Chemistry
All of the starting materials are commercially available. Commercial reagents were used without any purification. For UV irradiation, the high-pressure mercury UV lamp DRT-1000 was used. The products were isolated by MPLC: Buchi B‑688 pump; glass column C-690 (15 × 460 mm) with silica gel (particle size 0.015–0.040 mm); and UV detector Knauer K-2001. The analytical examples were purified by the Shimadzu HPLC system (model: LC-20AP) equipped with a UV detector (model: SPD 20A), using a Supelco C18 (5 µm, 20 × 250 mm) column using the MeOH:H2O (20:80, 50:50, 70:30) mobile phase by isocratic elution at a flow rate of 15 mL/min. The NMR spectra were recorded with an NMR instrument operating at 400 MHz (1H) and 100 MHz (13C). Proton spectra were referenced to TMS as an internal standard and, in some cases, to the residual signal of used solvents. Carbon chemical shifts were determined relative to the 13C signal of TMS or the used solvents. Chemical shifts are given on the δ scale (ppm). Coupling constants (J) are given in Hz. Multiplicities are indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br (broadened). The original NMR spectra of the relative compounds can be found in Supplementary Figure S1. High-resolution mass spectra (HRMS) were obtained with a time-of-flight (TOF) mass spectrometer (model Agilent TOF 6210) equipped with an electrospray source at atmospheric pressure ionization (ESI). The purity of the synthesized compounds was established based on the NMR and HRMS spectral information.
1-(1H-Indol-3-yl)-2,2-dimethylpropan-1-ol (2)
Compound 2 was obtained by a known method 39. For the synthesis, 3.0 mmol of 3-formylindole was taken with a product yield of 77%. 1H NMR (400 MHz, CDCl3): δ 8.15 (br. s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.20 (td, J = 7.5, 1.0 Hz, 1H), 7.16 (d, J = 2.4, 1H ), 7.12 (m, 1H), 4.83 (d, J = 2.0 Hz, 1H), 1.83 (d, J = 2.2 Hz, 1H ), 1,02 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 135.8, 127.0, 122.3, 121.9, 120.2, 119.6, 118.3, 111.0, 76.6, 36.4, 26.1.
2-(1H-Indol-3-yl)-3,3-dimethylbutanenitrile (3)
Compound 3 was obtained by a previously published method 40. Sodium cyanide was used to carry out the substitution reaction. For the synthesis, 3.4 mmol of 1-(3-indolyl)-2,2-dimethylpropanol was taken with a product yield of 40%. 1H NMR (400 MHz, CDCl3): δ 8.26 (br. s, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.23 (m, 2H), 7.17 (m, 1H), 3.9 (s, 1H), 1.13 (s, 9H). 13C NMR (100 MHz, CDCl3): 135.8, 126.7, 124.1, 122.4, 120.9, 120.1, 119.1, 111.3, 108.9, 40.8, 35.7, 27.6.
2-(1H-Indol-3-yl)-3,3-dimethylbutylamine (4)
Compound 3 was obtained by a previously published method 40. For the synthesis, 1.3 mmol of 2-(3-indolyl)-3,3-dimethylbutyronitrile was taken with a product yield of 23%. Due to instability, the product was introduced to the subsequent reaction without further purification. MS-ESI, m/z: [M+H]+ 217.11.
3-(3,3-Dimethyl-2-oxo-butyl)-3-hydroxy-indolin-2-one (7)
Pinacoline (6) (6.00 g, 59.9 mmol) and NaOH (1.15 g, 28.8 mmol) in 5 mL of H2O were successively added to a suspension of isatin (5) (5.00 g, 34.0 mmol) in EtOH (100 mL). Next, the reaction mixture was stirred on a magnetic stirrer heated to 50 °C. The reaction mixture was kept at a constant weak alkaline pH for 24 h. The progress of the reaction was monitored by TLC. After the appearance of traces of the by-product, the reaction was complete. Then, the mixture was evaporated under reduced pressure, and the residue was recrystallized from H2O. The crystals were filtered off, washed with cold H2O and dried. The obtained product was light beige crystals (2.30 g, 30%).
