Preparation of AzaC and AzadC for cell culture experiments

AzaC (Carbosynth NA02947) and AzadC (Carbosynth NA02969) were purchased and used without further purification. Both compounds were dissolved in Milli-Q H2O to a final concentration of 10 mM. Aliquots (10 µL) were made and stored at − 80 °C. Directly before usage, the required volume of 10 mM stock solution was thawed and diluted to 100 µM with Milli-Q H2O. The integrity of the compounds was checked on a regular basis using HPLC.

Cell culture MOLM-13

MOLM-13 cells (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures; AML cell line) were cultivated at 37 °C in water-saturated, CO2-enriched (5%) atmosphere. RPMI 1640 (Sigma-Aldrich R0883), containing 20% (v/v) fetal bovine serum (FBS) (Invitrogen 10500–064) and 1% (v/v) L-alanyl-L-glutamine (Sigma-Aldrich G8541), was used as growing medium. When reaching a density of 2 × 106 cells/mL, the cells were routinely passaged to a density of 0.25–0.4 × 106 cells/mL. Cells were tested at least once in two months for Mycoplasma contamination using Mycoplasma Detection Kit (Jena Bioscience PP-401L).

Treatment of MOLM-13 with AzaC or AzadC

For all experiments, cells were seeded at a concentration of 0.5 × 106 cells/mL unless stated otherwise and directly treated with AzaC and AzadC using the indicated concentration and incubation time during which the medium was not renewed. Untreated cells served as a negative control in all experiments unless indicated otherwise.

Isolation of gDNA and UHPLC-QQQ-MS

For the determination of mdC levels, 1.5 × 106 cells were seeded and treated with either 0.5 µM, 1.0 µM or 2.5 µM of AzaC or AzadC for 72 h. After 72 h of incubation time, the cells were harvested, gDNA was isolated and UHPLC-QQQ-MS measurements were performed as previously described in Traube et al. [42] with the modification that 3 µg of gDNA per technical replicate was digested using Nucleoside Digestion Mix (NEB M0649S, 2 µL of nuclease, 1.5 h at 37 °C).

RNA isolation for RT-qPCR

400 µL of the first flow-through of the gDNA analysis was used to isolate RNA. 300 µL of 95% ethanol was added, transferred to a Zymo-Spin IIC column (ZymoResearch C1011-50) and incubated for 1 min. After RNA binding, the column was centrifuged at RT, 1500 × g (2 min) and at 10 000 × g (30 s). The flow-through was discarded, and the column was washed with 800 µL RNA Wash Buffer (ZymoResearch R1003-3). To remove residual DNA contamination, DNA was digested according to the peqGOLD DNase I Digest kit (VWR 13-1091-01), followed by washing steps with 400 µL RNA Prep Buffer (ZymoResearch R1060-2) and 800 µL RNA Wash Buffer. The RNA was eluted using 53 µL of Milli-Q H2O, and the concentration was determined using a spectrophotometer.

cDNA synthesis and RT-qPCR

cDNA synthesis was performed with the iScript cDNA Synthesis Kit (Bio-Rad 1,708,891) according to the manufacturer’s protocol using 1 µg RNA per sample.

For subsequent RT-qPCR, the following oligonucleotides were used (Table 2).

Each primer pair (forward and reverse) was mixed and diluted to a final concentration of 1 µM. Subsequently, per 8 µL of diluted primers, 10 µL of iTaq Universal SYBR Green supermix (Bio-Rad 1,725,124) was added (RT-qPCR reaction mix). RT-qPCR was performed in 20 µL reactions with 2 µL of 10 ng/µL cDNA (20 ng per reaction) mixed with 18 µL of RT-qPCR reaction mix (final primer concentration 400 nm).

All samples were run in technical triplicates using the following PCR program on a qTOWER3/G cycler (Jena Biosciences) (Table 3).

Table 2 Primers used for RT-qPCR. HK = house keeperTable 3 PCR program used for RT-qPCR

An RT-qPCR assay for actin beta (ActB) transcripts was used as a housekeeping transcript reference to calculate ΔCt values. Fold change values were calculated with the ΔΔCt method.

