Vp 16

Stable A549 cells with H2AX knockdown and control stable A549 cells (CTR) were treated with VP16 (Sigma–Aldrich) (100 μM) for 48 h after overnight serum starvation. Then, RNA was isolated using the TRIzol reagent and analyzed using GeneChip miRNA 2.0 Array (Affymetrix, Santa Clara, CA, USA). Three biological replicates were used for each stable cell line. According to the data, the random variance model (RVM) t test was used to filter the differentially expressed miRNAs. After analysis of significance and false discovery rate (FDR) analysis, differentially expressed miRNAs were selected according to the p value threshold [4 (link)]. The miRNA expression data was deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE68233.

The human cell lines used in this study are described (Supplementary Table 4). Human hTERT RPE-1 cells (normal diploid karyotype, immortalised non-cancer) were obtained from ATCC and cultured at 37 °C, 5% CO₂ and 3% O₂ in Dulbecco’s Modified Eagle’s Medium DMEM/F-12 (Sigma), supplemented with 10% Fetal Calf Serum (FCS) and 90 Units/ml Penicillin-Streptomycin. For all experiments (CC-seq, Slot Blot, WB, IF, FACs) with asynchronous cell populations, RPE-1 cells were seeded at a density of (3.5 × 10³ cells/cm²l) and incubated for 72 h at 37 °C, to ensure subconfluent log-phase growth (~70% confluency) at the time of the experiment. For G1 cell populations, WT and TOP2B^−/− RPE-1 cells were seeded at a density of 5 × 10³ cells/cm² and incubated for 48 h at 37 °C in DMEM/F12 containing 10% FCS, prior to a further 24 h incubation in DMEM/F12 medium containing no FCS to ensure complete G1-phase arrest (verified by FACs, see below). For experiments with proteasome inhibitor and VP16 (CC-seq, Slot Blot), cells were preincubated with 5 µM MG132 (Sigma) for 90 min, trypsinised and incubated in suspension with 5 µM MG132 and 100 µM VP16 (Sigma) for 20 min at 37 °C. For experiments with VP16 alone (IF), adherent cells were treated with 100 µM VP16 for 20 min at 37 °C.

Antibodies used: anti-FLAG (F3165), anti-β-actin (A1978), anti-HDAC2 (H3159) from Sigma; anti-USP11 (ab109232), anti-H3 (ab1791), anti-PAR (ab14459), anti-MTA2 (ab50209), anti-GST (ab19256), anti-RNF20 (ab181032) from Abcam; anti-Mi-2β (sc-365638), anti-MTA2 (sc-55566), anti-HDAC1 (sc-7872), anti-HDAC2 (sc-7899), anti-Ku80 (sc-5280), anti-RbAp46/48 (sc-373873) from Santa Cruz Biotechnology; anti-MBD3 (14540), anti-H2AK119ub (8240), anti-H2BK120ub (5546), anti-γH2AX (9718P), anti-BMI1 (6964) from Cell Signaling Technology; anti-γH2AX (05-636), anti-H2AK15ub (MABE1119), anti-H2A (ABE327), anti-H2B (MABE15), anti-FK2 (04-263) from Merck Millipore; anti-H3ac (39139) from Active Motif; anti-HA (M180-3) from MBL; anti-BRCA1 (22362-1-AP), anti-USP44 (15521-1-AP), anti-Lamin B1 (66095-1-Ig) from Proteintech; anti-53BP1(612522) from BD; anti-PARP1 (A0942), anti-USP3 (A15769), anti-USP12 (A5201), anti-USP22 (A16297) anti-USP30 (A12862) from Abclonal; anti-USP43 (AP14283b) from ABGENT. VP16, CPT, MMC, MMS, PJ-34, 4-OHT, anti-FLAG M2 affinity gel, FLAG peptide were from Sigma, and phosphatase inhibitor was from Applygen, protease inhibitor cocktail was from Roche Applied Science. Protein A/G Sepharose CL-4B beads were from Amersham Biosciences, NuPAGE 4-12% Bis-Tris gel was from Invitrogen, and silver-stained kit was from Pierce.

Cells transfected with negative control or miR-3196 mimics/inhibtors for 24 h were starved overnight and then treated with VP16 (Sigma-Aldrich) for 48 h. The FCM assay was performed using the Annexin V FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions.

Drugs: VP-16, CDDP, and two inhibitors, A12B4C3 and DDRI-18, were obtained from Sigma-Aldrich (St. Louis, MO, USA), while the third inhibitor—YU238259—was synthesized using TriMen Chemicals (Lodz, Poland) as previously described [11 (link)]. VP-16 was dissolved in methanol in the 10 mM stock solution. CDDP was dissolved in water in the 5 mM stock solution. A12B4C3, DDRI-18, and YU238259 were prepared as 50 mM stock solutions in anhydrous dimethylsulfoxide (DMSO). The chemical structure of the drugs and inhibitors is shown in Figure 2. Other chemicals: Normal melting point (NMP) and low melting point (LMP) agarose, Triton X-100, Tris HCl, phosphate-buffered saline (PBS), NaCl, NaHCO3, NaOH, ethylenediaminetetraacetic acid (EDTA), 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), DNase-free RNAse were obtained from Sigma-Aldrich (St. Louis, MO, USA).

In total, 800 cells were seeded in 60 mm dishes in triplicate. Twenty-four hours after seeding, cells were treated with CPT (Sigma-Aldrich; Cat#C9911), VP16 (Sigma-Aldrich; Cat#E1383), formaldehyde (Sigma-Aldrich; Cat#F8775), HU (Sigma-Aldrich; Cat#H8627), or MMC (Sigma-Aldrich, Cat#M4287) at the indicated concentrations and for the indicated times. Cells were washed with PBS, supplemented with fresh media, and incubated for 10 to 14 days. Formed colonies were fixed and stained with Coomassie Blue. The numbers of colonies were counted, and the percentage cell survival was calculated.

The following four mutagens with different mechanisms of action were used to study the modulatory effect of BA on DNA damage: (i) methyl methanesulfonate (MMS; Sigma-Aldrich) dissolved in phosphate-buffered saline (PBS) at a concentration of 400 μM [11 (link)]. MMS is a direct-acting monofunctional alkylating agent that reacts with the DNA molecule by transferring methyl radicals [12 (link)]; (ii) doxorubicin (DXR; Eurofarma Laboratórios Ltda.) dissolved in sterile distilled water and used at a concentration of 0.3 μM [13 (link)]. DXR, one of the most potent broad-spectrum antitumor anthracycline antibiotics, is a free radical generator and a potent inhibitor of topoisomerase II [14 (link)]; (iii) (S)-(+)-camptothecin (CPT; Sigma-Aldrich) dissolved in DMSO (Sigma-Aldrich) at a concentration of 123.4 μM CPT acts by inhibiting topoisomerase I, an enzyme necessary for DNA replication [15 (link)]; (iv) etoposide (VP-16; Sigma-Aldrich) dissolved in DMSO at a concentration of 1.7 μM. VP-16 is a potent anticancer agent that inhibits topoisomerase II [16 (link)].