1H NMR (400 MHz, CDCl3): δ 8.46 (s, 1H), 7.30 (d, J = 7.4 Hz, 1H), 7.24 (td, J = 7.8, 1.0 Hz, 1H), 7.02 (td, J = 7.5, 0.6 Hz, 1H), 6.88 (d, J = 7.7 Hz, 1H), 4.82 (s, 1H), 3.33 (d, J = 17.6 Hz, 1H), 3.05 (d, J = 17.6 Hz, 1 H), 1.08 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 215.3, 178.6, 140.7, 130.4, 129.9, 124.0, 123.0, 110.5, 74.8, 44.6, 42.6, 25.9. HRMS-ESI, m/z: [M + H]+ calculated for C14H18NO3+ 248.1281, obtained 248.1291.
3-(3,3-Dimethyl-2-oxo-butylidene)indolin-2-one (8)
Compound 7 (2.00 g, 8.1 mmol) was dissolved in 5 mL of CH3COOH, and 4 drops of HCl (aq) were added to the solution. The reaction mixture was stirred on a magnetic stirrer heated to 75 °C. The progress of the reaction was monitored by TLC. Then, the mixture was poured into H2O and neutralized with NaHCO3; the solution was extracted with EtOAc (3 × 20 mL); and the extract was dried and evaporated under reduced pressure. The obtained product was orange crystals (1.84 g, 99%).
1H NMR (400 MHz, CDCl3): δ 8.36 (d, 7.8 Hz, 1H), 8.21 (br. s, 1H), 7.46 (s, 1H), 7.32 (td, J = 7.7, 0.9 Hz, 1H), 7.02 (td, J = 7.7, 0.8 Hz, 1H), 6.86 (d, J = 7.7 Hz, 1H), 1.29 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 206.5, 169.5, 143.1, 136.1, 132.6, 128.1, 125.7, 122.9, 120.7, 110.0, 44.8, 26.2. HRMS-ESI, m/z: [M + H]+ calculated for C14H16NO2+ 230.1176, obtained 230.1193.
3-(3,3-Dimethyl-2-oxo-butyl)indolin-2-one (9)
Compound 8 (0.98 g, 4.3 mmol) was dissolved in 30 mL of MeOH. A catalytic amount of 10% Pd/C was added to the solution. The reaction was carried out in a hydrogen atmosphere (6 bar). The mixture was stirred at room temperature for 18 h. The bright orange color of the solution disappeared. The mixture was then poured into H2O and extracted with EtOAc (3 × 20 mL). The extract was dried and evaporated under reduced pressure. The obtained product was light yellow crystals (0.93 g, 94%).
1H NMR (400 MHz, CDCl3): δ 8.30 (br. s, 1H), 7.19 (t, J = 7.7 Hz, 1H), 7.09 (d, J = 7.4 Hz, 1H), 6.97 (td, J = 7.3, 0.6 Hz, 1H), 6.88 (d, J = 7.8 Hz, 1H), 3.91 (dd, J = 8.5, 3.2 Hz, 1H), 3.31 (dd, J = 18.4, 3.3 Hz, 1H), 3.00 (dd, J = 18.3, 8.6 Hz 1H), 1.17 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 212.8, 180.0, 141.4, 129.7, 128.0, 124.2, 122.4, 109.6, 43.9, 41.4, 38.1, 26.4. HRMS-ESI, m/z: [M + H]+ calculated for C14H18NO2+ 232.1332, obtained 232.1332.
3-[2-Hydroxyimino-3,3-dimethyl-butyl]indolin-2-one (10)
Finely ground NH2OH·HCl (1.50 g, 21.5 mmol) and CH3COONa (1.76 g, 22.0 mmol) were added to a flat-bottomed flask. Next, 30 mL of MeOH was added, followed by the introduction of compound 9 (1.28 g, 5.2 mmol) into the mixture. The reaction mixture was stirred on a magnetic stirrer heated to 40 °C for 24 h. Then, the same amount of NH2OH·HCl and CH3COONa was added to the mixture, which was stirred with heating for another 24 h. At the end, the mixture was diluted with H2O and extracted with EtOAc (3 × 30 mL). The extract was dried and evaporated. The resulting product was a light cream powder (1.25 g, 97%).
1H NMR (400 MHz, CDCl3): δ 8.84 (br. s, 1H), 8.46 (s, 1H), 7.24 (d, J = 7.5 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 6.99 (td, J = 7.5, 0.5 Hz, 1H), 6.86 (d, J = 7.6 Hz, 1H), 4.43 (t, J = 8.6 Hz, 1H), 2.99 (dd, J = 13.9, 7.8 Hz, 1H) 2.68 (dd, J = 14.0, 9.6 Hz, 1H), 1.09 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 180.2, 163.9, 141.2, 129.5, 128.0, 125.3, 122.2, 109.4, 42.1, 37.7, 28.0, 27.2. HRMS-ESI, m/z: [M + H]+ calculated for C14H19N2O2+ 247.1441, obtained 247.1454.