Isolation of RNA for UHPLC-QQQ-MS

For the determination of m5C levels, 6.5 × 106 cells were seeded and treated with either 1.0 µM or 2.5 µM of AzaC or 0.5 µM of AzadC for 72 h. After 72 h of incubation time, the cells were harvested, counted and washed once with Dulbecco’s PBS (Sigma-Aldrich D8537-500ML). Afterward, 1.5 mL of TRI reagent (Sigma-Aldrich T9424-200ML) was added (1 mL for 5–10 Mio cells) to lyse the cells. After 5 min incubation time, 150 µL of 1-bromo-3-chloropropane (Sigma-Aldrich B9673) were added (100 µL per 1 mL TRI reagent) and samples were vortexed thoroughly, followed by an incubation time of 10 min. Afterward, cells were centrifuged at 4 °C, 12,000 × g for 15 min. The resulting red phase contained proteins, the interphase the DNA and the upper transparent phase the RNA. For RNA isolation, the upper phase was transferred into a new 1.5 mL tube and 750 µL of 2-propanol (500 µL per 1 mL of TRI reagent) was added. The samples were vortexed thoroughly and incubated for 10 min at RT, followed by centrifugation at 4 °C, 12,000 × g for 10 min. The supernatant was discarded, and 1.5 mL 75% (v/v) EtOH (1 mL per 1 mL TRI reagent) was added. After having thoroughly vortexed the sample, a centrifugation step at 4 °C, 12,000 × g for 5 min was performed. The supernatant was carefully discarded, and the pellet was allowed to dry at RT. When the pellet was just about to be completely dry, it was resuspended in 100 µL of Milli-Q H2O and the concentration was determined.

tRNA was further purified by size exclusion chromatography (SEC) (AdvanceBio SEC 300 Å, 2.7 μm, 7.8 × 300 mm for tRNA combined with BioSEC 1000 Å) according to our previously published protocol [43]. After purification, the RNA was precipitated and dissolved in 30 μL H2O. The RNA concentration of each sample was measured using an Implen nanophotometer.

RNA UHLPC-QQQ-MS

RNA (100 ng) in aqueous digestion mix (15 µL) was digested to single nucleoside and mixed with metabolically produced stable isotope labeled internal standard as previously described [43]. For quantitative mass spectrometry, an Agilent 1290 Infinity II equipped with a diode-array detector (DAD) combined with an Agilent Technologies G6470A Triple Quadrupole system and electrospray ionization (ESI-MS, Agilent Jetstream) was used.

Operating parameters: positive-ion mode, skimmer voltage of 15 V, cell accelerator voltage of 5 V, N2 gas temperature of 230 °C and N2 gas flow of 6 L/min, sheath gas (N2) temperature of 400 °C with a flow of 12 L/min, capillary voltage of 2500 V, nozzle voltage of 0 V and nebulizer at 40 psi. The instrument was operated in dynamic MRM mode (multiple reaction monitoring, MRM). For separation a Core-Shell Technology column (Synergi, 2.5 μm Fusion-RP, 100 Å, 100 × 2 mm column, Phenomenex, Torrance, CA, USA) at 35 °C and a flow rate of 0.35 mL/min were used in combination with a binary mobile phase of 5 mM NH4OAc aqueous buffer A, brought to pH 5.6 with glacial acetic acid (65 μL), and an organic buffer B of pure acetonitrile (Roth, LC-MS grade, purity ≥ 0.99.95). The gradient started at 100% solvent A for 1 min, followed by an increase to 10% over 3 min. From 4 to 7 min, solvent B was increased to 40% and was maintained for 1 min before returning to 100% solvent A and a 3 min re-equilibration period. The sample data were analyzed by MassHunter Quantitative Software from Agilent.

