BNC2 is a putative tumor suppressor gene in high-grade serous ovarian carcinoma and impacts cell survival after oxidative stress

Rs3814113 is the single-nucleotide polymorphism (SNP) showing the strongest association with high-grade serous ovarian carcinoma (HGSOC) incidence and is located in an intergenic region about 44 kb downstream of basonuclin 2 (BNC2) gene. Lifetime number of ovulations is associated with increased risk to develop HGSOC, probably because of cell damage of extrauterine Müllerian epithelium by ovulation-induced oxidative stress. However, the impact of low-penetrance HGSOC risk alleles (e.g. rs3814113) on the damage induced by oxidative stress remains unclear. Therefore, the purpose of this study was to investigate whether rs3814113 genetic interval regulates BNC2 expression and whether BNC2 expression levels impact on cell survival after oxidative stress. To do this, we analyzed gene expression levels of BNC2 first in HGSOC data sets and then in an isogenic cell line that we engineered to carry a 5 kb deletion around rs3814113. Finally, we silenced BNC2 and measured surviving cells after hydrogen peroxide (H2O2) treatment to simulate oxidative stress after ovulation. In this paper, we describe that BNC2 expression levels are reduced in HGSOC samples compared with control samples, and that BNC2 expression levels decrease following oxidative stress and ovulation in vitro and in vivo, respectively. Moreover, deletion of 5 kb surrounding rs3814113 decreases BNC2 expression levels in an isogenic cell line, and silencing of BNC2 expression levels increases cell survival after H2O2 treatment. Altogether, our findings suggest that the intergenic region located around rs3814113 regulates BNC2 expression, which in turn affects cell survival after oxidative stress response. Indeed, HGSOC samples present lower BNC2 expression levels that probably, in the initial phases of oncogenic transformation, conferred resistance to oxidative stress and ultimately reduced the clearance of cells with oxidative-induced damages.

In HGSOC, the most important risk factor is the occurrence of the disease in a first-degree relative; 9 this excess of risk is because of both environmental (e.g. lifetime number of ovulations, body mass index and so on) and genetic factors. Concerning environmental factors, lifetime number of ovulations was one of the first to be described, 10 and rodent models of repeated superovulation confirmed the insurgence of modifications resembling early transformation. 11,12 The explanation of the association between lifetime number of ovulations and HGSOC incidence may reside in cell damage by ovulation-induced oxidative stress. 11,13 Concerning genetic risk factors, which have the greatest impact, less than half of the excess of risk is due to BRCA1/2 high penetrance mutations, whereas the remaining risk is probably because of low-to moderate-penetrance risk alleles. 14 Low-to moderatepenetrance risk alleles (e.g. single-nucleotide polymorphism (SNP)) can be identified by genome-wide association studies (GWAS) 15 and ultimately give novel molecular insights on tumor biology. [16][17][18] SNP rs3814113 has the strongest association with HGSOC incidence 14 and is located in an intergenic region of human 9p22.2 locus,~44 kb downstream from the transcription starting point (TSS) of basonuclin 2 (BNC2) gene and 200 kb upstream from TSS of centlein (CNTLN) gene. BNC2 transcribes a zinc-finger protein highly conserved during evolution, 19 with about 200 transcript isoforms, 20 and BNC2-knockout (KO) mice die within 24 h after birth. 21 Preliminary evidences pointed to BNC2 as the coding transcript that could be responsible for the association between 9p22.2 locus and HGSOC: BNC2 promoter is more frequently methylated in peripheral lymphomonocytes of EOC patients compared with healthy subjects, 22 BNC2 expression levels are reduced in EOC cell lines compared with primary human ovarian surface epithelial cell cultures, 23 and BNC2 was described as a presumptive tumor suppressor gene in bladder, esophageal and glioblastoma tumors. [24][25][26] The impact of low-penetrance HGSOC risk alleles (e.g. rs3814113) on cell damage induced by oxidative stress remains unclear. For instance, acquired genetic modifications, which reduce cell death after oxidative stress, may offer fallopian serous epithelial cells the advantage to survive despite carrying genetic damages caused by oxidative stress exposure. 12 Therefore, the purpose of this study was to investigate whether rs3814113 genetic interval regulates BNC2 expression and whether BNC2 expression levels impact cell survival after oxidative stress. To do this, first we engineered an isogenic cell line to carry a 5 kb deletion around rs3814113 and measured BNC2 levels; next, we silenced BNC2 expression levels, and after hydrogen peroxide (H 2 O 2 ) treatment, which simulates oxidative stress after ovulation, 11 we measured cell survival.