1-(1H-Indol-3-yl)-3,3-dimethyl-butan-2-amine (11)
Compound 10 (0.74 g, 3.0 mmol) was dissolved in 20 mL of MeOH. A catalytic amount of PtO2 was added to the solution. The reaction was carried out in a hydrogen atmosphere (6 bar). Thereafter, the mixture was stirred at room temperature for 48 h. Afterwards, the precipitate that formed was filtered off and dried. Due to its instability, the resulting product was immediately introduced to the next stage of the synthesis. NaBH4 (0.57 g, 15.1 mmol) was added to 10 mL of freshly distilled THF, which was in a flat-bottomed conical flask with a stirrer. The flask was placed in an ice bath; its contents were cooled to 0 °C, after which freshly distilled BF3·OEt2 (2.043 mL, 16.6 mmol) was added in portions. The ice bath was removed, and the reaction mixture was stirred for another 15 min at room temperature. The previously obtained product was added to the solution. The mixture was heated to reflux. After 2 h, the mixture was cooled to room temperature, after which a 10% HCl solution was added to it to a fivefold dilution. The mixture was again heated to reflux and after 2 h was cooled to room temperature. The mixture was then neutralized with Na2CO3 (aq) and extracted with EtOAc (3 × 30 mL). The extract was dried, evaporated under reduced pressure, and immediately introduced to the next reaction. Product 11 was not isolated or characterized due to low stability.
1-(2'-Iodobenzoyl)-3-tert-butyl-β-carboline (12)
2’-Iodoacetophenone (0.5 mmol) and iodine (0.09 g, 0.4 mmol) were added to 2 mL of DMSO, and the resulting solution was heated at 110 °C for 1 h. Afterwards, tryptamine 11 (0.5 mmol) was added to the solution, and this solution was stirred at the same temperature for 3–4 h until the completion of the reaction (monitored by TLC). Then, the reaction mixture was cooled to room temperature followed by the addition of H2O (50 mL) and extraction with EtOAc (2 × 25 mL). The extract was washed with 10% Na2S2O3, dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by MPLC using benzene as an eluent to give the desired product 12 (yellow solid, 6 %).
1H NMR (400 MHz, CDCl3): δ 10.20 (br. s, 1H), 8.20 (s, 1H), 8.18 (dd, J = 8.0, 0.5 Hz, 1H), 7.67 (d, J = 8.0, 0.8 Hz, 1H), 7.61–7.58 (m, 3H), 7.44 (td, J = 7.5, 1.0 Hz, 1H), 7.38–7.31 (m, 2H), 1.35 (s, 9H). 13C (100 MHz, CDCl3): δ 198.2, 158.4, 141.4, 140.8, 135.1, 133.4, 132.6, 132.5, 130.7, 130.2, 129.0, 126.3, 121.7, 121.1, 120.6, 120.4, 114.4, 111.9, 37.5, 30.5. HRMS-ESI, m/z: [M + H]+ calculated for C22H20IN2O+ 455.0615, obtained 455.0624.
1-(2'-Bromobenzoyl)-4-tert-butyl-β-carboline (13)
A solution of 2'-bromoacetophenone (28 mg, 0.14 mmol) and iodine (28 mg, 0.11 mmol) in DMSO (1 mL) was heated at 110 °C for 1 h. Next, 2-(3-indolyl)-3,3-dimethylbutylamine (30 mg, 0.14 mmol) in DMSO (1 mL) was added to the solution and stirred at 110°C for 4 h. The reaction mixture was cooled, then poured to water and N2S2O3 was added. The formed precipitate was filtered off, and the filtrate was discarded. The precipitate was washed with ethyl acetate and the solvent was evaporated under reduced pressure. The residue was purified by MPLC using benzene as eluent. The product is a yellow solid (22 mg, 38%).
1H NMR (400 MHz, CDСl3): δ 10.88 (br. s, 1H), 8.63 (s, 1H), 8.47 (d, J = 8.3 Hz, 1H), 7.69 (t, J = 7.5 Hz, 2H), 7.63 (t, J = 7.5 Hz, 1H), 7.53 (dd, J = 7.6, 1.7 Hz, 1H), 7.47 (td, J = 7.5, 1.0 Hz, 1H), 7.40 (m, 2H), 1.75 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 198.2, 145.5, 141.2, 140.7, 137.5, 136.9, 133.8, 133.0, 131.0, 129.5, 128.7, 128.3, 127.4, 126.8, 120.5, 119.9, 119.8, 112.2, 35.2, 29.5; HRMS-ESI, m/z: [M]+ calcd. for C22H19BrN2O, 407.3031; found, 407.3027.