Isolation of nuclear proteins for western blotting

For the preparation of nuclear extracts, 2.5 × 106 cells were seeded and treated with 0.5 µM, 1.0 µm or 2.5 µM of AzaC or AzadC for 48 h. After treatment, the cells were harvested and nuclear extracts were prepared as previously described by Dignam et al. [44] with the modification that every buffer was supplemented with Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich P5726) and Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich P0044), 1:100 each. Afterward, the protein concentration was determined using a Bradford assay (Bio-Rad #5,000,006) as described by the manufacturer. SDS loading buffer (final concentration 50 mM Tris pH 6.8, 100 mM DTT, 2% (w/v) SDS, 10% (v/v) glycerol, 0.1% (w/v) bromophenol blue) was added, and the samples were incubated for 5 min at 92 °C before being stored at − 20 °C. Before loading the samples on a polyacrylamide gel, the samples were heated for additional 2 min at 92 °C and vortexed thoroughly.

Isolation of whole proteome for western blotting

For the preparation of whole proteome, 2.5 × 106 cells were seeded and treated with 0.5 µM, 1.0 µm or 2.5 µM of AzaC or AzadC for 72 h. After treatment, the cells were harvested and washed once with ice-cold PBS. Afterward, cells were lysed in RIPA buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.1% SDS, 2 mM MgCl2, 0.5 mM DTT, EDTA-free protease inhibitor cocktail tablet (Roche 43,203,100), 1% phosphatase inhibitor cocktail 2 (Sigma P5726-1ML), 1% phosphatase inhibitor cocktail 3 (Sigma P0044-1ML), DNase I, Benzonase) and incubated for 1.5 h on ice. Afterward, the lysate was centrifuged at 4 °C, 12,000 × g for 15 min, the supernatant was transferred into a new tube and the protein concentration was determined using a Bradford assay (Bio-Rad #5,000,006) as described by the manufacturer. SDS loading buffer (final concentration 50 mM Tris pH 6.8, 100 mM DTT, 2% (w/v) SDS, 10% (v/v) glycerol, 0.1% (w/v) bromophenol blue) was added, and the samples were incubated for 5 min at 92 °C before being stored at − 20 °C. Before loading the samples on a polyacrylamide gel, the samples were heated for additional 2 min at 92 °C and vortexed thoroughly.

Western blotting

15 µg of nuclear extract in SDS loading buffer or 30 µg of total protein extract was loaded on a 4–15% precast polyacrylamide gel (Bio-Rad #4561083EDU), and Color-coded Prestained Protein Marker, Broad Range (10–250 kDa) (New England Biolabs P7719S or Bio-Rad 1,610,374) was used as a protein standard. The gel was run at constant 150 V for 60 min in SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS). For blotting, we used a PVDF blotting membrane (GE Healthcare Amersham Hybond P0.45 PVDG membrane 10,600,023) and pre-cooled Towbin blotting buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol, 0.038% (w/v) SDS). The membrane was activated for 1 min in methanol, washed with Milli-Q water and equilibrated for additional 1 min in Towbin blotting buffer; the Whatman gel blotting papers (Sigma-Aldrich WHA 10,426,981) were equilibrated for 15 min in Towbin buffer and the precast gel was equilibrated for 5 min in Towbin buffer after the run. Western blotting (tank (wet) electro transfer) was performed at 4 °C for 9 h at constant 35 V or for 4 h at constant 50 V. After blotting, the PVDF membrane was blocked for 0.5—1 h at room temperature using 5% (w/v) milk powder in TBS-T (20 mM Tris pH = 7.4, 150 mM NaCl, 0.1% (v/v) Tween-20). The primary antibodies were diluted in 5 mL of 5% (w/v) milk powder in TBS-T. The blocking suspension was discarded, and the diluted primary antibodies were added for 12 h at 4 °C and shaking. After incubation, the primary antibodies were discarded, and the membrane was washed three times ten minutes with TBS-T. HRP-conjugated secondary antibodies were diluted in 5% (w/v) milk powder in TBS-T and added for 1 h at room temperature under shaking. Afterward, the membrane was washed two times with TBS-T and one time with TBS (TBS-T without Tween-20) before SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific 34,077) was used for imaging. Western blots were imaged using Amersham Imager 680 (auto exposure mode).

