Lipofectamine RNAiMAX, TRIzol, Propidium Iodide, RNase were purchased from Invitrogen Corp (Carlsbad, CA, USA). siRNA was obtained from Qiagen (Hilden, Germany). Cell culture reagents and flasks were purchased from HiMedia (France) and Corning Inc. (Corning, New York, USA). SYBR Green was purchased from Bio-Rad (Hercules, California). Antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA), Cloud-Clone Corp. (Houston, USA). Cell Titer-Glo reagent was purchased from Promega Corp. (Madison, Wisconsin, USA).

MDA-MB-231 cell was purchased from National Centre for Cell Science (Pune, India). The cells were cultured in L-15 medium supplemented with 10 % Fetal Bovine Serum (FBS), penicillin (100 unit/ml) and streptomycin (100 µg/ml). The cell culture was maintained at 37 °C in humidified air containing 5 % CO₂.

We used functionally verified siRNA directed against human CSNK2β (NM_001320), purchased from Qiagen with no potential off-targets prevalidated in HeLa cell line (Catalog Number: SI00605185). Also, predesigned negative control siRNA (Scramble) from Qiagen was used in our experiments (Catalog Number /ID: 1027310). Cells were cultured in 6 well plates one day before siRNA transfection. We used 25 nanomolar of each siRNA and made complex in Opti-MEM media. Similarly, the complex of Lipofectamine RNAiMAX (4 µl/each well) and Opti-MEM was made and incubated for 5 minutes at room temperature. After that, both the complexes were mixed in 1:1 proportion and incubated for 25 minutes at room temperature. Cells were treated with Opti-MEM-siRNA-Lipofectamine complex and incubated at 37 °C for 72 hours. The sequences used in siRNA and the target sequence for the genes in our study are mentioned below (Table 1(Tab. 1)).

Cell viability was assessed with CellTiter-Glo (CTG) assay (Promega, Madison, WI). Briefly, MDA-MB-231 cells were seeded in 96 well white cell culture plate at a density of 3000 cells per 180 µl of medium per well with 20 µl of siRNA complexes for CSNK2β and Scramble and incubated at 37 °C, 5 % CO₂ for 24 hours. On the next day, the media containing the complex was changed with the fresh media and further incubated till 96 hours. The cells were treated in quadruplets with respective siRNAs. The reagents were prepared according to the manufacturer's protocol. After incubation, 100 µl of fresh media was added to each well followed by 100 µl of reagent and kept on a shaker for 2 minutes to induce the cell lysis. The plate was incubated for 10 minutes at room temperature to stabilize the luminescence signal. Luminescence was measured using a microplate ELISA reader (Bio Tek, Winooski, Vermont, US).

MDA-MB-231 cells were transfected with Scramble and CSNK2β siRNA and incubated for 48 hours. After the cells were trypsinized, collected and counted, 500 cells/well were taken from each Scramble and CSNK2β transfected samples and seeded in 6 wells plate. After every two days, the medium was changed, and the cells were grown for three weeks. Then the cells were washed with Dulbecco's Phosphate Buffered Saline (DPBS), fixed with 3.7 % formaldehyde for 10 min and stained with 0.4 % crystal violet. The cells were washed with DPBS for 2-3 times and allowed to dry. The colonies were counted using Image J software.

MDA-MB-231 cells were plated in 6 well plates (4×10⁵ cells/well) and transfected with Scramble and CSNK2β siRNA as mentioned above. After the cells reached 90 % confluence, a wound was made in the center of the plate by scrapping the cells monolayer with 10µl tip. The cells were washed with sterile DPBS to remove the detached cells. The width of the wound area was measured at different time points using an inverted microscope. Densitometry analysis of wound area was carried by using Image J software.

Transwell chambers (Corning) were used for invasion assay. MDA-MB-231cells were transfected with CSNK2β siRNA and post 48 hours of transfection, cells (5 X 10⁴) were harvested and transferred to the upper chamber of transwell, pre-coated with matrigel, in 200 µL of serum free L-15 media whereas lower chamber consisted of complete media serving as a chemoattractant for the cells. Following 24 hours of incubation, cells in the upper surface of chambers were removed with a cotton swab, and the cells invaded through matrigel at the lower surface were fixed in 70 % ethanol and stained with 0.4 % crystal violet. Quantification of invaded cells was done by counting in six random fields.