Results
BNC2 is in linkage with rs3814113 and is a putative tumor suppressor in HGSOC. To consolidate that BNC2 is the principal actor of the association between human 9p22.2 locus and HGSOC incidence, we used two independent strategies. First, we searched for SNPs in linkage disequilibrium (LD) with rs3814113 by using SNAP bioinformatics tool, 27 and we discovered that rs3814113 is correlated (r 2 ≥ 0.8) to other 11 SNPs located within 6.8 kb upstream (Figure 1a and Supplementary Table 1). Yet, recombination rates suggested a larger haplotypic block, which includes the first exon and intron of BNC2, embracing one of BNC2 promoters, and includes additional SNPs that correlated to rs3814113 with an r 2 ≥ 0.5 (Figure 1a and Supplementary  Table 1). Within the identified haplotypic block, rs3814113 was the only SNP present in the Illumina SNP-chromatin immunoprecipitation (ChIP) array used in the GWAS study that initially correlated 9p22.2 region with EOC predisposition. 14 In the second strategy, we searched for genes within the 9p22.2 locus differentially expressed between tumor-free control and HGSOC samples. To do this, we interrogated the gene data set by Tone et al., 28 which analyzed microdissected non-malignant fallopian tube epithelium (FTE) and HGSOC samples using a high-density Affymetrix gene expression array (Affymetrix Human Genome U133 Plus 2.0 Array, Santa Clara, CA, USA). We correlated signal intensity levels of microarray probes located within ± 4 Mb from rs3814113 (Supplementary Figure S1A) with the sample type (i.e. FTE or HGSOC samples), and we found that BNC2 locus was the region with the greatest number of probes inversely correlated with HGSOC (i.e. lower signal intensity in HGSOC compared with non-malignant FTE samples) (Supplementary Figure S1B and Figure 1b). Instead, CNTLN, which is the closest gene downstream of rs3814113, was not differentially expressed between the groups (Figure 1b). We confirmed these results in a second publically available data set 29 ( Figure 1c). Meanwhile, we collected at our Institute an original cohort of HGSOC and tumor-free control samples (fallopian tube and ovarian), in which we confirmed by quantitative realtime PCR (qRT-PCR) that BNC2 expression was reduced in HGSOC ( Figure 1d). Therefore, both LD and gene expression data analyses pointed to BNC2 as the most likely candidate genetic element involved in HGSOC predisposition.
Next, we measured BNC2 transcript expression in a panel of 16 EOC cell lines (Figure 2a). By grouping EOC cells into likely and unlikely HGSOC, 30 we did not observe any significant difference in BNC2 expression levels between the two groups. In seven likely HGSOC cell lines, BNC2 protein expression was proportional to mRNA expression and showed several isoforms between 100 and 150 kDa ( Figure 2b); moreover, BNC2 was localized exclusively in the NP40 nuclear insoluble fraction (Supplementary Figures S2A  and B) in agreement with Vanhoutteghem and Djian. 19 Interestingly, of the 12 EOC cell lines that we genotyped for rs3814113, all carried the T allele in homozygosis (data not shown), which is the allele associated with increased EOC predisposition in the general population, 14 whereas according to dbSNP build 141, 31 the expected frequency for rs3814113 in the general population is 58% for T and 42% for C.
According to Vanhoutteghem and Djian 20 and Ghanbari et al., 32 BNC2 has several putative promoters and enhancers, which is also confirmed by the Encode project 33 (Supplementary Figure S3); however, histone marks enriched in promoters and enhancers (H3K4me3 and H3K27Ac, respectively) have never been described in HGSOC cell models. Therefore, we performed ChIP for these histone  Figure 2c). Moreover, in both COV318 and OVCAR4, the region labeled as BNC2 Prom2 had the highest enrichment, in agreement with Ghanbari et al. 32 To sum up, these data indicate that the genomic region in LD (r 2 ≥ 0.5) with rs3814113 includes one of the BNC2 promoters and that BNC2 is downregulated in HGSOC samples compared with non-malignant FTE samples.