For imaging the same blot multiple times using different antibodies, the membrane was directly stripped after imaging. To this end, the membrane was put in TBS-T and the buffer was heated in a microwave until boiling. Afterward, the buffer was discarded and the procedure was repeated in total three times. After stripping, the membrane was blocked again using 5% (w/v) milk powder in TBS-T and the protocol followed the above-described procedure.

Primary antibodies

Anti-phospho-Histone-H2AX (γH2AX, Ser-139) antibody, Millipore 05-636-1 clone 7BW301, mouse monoclonal antibody, 1:1000

Anti-Histone-H3 antibody, Cell Signaling Technology 4499S clone D1H2, rabbit monoclonal antibody, 1:1000

Anti-c-MYC antibody, ptglab 67,447-1-Ig, mouse monoclonal antibody, 1:500

Anti-HOXA9 antibody, ptglab 18,501-1-AP, rabbit polyclonal antibody, 1:1000

Anti-BCL-2 antibody, ptglab 12,789-1-AP Lot 00,087,156, rabbit polyclonal antibody, 1:1000

Anti-phospho-TP53 (Ser15) antibody, ptglab 28,961-1-AP, rabbit polyclonal antibody, 1:1000

Secondary antibodies

HRP-conjugated anti-mouse IgG, Sigma-Aldrich AP130P, 1:5000

HRP-conjugated anti-rabbit IgG, Sigma-Aldrich A0545, 1:5000

Brightfield microscopy

For the brightfield microscopy images, cells were imaged directly in the medium after 72 h treatment with 0.5 µM, 1.0 µM or 2.5 µM of either AzaC or AzadC using an EVOS FL microscope (ThermoFisher) in the transmission mode and 40 × magnification.

MTT assay

For the MTT assay, 5 × 104 cells were seeded in 100 µL RPMI/20% FBS medium that did not contain phenol red. The assay was performed as described previously [45]. Each time point (24 h, 48 h or 72 h) included samples for untreated cells and cells that were treated with 0.5 µM, 1.0 µM or 2.5 µM of either AzaC or AzadC. Each time point was measured individually and after measuring the absorption at 570 nm, the average of the technical replicates was calculated and the absorption of the AzaC- or AzadC-treated cells was set in relation to the absorption measured for the untreated cells, resulting in the relative metabolic activity. Three biologically independent experiments were performed per time point, and each sample was measured in technical quadruplicates.

Flow cytometry analysis for monitoring proliferation

To measure proliferation, EdU Flow Cytometry 488 kit (baseclick BCK-FC488-50) was used according to the manufacturer’s protocol. In brief, MOLM-13 (1 × 106 per condition, 0.5 × 106/mL) were treated with 0.5 µM, 1.0 µM or 2.5 µM of either AzaC or AzadC for 48 h, before EdU (final concentration 10 µM) was added for two additional hours. Untreated cells without EdU served as unstained control, and untreated cells with EdU served as untreated control. Afterward, the cells were harvested and washed with DPBS, including 1% (w/v) BSA, before fixation and permeabilization. Then, click chemistry was performed as described in the manual. Prior to flow cytometry measurement, the cells were filtered through a 35-µm strainer. For the analysis, BD FACSCanto™ and FlowJo Single Cell Analysis Software (v10.8.0) were used. Gates were set once for the control sample and then applied to all other samples.

Flow cytometry analysis for monitoring cell death

Previous to flow cytometry analysis, cells were treated with 0.5 µM, 1.0 µM or 2.5 µM of either AzaC or AzadC for 72 h, harvested and washed twice with DPBS. Untreated cells served as a control. Afterward, cells were counted and 2 × 105 cells per sample were transferred into a new tube. Apoptosis and necrosis were determined by using the FITC Annexin V Apoptosis Detection Kit (BioLegend 640,914) and SYTOX™ Red Dead Cell Stain (ThermoFisher S34859). To this end, cells were resuspended in 100 µL of Annexin V binding buffer supplemented with 1 µL of FITC-conjugated Annexin V, gently vortexed and incubated at RT for 15 min in the dark. Afterward, cells were put on ice and 100 µL of cell suspension (2 × 105 cells) were filtered through a 35 µm strainer. 0.2 µL of Sytox was added just before the measurement. For the analysis, BD FACSCanto™ and FlowJo Single Cell Analysis Software (v10.8.0) were used. Gates were set once for the control sample and then applied to all other samples.