2.5x10⁵ cells were seeded in 6 well plates and transfection was done as mentioned before. The cell cycle analysis was performed 48 hours post-transfection using BD FACS Verse ™. The cells were pooled out and fixed with ice-cold 70 % ethanol and kept overnight at 4 °C. On the next day, the cells were washed with DPBS for two times. The cells were further digested with RNase A (50 U/ml) at 37 °C for 1 hour and then stained with propidium iodide (20 μg/ml) at room temperature for 20 min. The samples were acquired by flow cytometer and results were analyzed by ModFit software.

MDA-MB-231 cells were cultured in 6 well plates. On the next day, cells were transfected with Scramble and CSNK2β siRNA and incubated for 72 hours. After incubation, the cells were washed twice with ice-cold DPBS and then lysed with sodium dodecyl sulfate (SDS) lysis buffer. The samples were heated for 5 minutes at 100 °C. The samples were run on SDS-PAGE, transferred to polyvinylidene (PVDF) membrane (90 volts for 2 hours). The membranes were blocked using 5 % skim milk in Tris-buffered saline with 0.1 % tween-20 (TBST) for 2 hours at room temperature. The blots were incubated with primary antibodies overnight at 4 °C on a rocker followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibodies. The blots were developed using enhanced chemiluminescence (ECL, Biorad). β actin was used as a loading control. The list of antibodies with their working dilution is mentioned in Table 2(Tab. 2).

Cells were transfected for 72 hours with the respective siRNAs and RNA was extracted using TRIzol lysis reagent using manufacturer's protocol. 2 µg of RNA was taken and pre-treated with DNase followed by cDNA synthesis with oligoDT primers. The thermocycler condition was 65 °C, 5 min, 42 °C, 60 min and 72 °C (final extension). Real time PCR was done with iTaq SYBRgreen mix (Biorad) using ABI 700 (Invitrogen). The Real time PCR thermocycler condition was as Hold stage 95 °C (10 min), PCR Stage (40 cycles) 95 °C (15 sec), 60 °C (1 min) and melt curve stage 95 °C (15 sec), 60 °C (1 min), 95 °C (15 sec). Gapdh was used as a housekeeping gene. mRNA expression analysis was done by the delta-delta-Ct method. The expression of each gene in CSNK2β siRNA treated sample was compared with Scramble. The sequences of primers used are mentioned in Table 3(Tab. 3).

1.5x10⁵ cells were seeded in 6 well plates. On the next day, the cells were transfected with Scramble and CSNK2β siRNA and incubated for 72 hours. Then the cells were stained with Hoechst 33342 for 15 minutes in the dark. Cells were once washed with DPBS and the images were taken using Nikon fluorescent microscope.

Cell apoptosis assay was performed by flow cytometry using an Annexin V-FITC/ propidium iodide (PI) apoptosis detection kit (BD Biosciences). Cells were seeded in triplicates in a 6-well plate at the density of 2x10⁵ cells per well. After 48 hours of transfection with Scramble and CSNK2β siRNA, the cells were harvested and the Annexin V-FITC/PI assay was performed according to the manufacturer's instruction. A total of 30,000 cells were analyzed using FACSuite software. The percentage of cells in different phases of apoptosis was examined.

Intracellular reactive oxygen species generation was assessed by fluoroprobe CM-H2DCFDA (Invitrogen). 1.5x10⁵ cells were cultured in 6 well plates and transfected with the Scramble and CSNK2β siRNA for 72 hours. The cells were treated with 10 µM CM-H2DCFDA for 30 min in the dark at 37 °C. The wells were once washed with DPBS and the images were captured using a fluorescence microscope (Nikon Ti).

Data were represented as a mean ± standard deviation. The level of significance between the two groups was calculated by Student's t-test. P < 0.05 was considered statistically significant.

Mutational status of CSNK2β across different cancers was carried out using cBioPortal (https://www.cbioportal.org/). Kaplan-Meier curves of relapse-free survival for breast cancer patients were evaluated by Kaplan-Meier plotter (KM-potter, https://kmplot.com/analysis/). Cancer and normal gene expression profiling of CSNK2β were examined by GEPIA (http://gepia.cancer-pku.cn/) and MERAV (http://merav.wi.mit.edu/) online servers.