A human genomic region including rs3814113 regulates BNC2 expression. Interestingly, rs3814113 resides inside AK024561 genomic locus, a putative non-coding RNA transcript that was cloned in human adipose tissue according to GenBank records ( Figure 3a). Two probes of the Affymetrix U133 Plus 2.0 Array hybridize to AK024561 (i.e. 216730_at and 216742_at) (Supplementary Figure 3), but both show low expression signals and no difference between non-malignant FTE and HGSOC samples in Tone et al. 28 (Figure 1b and Supplementary Figure S1B). We evaluated expression levels of AK024561 in a panel of 17 different non-malignant tissues, including peripheral blood mononuclear cells (PBMCs) collected from healthy donors, and in a panel of HGSOC samples, fallopian tube and ovary tumor-free samples. AK024561 was strongly expressed only in the testis 20 and moderately expressed in the adrenal gland and skeletal muscle (Figure 3b). Only in 4 out of 10 HGSOC tumor samples, we could detect a faint band, whereas we did not F Figure 1 Rs3814113 at chromosome 9p22.2 is in linkage with BNC2, a putative tumor suppressor gene in HGSOC. (a) Regional LD plot generated using the SNPAnnotation and Proxy Search web interface using rs3814113 as target SNP (https://www.broadinstitute.org/mpg/snap/ldsearch.php). The left-hand y axis shows values for r 2 (correlation coefficient, a measure of LD; 0.8 was set as threshold), whereas the right-hand y axis shows recombination rate values in centiMorgans (units of recombination) per million bases (cM/Mb). Diamond labels correspond to SNP (black diamond is rs3814113, gray filled diamonds represent SNPs with r 2 ≥ 0.8 with rs3814113, empty filled diamonds have an r 2 o0.8). Higher peaks of recombination rate indicate an association between rs314113 and the end of BNC2 genetic locus. (b) Analyses of gene expression for Affymetrix probes surrounding rs3814113 in the 9p22.2 locus from the GSE10971 data set. Affymetrix probes are indicated by a number followed by '_at'. Laser capture microdissected nonmalignant distal FTE n = 24; HGSOC, n = 13. BNC2, intergenic region surrounding rs3814113, and CNTLN loci were represented in this array by 8, 2 and 4 probes, respectively. Bars are mean ± S.E. (c) Analyses of BNC2 and CNTLN expression in the GSE26712 data set. Non-malignant ovarian surface epithelium (OSE), n = 10; HGSOC, n = 185. In this Affymetrix array, BNC2 and CNTLN were represented only by one probe (220272_at and 22095_at, respectively). Lines are mean ± S.E.  Figure S3). H3K4me3 (top panels) and H3K27Ac (bottom panels) = histone H3 trymethylated on lysine 4 and acetylated on lysine 27, respectively. BNC2 intron 2 (Intr 2) was set as 1 Bnc2 impacts oxidative stress outcome in EOC cells L Cesaratto et al detectable expression levels except COV318, which had an extremely low signal (data not shown). Consistently, we did not observe histone mark enrichment in the intergenic regions including AK024561 locus and nearby enhancers (intergenic enhancers 1, 2 and 3), except for a H3K27Ac enrichment in OVCAR4 intergenic enhancers 1 and 3 ( Figure 2c).
To investigate whether AK024561 locus may still regulate BNC2 expression, we deleted 5 kb surrounding the SNP (between guide 6 and guide 9 in Figure 3a) using CRISPR-Cas9 system. 17,34 Unfortunately, we could not obtain KO cell clones in COV318, which is the only cell line, among the ones that we tested, that expressed AK024561 (data not shown), because of the low efficiency of COV318 to grow as single-cell clones; therefore, we opted for 293FT because of very high efficiency of transfection and of single-cell clone recovery. We recovered five clones with homozygous deletion of the targeted region (KO clones) (Figure 3c and Supplementary Figure S4B), and we compared them with four wild-type (WT) clones, which had either point mutations in guide RNA target We measured BNC2 and CNTLN transcript expression levels in WT and KO clones; given the multiplicity of BNC2 isoforms, 20 we designed three qRT-PCR primer pairs that target exon boundary 1-2, 2-2a and exon 6 20 (Supplementary Figure S3). By these means, we observed that the deletion of the 5 kb region comprising rs3814113 almost halved expression levels of BNC2 but not of CNTLN (Figure 3d). Unfortunately, 293FT expression levels of BNC2 protein were almost undetectable and it was not possible to observe any difference between WT and KO clones. Of note, in 293FT cells, rs3814113 is heterozygous (C/T) and AK024561 was never detected both in whole and nuclear RNA (data not shown).