6-tert-Butylfascaplysin (6-TB)
A solution of β-carboline 12 (0.05 mmol) in 10 mL of acetonitrile was irradiated with UV for 30–90 min. The solution was evaporated, the residue was washed from the starting β-carboline with acetonitrile or benzene, depending on the solubility of the resulting fascaplysin. For re-irradiation, the non-reacted β-carboline solution was evaporated, dissolved in 10 mL of acetonitrile, and irradiated again. The end of the reaction was monitored by TLC. After filtration, the fascaplysin was washed with EtOH, then evaporated under reduced pressure and dried. Then, the product was dissolved in H2O, and an aqueous solution of Na2CO3 was added. The resulting dark green precipitate of the deprotonated form of the product was filtered, washed with water, and washed off with an aqueous solution of HCl. The resulting solution was evaporated and dried. The product is a red solid (93%).
1H NMR (400 MHz, CD3OD): δ 9.00 (s, 1H), 8.53 (d, J = 8.0 Hz, 1H), 8.43 (d, J = 8.6 Hz, 1H), 8.08 (d, J = 6.8 Hz, 1H), 7.96 (t, J = 7.6 Hz, 1H), 7.85 (t, J = 7.6 Hz, 1H), 7.77–7.71 (m, 2H), 7.48 (t, J = 7.5 Hz, 1H), 1.95 (s, 9H). 13C NMR (100 MHz, MeOH-d4): δ 182.9, 149.1, 148.8, 141.5, 136.1, 134.7, 131.5, 130.5, 125.5. 125.3, 124.8, 124.4, 124.2, 122.9, 119.6, 119.2, 113.2, 112.0, 36.6, 30.4. HRMS-ESI, m/z: [M]+ calculated for C22H19N2O+ 327.1492, obtained 327.1486.
7-tert-Butylfascaplysin (7-TB)
1-(2'-Bromobenzoyl)-4-tert-butyl-β-carboline (17 mg, 0.04 mmol) was heated at 220 °C for 30 min. The product was washed with EtOAc until coloring ceased. The red precipitate was dissolved in hot water and filtered. Sodium carbonate was added to the solution, and a green precipitate formed. The precipitate was filtered, dissolved with acidified EtOH, the substance went to solution, and the color became red. The solvent was evaporated under reduced pressure. The product is a red solid (14 mg, 93%).
1H NMR (400 MHz, CD3OD): δ 9.00 (s, 1H), 8.63 (d, J = 8.6 Hz, 1H), 8.50 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 7.3 Hz, 1H), 7.97 (t, J = 7.6 Hz, 1H), 7.88 (m, 2H), 7.75 (t, J = 7.5, 1H), 7.59 (m, 1H), 1.87 (s, 9H); 13C NMR (100 MHz, CD3OD): δ 181.8, 147.7, 147.4, 147.1, 138.0, 136.7, 133.1, 132.4, 131.3, 128.6, 125.3, 124.3, 122.8, 122.7, 120.7, 118.6, 115.3, 113.5, 35.6, 27.9; HRMS-ESI, m/z: [M]+ calcd. for C22H19N2O, 327.3985; found, 327.3981.
Biology
Reagents and antibodies
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma (Taufkirchen, Germany), annexin-V-FITC from BD Bioscience (San Jose, CA, USA); tariquidar (p-glycoprotein inhibitor) from MedChemExpress (Monmouth Junction, NJ, USA); calcein-AM from BIOZOL (Eching, Germany); VE-822 (ATR inhibitor), LY2603618 (Chk1 inhibitor), KU-60019 (ATM inhibitor), and PV1019 (Chk2 inhibitor) from LC Laboratories (Woburn, MA, USA); Anisomycin – from NeoCorp (Weilheim, Germany). Docetaxel, and doxorubicin – from a Pharmacy of the University Hospital Hamburg-Eppendorf (Hamburg, Germany). Primary and secondary antibodies used are listed in Supplementary Table S1.