Proteomics

MOLM-13 cells were incubated for 72 h with 0.5 μM AzadC, 1 μM or 2.5 μM AzaC in 4 replicates each. Untreated cells (n = 4) served as a control. The cells were harvested and washed twice with PBS. Sample preparation followed in principle a previously published protocol using filter-assisted sample preparation (FASP) [46]. Lysis was achieved by adding 1 mL 100 mM Tris/HCl pH 8.5, 8 M urea, incubation at 95 °C for 5 min and subsequent sonication at 20% intensity for 20 s using a rod sonicator. The suspension was centrifuged at 14,000 × g for 5 min, and the resulting supernatant was transferred into a new reaction tube. A BCA assay was conducted to determine the protein concentration, before a defined volume (e.g., 700 μL) was taken from each sample and mixed with TCEP (10 mM final concentration) and 2-chloroacetamide (40 mM final concentration) before incubation at 95 °C for 5 min. 30 μg of each sample was filled up to 150 μL with 100 mM Tris/HCl pH 8.5, 8 M urea and transferred onto a 30 kDa molecular weight cutoff column (Microcon-30, Merck Millipore). 150 μL 100 mM Tris/HCl pH 8.5, 8 M urea was added to the column before centrifugation at 14,000 × g for 15 min. The flow-through was discarded, and the columns were washed twice by addition of 100 μL 50 mM ammonium bicarbonate and subsequent centrifugation at 14,000 × g for 10 min. The filter units were then placed into a new collection tube before addition of 100 μL 50 mM ammonium bicarbonate containing trypsin (1:100 trypsin/protein) and incubation at 37 °C overnight. Next, the samples were centrifuged at 14,000 × g for 15 min followed by washing the column twice with 40 μL 50 mM ammonium bicarbonate and centrifugation at 14,000 × g for 10 min. The eluate was acidified with 5% formic acid to a pH of 1–2 and purified using C18 cartridges (Sep-Pak tC18 1 cc, 50 mg, Waters) and a vacuum manifold. For this, the cartridges were washed with 1 mL MeCN, 1 mL 80% MeCN, 0.5% formic acid and thrice with 1 mL 0.5% formic acid, applying vacuum after each step. Then, the acidified samples were loaded onto the cartridges without application of vacuum following three washing steps using 1 mL 0.5% formic acid. Clean collection tubes were inserted into the vacuum manifold, and the peptides were eluted by addition of 250 μL 80% MeCN, 0.5% formic acid without applying vacuum before another 250 μL of this buffer was added while applying vacuum. Finally, the solvent was evaporated and the purified peptides were resuspended in 2% MeCN, 0.1% formic acid.

2 μg of each sample was submitted to a LC-MS analysis using an Ultimate 3000 RSLCnano UHPLC (Thermo Fisher Scientific) coupled to an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) with the FAIMS Pro interface attached. First, the peptides were separated by reverse-phase chromatography using a pre-concentration setup. For this, the samples were bound to a pre-column (Acclaim C18 PepMap100, 300 μm i.d., 5 mm length; Thermo Fisher Scientific) and then eluted onto the analytical column (SilicaTip Emitter, 75 µm i.d., 8 µm tip, 15 cm length; New Objective; packed in-house with ReproSil-Pur 120 C18-AQ, 1.9 µm, 120 Å; Dr. Maisch GmbH), which was heated to 40 °C using a column oven by Sonation. The separation was achieved at a flow rate of 0.3 μL/min and application of a gradient between solvent A (0.1% formic acid in H2O) and solvent B (0.1% formic acid in MeCN) going from 7 to 24.8% B in 99 min and from 24.8 to 35.2% B in 21 min. The ionization was carried out by applying a voltage of 2.0 kV to the column.