To understand the expression status of CSNK2β in different cancers, we used the datasets retrieved from cbioportal (Cerami et al., 2012[5]) and compared the genomic alterations in multiple tumors. CSNK2β showed a high degree of amplification in most cancer types followed by deletion and mutation (Figure 1A(Fig. 1)). The TCGA data retrieved from cbioportal shows about 14 % gene amplification of CSN2β in breast cancer. Next, we checked the expression status of CSNK2β in different cancers using the GEPIA database (Tang et al., 2017[52]). Among 32 different cancers showed in the panel, expression is significantly higher in bladder urothelial carcinoma, breast invasive carcinoma and mesothelioma (Figure 1B(Fig. 1)). We also observed the distribution of CSNK2β gene expression between breast tumor tissue and adjacent tissue using MERAV database (http://merav.wi.mit.edu/) (Shaul et al., 2016[46]). As shown in Figure 1C(Fig. 1), the expression of CSNK2β is upregulated in breast tumors. To evaluate whether CSNK2β could be a prognostic factor for breast cancer patients, we examined mRNA expression status of CSNK2β in a large cohort of datasets containing 3951 breast cancer patients with online KM-Plotter software (http://kmplot.com/analysis) (Györffy et al., 2010[20]) and analyzed the correlation between CSNK2β expression and patient survival. Kaplan-Meier analysis using KM-plotter revealed that a group with higher CSNK2β expression have significantly poorer relapse-free survival (RFS) (p<0.001, longrank) (Figure 1D(Fig. 1)). The correlation of CSNK2β gene expression and RFS was evaluated using microarray data set containing breast cancer expression profiling from Gene Expression Omnibus (GEO). Kaplan-Meier estimates of RFS were calculated by splitting the patients into high- and low-expressing groups. The hazard ratio, log-rank P-value and number of breast cancer patients in each group are shown on the KM plot. Together, these results suggest that CSNK2β is clinically important for cancer pathogenesis and correlates with patient outcome.

To study the knocking down effect of CSNK2β in vitro, we silenced the expression of CSNK2β using the siRNA approach and evaluated the CSNK2β protein level. The result showed the silencing of CSNK2β was efficient compared to scrambled siRNA (SCR) (Figure 1E(Fig. 1)), Next, cell viability was measured in 96 hours of post-transfection by using CTG substrate. The viability of CSNK2β silenced MDA-MB-231 cells was significantly lower as compared to Scramble siRNA (p< 0.001) (Figure 1F(Fig. 1)). These results suggested that CSNK2β play a crucial role in cell proliferation of MDA-MB-231 cells. Clonogenic assay which mimics the tumor formation in vivo was carried out to observe the long-term survival of cells, which were transfected with CSNK2β +/- siRNAs. Silencing of CSNK2β significantly reduced the number of colonies as compared to Scramble siRNA transfected cells (p< 0.001) (Figure 1G, H(Fig. 1)). This result further confirmed the pivotal role of CSNK2β in the proliferation of MDA-MB-231 cells.

ROS possesses double edge sword property both as, oncogenic- maintaining sustained and increased proliferation of cancer cells, as well as, tumor suppressor- leading to cell death when an aberrant increase in intracellular ROS arises due to any kind of stress. It is a good idea to find oxidative stress modulators as an anti-cancer strategy (Wang and Yi, 2008[53]; Gurer-Orhan et al., 2018[19]). In our experiment, we found the increased production of ROS in CSNK2β siRNA transfected cells compared to scramble siRNA transfected cells (Figure 2A(Fig. 2)). This data suggested that CSNK2β plays a crucial role in attenuating the intracellular ROS production in MDA-MB-231 cells for its sustained proliferation.

Next, we checked the status of the cell cycle phase following knockdown of CSNK2β. After 48 hours of transfection, the percentage of cells in the different phases of the cell cycle were analyzed by flow cytometry. The percentage of cells in G2/M phase was significantly increased in CSNK2β transfected samples (22.806 %) than in Scramble siRNA transfected samples (13.266 %) (p < 0.01). At the same time, the percentage of cells in G0/G1 and S phase was reduced (Figure 2 B, C(Fig. 2)). These results suggest that knockdown of cells with CSNK2β inhibits the progression of cells via blocking the cell cycle at G2/M phase.

The significant arrest of MDA-MB-231 cells in G2/M phase led us to check the effect of CSNK2β knockdown on cell apoptosis. We found that silencing of CSNK2β caused change in cellular morphology and condensation of nuclei - a prominent feature of cells undergoing apoptosis (Figure 2D(Fig. 2)). