glass-bottom dishes was performed 10–14 days after viral transduction. To this end, cells on 35-mm MatTek glass-bottom dishes were washed 2 times with 2 mL of DPBS buffer (Gibco, Life Technologies Limited, Paisley, UK). The cells were then alkylated by incubation with 1 mL of 1 mM NEM in DPBS buffer for 10 min at room temperature. The alkylated cells were then washed with 2 mL of DPBS buffer and fixed at room temperature for 15–20 min with 2 mL of 4% paraformaldehyde in PBS. The fixed cells were then washed 2 times with 2 mL of DPBS and then stored in DPBS at 4 °C.

Sodium alginate powder (W201502, Sigma Aldrich, Darmstadt, Germany) was dissolved in Ca-free and Mg-free DPBS buffer (14190144, Gibco, Waltham, MA, USA), and its final concentration was adjusted to 0.8% w/v [32 (link)]. Caki cells (2 × 10⁴) and MSC cells (2 × 10⁴) were mixed in PBS and infused from the middle inlet of the microfluidic device. Alginate, 0.1 M CaCl₂ solution, mineral oil (M8410, Sigma Aldrich, Darmstadt, Germany) with 5 wt% SPAN 80, and cell solution were infused from the five inlets of the microfluidic device at 20 µL/min, 20 µL/min, 50 µL/min, 50 µL/min, and 10 µL/min, respectively, using a multi-channel home-built syringe infuser [33 (link)] and a commercial syringe pump (NE-1600, New Era, New York, NY, USA). The cell-laden alginate hydrogel beads were kept in sterile conditions in a 24-well plate and washed with sterile PBS. Alginate beads containing Caki and MSC cells were grown in McCoy and DMEM/F-12 mixed medium (ratio 2:1) at 37 °C and 5% CO₂ conditions. Cells were followed for 21 days and replaced with fresh medium every 4 days. In addition, some of the alginate-capsulated Caki and MSC cells were grown in a cell medium containing 2 µM cisplatin on the 1st, 7th, 14th, and 21st days. As a cell control group, Caki cells were used with and without cisplatin, and the experiment results were compared to these cells.

Alexa488NeuN+, DAPI+, and Alexa488NeuNDAPI+ nuclei were sorted using a BD FACSAria III flow cytometer. Parameters and data analysis were established using BD FACSDiva software version 8.0.1 (BD Biosciences, San Jose, CA, USA). Alexa488NeuN antibody fluorescence was detected with the 488 nm laser and a 530/30 filter and DAPI fluorescence was detected using the 407 nm laser and a 450/40 filter. Nuclei were sorted using a 100 μm nozzle at 20 psi with a drop-drive frequency of 29.2 kHz and collected in 1.5 mL Eppendorf tubes with DPBS buffer (14287-080, Gibco).

HAP1 WT, NAA30-KO, NAA35-KO, and NAA38-KO cells were grown in 10-cm dishes to 70–80% confluency. Cells were washed twice in ice-cold DPBS buffer (Gibco), detached by scraping in ice-cold DBPS with 1 × cOmplete protease inhibitor (Roche), and collected by centrifugation 16,000 × g, 15 s, 4 °C (twice). Cell pellets were flash frozen in liquid nitrogen and stored at −80 °C until further processing. For each cell line, four samples per proteome study were used. The N-terminal acetylation status was determined by positional proteomics using strong cation exchange (SCX) enrichment, while protein abundance was determined by label-free quantitative (LFQ) shotgun proteomics. Detailed information is given below.