Taken together, these findings indicate that the genetic region encompassing rs3814113 regulates BNC2 but not CNTLN expression levels, and that this regulation does not depend on AK024561 transcription.
Oxidative stress reduces BNC2 expression in vitro and in vivo. The incessant ovulation model of EOC origin lies on the hypothesis that fallopian tubes and ovaries are exposed to increased levels of reactive oxygen species (ROS) during ovulation cycle, which reiterated over time may generate the soil for EOC insurgence. 4,10,12 Therefore, to investigate whether BNC2 may be involved in the incessant ovulation model, we evaluated BNC2 expression levels following oxidative stress. To simulate postovulation environment, we treated OV90, COV318 and COV504 with increasing doses of H 2 O 2 and evaluated BNC2 levels at early and late time points, after 3 h and 24 h, respectively. 35 Effectiveness of H 2 O 2 cell treatment was verified by phosphorylation of H2AX with no evident disparities among cell lines (Figure 4a and Supplementary Figure 5). While in OV90 BNC2 levels remained undetectable even after H 2 O 2 treatment (data not shown), in COV318 and COV504, we observed a dosagedependent reduction of BNC2 protein levels at 24 h after treatment, but not at earlier time points. To understand whether BNC2 reduction occurred both at the transcriptional and protein level, we measured BNC2 by qRT-PCR and found that, 5 h following H 2 O 2 exposure, BNC2 transcript levels were already halved, suggesting a regulation of BNC2 at the transcriptional/mRNA level rather than at the protein stability level (Figure 4b). To address deeper the molecular mechanism by which BNC2 is regulated in an oxidative stress condition, we investigated the role of epigenetic modifications in BNC2 expression at different time points after H 2 O 2 exposure. To do this, we performed a time-course experiment in COV318 treated with 0.06 mM of H 2 O 2 , which is the lowest dosage at which we previously observed a reduction of BNC2 protein expression levels, and we confirmed that BNC2 expression decreases both at mRNA and protein levels (Figures 4c and d). As BNC2 promoter contains three CpG islands (Supplementary Figure S3) and BNC2 promoter methylation has been previously found associated with EOC, 22 we analyzed methylation status of CpG island 141 (chr9:16870124-16872020 hg19) and CpG island 21 (chr9:16828936-16829169 hg19) and did not observe any methylation both in untreated and treated cells (data not shown). Concomitantly, we evaluated activating and repressing histone modifications (H3K4me3 and H3K27me3, respectively) in the same time-course experiment. Unexpectedly, activating H3K4me3 modification increased at earlier time points and eventually decreased at 24 h (Figure 4e), whereas we did not find any enrichment of repressing H3K27me3 modification (data not shown). We also wondered whether BNC2 repression persisted longer than 24 h after H 2 O 2 exposure; indeed, we found BNC2 downregulation both at mRNA and protein levels lasting up to 1 week post-treatment (Figures 4f and g), likely due to a decrease of activating H3K4me3 enrichment and an increase of suppressing H3K27me3 in BNC2 promoters and enhancers (Figure 4h). Enrichment of H3K27me3 modification in H 2 O 2 -treated COV318 cells was similar to OV90, which constitutively do not express BNC2 (Figure 4h). According to our results, decrease of BNC2 expression after H 2 O 2 exposure is not due to methylation and histone modifications at early time points, whereas at later time points, histone modification is involved in BNC2 transcription repression.
To study the effect of ovulation on BNC2 expression levels in vivo, we induced superovulation in prepubertal mice and measured BNC2 protein levels in murine oviducts. In this model, BNC2 displayed several protein isoforms similarly to COV318, and its protein expression levels decreased in ovulated mice compared with control mice (Figure 4i).
To sum up, these evidences suggest that BNC2 expression levels decrease both in vitro and in vivo after oxidative stress or ovulation, respectively.   (Figure 5a). With this observation in mind, we silenced BNC2 levels by shRNA in three different BNC2-expressing HGSOC cell lines and in BNC2-null OV90 cell line. We tested five short hairpins spanning different regions of BNC2 (Supplementary Figure S3), and used the ones with a strong and intermediate  Figure 6B), which are null for BNC2, indicating that the phenotype that we described was BNC2-specific. Cells silenced with sh5, which always displayed the smallest BNC2 reduction, were also the ones with the smallest increase in cell survival, implying that the effect that we observed was dependent on BNC2 expression levels.