Cell lines and culture conditions
The human prostate cancer LNCaP, 22Rv1, PC-3, and DU145 cell lines and human prostate non-cancer PNT2 cells were purchased from ATCC (Manassas, VA, USA). Human non-cancer human embryonic kidney HEK 293T cells and human fibroblast MRC-9 cells were purchased from ECACC (Salisbury, UK). The human prostate cancer docetaxel-resistant PC3-DR cell line was generated by long-term incubation with docetaxel as previously described 43 and were kindly provided by Prof. Z. Culig, Innsbruck Medical University, Austria. The cells lines were recently authenticated by Multiplexion GmbH (Heidelberg, Germany). Cells were cultured as previously described for maximum 3 months and were regularly examined for mycoplasma infection.
Trypan blue exclusion assay
The trypan blue exclusion assay was performed to access membrane integrity. Cells were plated in 6-well plates (2×105 cells/well) per well in 2 mL/well, incubated overnight, and treated with drugs at indicated concentrations in fresh medium. Cells were incubated for 48 h, harvested using trypsinization, stained with trypan blue dye, and trypan blue-negative alive cells were conducted using the automatic Beckman Coulter Vi-CELL (Beckman Coulter, Krefeld, Germany).
DNA intercalation assay
The DNA-intercalating activity of the compounds was assessed using thiazole orange (TO) displacement method from double-stranded DNA as reported previously 37. In brief, for this analysis we used mixture containing 1 µM of double-stranded calf thymus DNA and 2 µM of thiazole orange (TO) in water. The solutions of tested compounds in DMSO were added to the mixture ensuring that the maximum DMSO concentration in the samples do not exceed 0.02%. Propidium iodide (PI) was used as a positive control. The samples were incubated for 7 min at room temperature, and the TO fluorescence was measured using a TECAN Spark multimodal plate reader (Tecan Group Ltd., Männedorf, Switzerland). The effective concentration (EC50) was calculated using GraphPad Prism software v.9.1.1 (GraphPad Software, San Diego, CA, USA).
p-gp activity assay
The assessment of p-glycoprotein (p-gp) activity was done using calcein-AM as previously reported 59. Cells were plated in black 96-well plates with clear bottoms at a density of 10×103 cells/well in 100 µL/well, incubated overnight, and the medium was replaced with 50 µL of a drug solution in PBS. Following 30 min incubation, 50 µL of calcein-AM solution in PBS was added to each well to achieve a final concentration of 1 µM. The plates were then incubated for an additional 15 min, and the green fluorescence was immediately measured using an Infinite F200PRO reader (TECAN). The viability of cells treated in the same conditions with the drug was accessed using the MTS assay as previosuly reported 59.
MTT assay
Cell viability was assessed using the MTT assay. Cells were plated in 96-well plates (6×103 cells/well in 100 μL/well, incubated overnight, and the medium was replaced with fresh drug-containing medium containing and incubated for 48 h unless otherwise indicated. Then MTT reagent was added to each well and the plates were then incubated for another 2-4 h. The media was removed, and the plates were dried overnight, and 50 μl/well of DMSO was added to dissolve the formazan crystals. The absorbance of these solutions was measured using an Infinite F200PRO reader (TECAN, Männedorf, Switzerland). The inhibition concentration (IC50) was calculated using GraphPad Prism software v.9.1.1 (GraphPad Software, San Diego, CA, USA).
Functional kinome profiling Functional kinome profiling was performed as previously reported 60. In brief, the PamStation®12 system (PamGene International, ´s-Hertogenbosch, The Netherlands) and STK-PamChip® array designed for the analysis of serine-/threonine kinase activities were used. Each array comprises 140 distinct peptide phospho-sites that serve as analogs for the substrates of corresponding serine-/threonine kinases. The whole cell lysates were prepared using M-PER Mammalian Extraction Buffer (Pierce, Waltham, Massachusetts, USA), supplemented with protease and phosphatase inhibitor cocktails, following 2 h incubation of the cells with 7-TB. A mixture of total protein extract and ATP was prepared and applied in the array, and the phosphorylation of specific peptide sequences was then detected using primary anti-phospho-Ser/Thr antibodies, secondary immunoglobulin-FITC antibody (PamGene International). The signals were recorded using a CCD camera and Evolve software v. 1.0 (PamGene International), and the quality of the signals was monitored. The final signal intensities were log2-transformed and analyzed using the BioNavigator software v. 6.0 (BN6, PamGene International).