The eluting peptides were analyzed at alternating FAIMS CV voltages of − 50 V and − 70 V in Standard Resolution mode with the total carrier gas flow set to static and 3.5 L/min. To take peptide charge-state distributions into account, the data-dependent acquisition at − 50 V was run for 1.7 s, while the corresponding cycle time at − 70 V was shortened to 1.3 s. The rest of the parameters were kept identical for the two CV values, starting with a full mass orbitrap scan in profile mode at a resolution of 240,000 using a mass range of 375–1500 m/z and a RF lens level of 30%. The AGC target was set to Standard, and the maximum injection time was set to 50 ms. Following this scan, multiple data-dependent MS2 scans were carried out for the cycle time specified above using the filters MIPS (Peptide), Intensity (intensity threshold: 1.0e4), Charge State (include charge states 2–6, don’t include undetermined charge states) and Dynamic Exclusion (exclude for 40 s after one selection with a window of ± 10 ppm, also exclude isotopes and other charge states). The most intense ions were individually selected using an isolation window of 1,2 m/z, subjected to fragmentation at a normalized HCD energy of 30% and analyzed in the ion trap at Rapid scan rate with a Normal mass range, the AGC target set to Standard and a maximum injection time of 50 ms.

Identification and label-free quantification (LFQ) of peptides and proteins was accomplished using the MaxQuant software version 2.0.3.0 [47, 48]. To prepare the .RAW-files for analysis, they were split into two files containing either the spectra obtained at a CV of − 50 V or − 70 V. This was accomplished by the FAIMS MzXML Generator software developed by the Coon lab [49]. During the MaxQuant analysis, these two files were handled like fractions. Next, a database containing the human proteome was chosen. As protease, Trypsin was chosen and a maximum of two missed cleavages were allowed. Carbamidomethylation of C was set as static modification, while oxidation of M and phosphorylation of YST were set as dynamic modification. Peptide mass deviations were set to 20 ppm in the first search and 4.5 ppm in the second search, respectively. The minimal peptide length was set to 6, and the PSM and protein FDRs were both set to 0.01. A reverse of the database was chosen as decoy database. The LFQ option was checked and the minimum ratio count was set to 2. The feature “match between runs” was checked, and a match time window of 0.7 min as well as an alignment time window of 20 min was defined. MaxQuant results were analyzed using Perseus (v 1.6.14) [50]. In short, contaminants that were assigned by MaxQuant were filtered and LFQ intensities were log2 transformed (Perseus step1). The four biological replicates (cells that were treated the same way) were grouped, and afterward, the data were filtered so that only proteins remained in the data set which were detected in at least three biological replicates of at least one group to ensure high quality data (Perseus step2). Afterward, the remaining missing values were imputed using imputation from Gaussian distribution (per column) with the default settings (Perseus step3). Last, the volcano plot option was used to display pairwise comparisons (Perseus step4).

IPA

Proteomics data were further analyzed with the use of QIAGEN IPA (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA). For IPA [31], processing of the proteomics data was processed until the end of Perseus step 2. Before imputation, however, proteins were checked groupwise and if a protein was not detectable in any of the four biological replicates of one group, indicating that the protein was not present in this group, an artificial log2 transformed LFQ intensity of 1 was assigned. Afterward, the remaining missing values were imputed with the values that were obtained from the initial imputation of Perseus step 3. Last, treated samples were pairwise compared to the untreated control using the volcano plot option and the obtained p values and log2 fold changes were used for IPA. In IPA, core analysis was performed for each comparison with the setting “human” as species and a p value cutoff of 0.05.

Statistical analysis

Statistical analysis, except for the proteomics data where Perseus was used, was performed using GraphPad Prism (v 9). Per biological replicate, the mean of the technical replicates was calculated where applicable and only the biological replicates were taken into account for the statistical analysis.

HL-60 cells

Maintenance and treatment of HL-60 (ATCC) as well as the described experiments were equally performed as with MOLM-13.