In conclusion, these evidences indicate that low BNC2 expression levels have a protective role in response to H 2 O 2 , which is in line with reduced expression levels of BNC2 in HGSOC compared with control samples (Figures 1b-d).

Discussion
Our study showed that the first intron and exon of BNC2 and one of its promoters are in linkage with rs3814113, and that BNC2 expression levels were reduced in HGSOC samples compared with control samples. Moreover, we observed that deletion of 5 kb surrounding rs3814113 decreased BNC2 expression levels in an isogenic cell line and, finally, that silencing of BNC2 expression levels increased HGSOC cell survival after H 2 O 2 treatment.
Altogether, our findings give new insights about the mechanism at the basis of HGSOC and 9p22.2 association, 14 and in particular they suggest that the intergenic region located around rs3814113 regulates BNC2 expression, which in turn affects cell survival after oxidative stress response. Indeed, HGSOC samples present lower BNC2 expression levels that, So far, few groups have published results regarding the role of BNC2 in cancer, but all suggested a putative tumor suppressor function of BNC2, [24][25][26] in agreement with our findings. Ramus et al. 36 reported that rs3814113 minor allele (C) is also associated to a lower risk of EOC in BRCA1/2mutated populations. When we analyzed the gene expression data set from Tone et al., 28 which contains both BRCA1/2 WT and mutant control samples, we did not observe any difference in BNC2 expression levels between the two groups (data not shown). Ultimately, these evidences may suggest that BNC2 locus and BRCA1/2 loci are two independent risk factors in EOC onset.
Our study presents some limitations: for instance, (1) we used cell lines derived from HGSOC and not cell lines derived from normal tissues (e.g. normal fallopian tubes); nevertheless, we confirmed our in vitro findings in the normal oviducts obtained from a mouse model of superovulation. (2) We did not investigate in our cell model the allele-specific impact of rs3814113 on BNC2 expression, as we expected that it would have taken a GWAS-size clones to observe any effect. Instead, we took advantage of an isogenic cell line knocked out for 5 kb interval surrounding rs3814113 to obtain a stronger effect. Despite having found a link between rs3814113 genomic region and BNC2 expression, we cannot exclude that other genetic elements that are located in the haplotypic block of rs3814113 participate in BNC2 regulation.
(3) Finally, even though we described that silencing of BNC2 expression affects survival after H 2 O 2 treatment, despite our attempts, we could not generate cells overexpressing BNC2 to rescue silencing experiments and we did not dissect in greater detail the molecular mechanism at the basis of this phenotype. Precisely, our results on BNC2 localization in agreement with 19 suggest that BNC2 is localized in the nucleus more firmly compared with transcription factors and is a putative chromatin-modulating protein; therefore, ChIP-seq would be a good approach to study BNC2 binding regions.
EOC and breast cancer share some common risk factors (e.g. BRCA1/2 status); therefore, it might be expected that BNC2 has a role also in breast cancer onset. We are not aware of BNC2 expression levels in normal and tumor breast samples; however, there is no report of any association between rs3814113 and breast cancer risk, in both the general population 15 and BRCA1/2 mutant carriers, 36 ultimately suggesting that BNC2 is not involved in breast cancer.
According to our results, BNC2 seems to be a good candidate to develop new molecular tools to select healthy subjects with increased risk of EOC. Despite having demonstrated that the 5 kb region around rs3814113 regulates BNC2 expression, it is unlikely that we can use rs3814113 genotyping as an indicator of BNC2 expression 14 and novel strategies should be undertaken. At the same time, we showed that BNC2 silencing affects cell survival after oxidative stress, which suggests that modulating BNC2 levels pharmacologically may be an intriguing strategy to reduce the excess of EOC risk in selected populations (e.g. BRCA1/2 mutation carriers). Ultimately, if we consider that the discovery of high penetrance risk alleles (BRCA1/2 mutations) not only helped to identify subjects at higher risk but it was also helpful to select EOC patients benefiting the most from poly(ADP-ribose) polymerase inhibitors, 5 the same scenario may also be true for BNC2. Hence, understanding how to target BNC2 and/or the associated stress response pathway could lead to the development of alternative therapeutic approaches for patients with HGSOC.