Western blotting
Western blotting was performed as discribed previously 61. In brief, 106 cells were seeded per dish in Petri dishes, incubated overnight and treated with the drug for 48 h unless otherwise stated. Cells were harvested by scratching, washed and lyzed using RIPA buffer containing protease and phosphotase inhibitors. Proteins were saparated using SDS-PAGE and Mini-PROTEAN® TGX Stain-FreeTM gels (Bio-Rad, Hercules, CA, USA), transferred onto PVDF membranes and visualized using appropriate primary and secondary antibodies. The signals were detected using ECL chemiluminescence system (Thermo Scientific, Rockford, IL, USA). The antibodies used are listed in Supplementary Table S1. The original images obtained were cropped and the figures were composed using MS PowerPoint software v. 2310, build 16924,20150 (Microsoft Inc., Redmond, WA, USA). The original uncroped images are represented in Supplementary Figure S2.
Colony formation assay
Colony formation assay was conduced as previously described 62. Briefly, 22Rv1, DU145 or PC-3 cells were seeded in 6-well plates with a denisty of200 cells/well in 2 mL/well, and incubated overnight. On the next day the tested drugs were added to the cells at the indicated concentrations an the cells were incubated for a period of 14 days to allow colony formation. The resulting colonies were fixed using 70% ethanol, stained with 0.1% crystal violet, and counted by eye.
Flow cytometry analysis
22Rv1 cells were plated and as described above for Trypan blue exclusion assay and incubated overnight. Then, the cells were pre-treated with 100 µM z-VAD(OMe)-fmk for 1 hour and then treated with the tested drugs for another 48 h. Cells were collected using trypsination, stained with annexin-V-FITC and propidium iodide, and analyzed using a FACS Calibur instrument (BD Bioscience, San Jose, CA, USA) as previously reported 61. The generated data were analyzed using the Cell Quest Pro software version 5.2.1 (BD Bioscience).
Immunofluorescence
Cells were placed on coverslips and treated for indicated times. The following concentrations of 6-TB and 7-TB were used, respectively: 0.5 mM and 0.1 mM for PC3 cells, 0.25 mM and 0.05 mM for 22Rv1 cells, and 1 mM and 0.2 mM for DU145 cells. After the treatment, cells were washed once with cold PBS, then fixed using 4% paraformaldehyde/PBS for 10 min. Following fixation, the cells were permeabilized using 0.2% Triton X-100/PBS on ice for 5 min. The slides were incubated with primary anti-phospho-S139-H2AX and anti-53BP1 antibodies for 1 h at room temperature. The cells were washed three times with cold PBS and incubated for 1 h with secondary anti-mouse Alexa-fluor594 and anti-rabbit Alexa-fluor488 antibodies. Nuclei were additionally stained with DAPI (10 ng/mL) and the slides were mounted using Vectashield mounting medium (Vector Laboratories, Newark, CA, USA). Fluorescence microscopy was performed using a Zeiss AxioObserver.Z1 microscope, equipped with objectives of ×20 (resolution 0.44 µm) and Plan Apo 63/1.4 Oil DICII (resolution 0.24 µm), and filters including Zeiss 43, Zeiss 38, and Zeiss 49 (Carl Zeiss, Göttingen, Germany). Z-stacks of semi-confocal images were captured using the Zeiss Apotome, Zeiss AxioCam MRm, and Zeiss AxioVision Software (Carl Zeiss, Göttingen, Germany). The quantification of DSBs was performed using ImageJ, applying DAPI-based image masks, and the results were normalized to values per individual nucleus. The antibody used are listed in the Supplementary Table S1.
Fiber assay
Cells were seeded and treated with investigated drugs for 24 h as described above for the Immunofluorescence assay. The cells were washed with PBS and sequentially pulse-labeled with CldU (25 μM) and then with IdU (250 μM), for 20 min each. Following labeling the cells were harvested and DNA fibers were spread and stained according to a previously described method 63. The stained DNA fibers were then analyzed using an Axioplan 2 fluorescence microscope (Zeiss, Oberkochen, Germany). Analysis of the CldU and IdU tracks on these fibers was conducted using ImageJ software. The minimal number of replication forks analyzed per sample was 300.
Data and statistical analysis
Statistical analyses were conducted using GraphPad Prism software version 7.05 (GraphPad Prism software Inc., La Jolla, CA, USA). The data are expressed as mean ± standard deviation (SD). For comparing two groups, the unpaired Student’s t-test was used, while for comparisons among multiple groups, one-way ANOVA followed by Dunnett's post-hoc tests was applied. Specifically, all experiments were carried out in triplicate (n = 3, representing biological replicates) unless otherwise stated. The difference between the groups was considered as statistically significant if p < 0.05.