Materials and Methods
Cell lines, cell cultures, lentiviral transduction and transfections. Cell lines used in this work are listed in Supplementary Table 2, which includes cell type, source and culture media. No antibiotics were routinely used for cell culture. Cells have been authenticated in 2012 by BMR Genomics (Padova, Italy) according to Cell ID System (Promega, Madison, WI, USA) and using Genemapper ID version 3.2.1, to identify DNA STR profiles. Cells were expanded and have been used only for a short number of passages after authentication. Cell lines were maintained at 37°C under 5% CO 2 in humidified incubators and routinely tested using MycoAlert Detection Kit (Lonza, Cologne, Germany) for mycoplasma contamination. Only mycoplasma-negative cells were used for experiments.
BNC2-silenced ovarian cancer cell lines were generated by lentiviral transduction. Briefly, 293FT cells were co-transfected with pLKO.1 puro vectors encoding for either human BNC2-shRNAs (sh1_TRCN0000108177, sh2_TRCN0000327841 sh3_TRCN0000108179, sh4_TRCN0000108175, sh5_TRCN0000108176, MISSION shRNAs from Sigma-Aldrich (St. Louis, MO, USA)) or non-target shRNA control (SHC016; Sigma-Aldrich), together with ViraPower Packaging Mix (pLP1, pLP2 and pLP/VSV-G) (Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA). Viral supernatants were collected 72 h later and transducing units per ml were determined by limiting dilution titration in HCT116 cells. An MOI of~5 was used for transducing ovarian cancer cells. After 5 days of selection with puromycin, BNC2-silenced cells were immediately used for in vitro experiments, protein and RNA extraction. Polybrene (Sigma-Aldrich) at a final concentration of 8 μg/ml was used to increase transduction efficiency.
Cell transfections were performed using Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's instructions. CRISPR-Cas9 system. KO of AK024561 in 293FT cells was generated by CRISPR-Cas9 system, according to Ran et al., 34 using two guide RNAs (guides 6 and 9; Supplementary Table 3) that delimit a region of 5 kb surrounding the AK024561 non-coding transcript (Figure 3a). To this end, guide RNAs were cloned into pSpCas9(BB)-2A-GFP (PX458) vector (cat. no. 48138; Addgene, Cambridge, MA, USA) and transfected into cells. To evaluate guide RNA-directed Cas9 cutting efficiency, genomic DNA was extracted using Gentra Puregene Cell Kit (Qiagen Sciences, Germantown, MD, USA), according to the manufacturer's instructions, and was subjected to Surveyor Assay using Surveyor Mutation Detection Kit (IDT Integrated DNA Technologies, Leuven, Belgium), as per the manufacturer's instructions (Supplementary Figure 4A).
The 293FT CRISPR-modified cell clones were grown following GFP single-cell sorting by FACS, then genomic DNA was extracted and clones were screened by PCR, using two primers, AK024561 5+3 and Screen 6F+7R (Figure 3a and  Supplementary Table 3), which for KO clones were expected to yield no product and a 1.6 kb fragment, respectively (Figure 3c and Supplementary Figure S4). Genetic deletion was confirmed by direct Sanger sequencing, using BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequences of all the clones that we used are reported in Supplementary Material.
Cell treatments. H 2 O 2 treatment was performed as follows: cells plated 24 h earlier in a 6-or 12-well format were exposed for 1 h to H 2 O 2 (0-4 mM range) in serum-free medium. After two washes with PBS, H 2 O 2 containing medium was removed and replaced with complete medium. Cell viability was assed 24 h later by MTT staining (M2003; Sigma-Aldrich). MTT stock solution was added to cells at a final concentration of 0.28 mg/ml and incubated for 1 h at 37°C. Supernatant was then discarded and cells were air-dried. Reduced MTT, measure of cellular metabolic activity and index of cell viability was dissolved by adding 1 or 2 ml of DMSO, depending on plate format. Two hundred microliters of DMSO were transferred to a 96-well and absorbance was measured at 570 nm with Infinite M1000 Microplate Reader (Tecan, Männedorf, Switzerland).
Bnc2 impacts oxidative stress outcome in EOC cells L Cesaratto et al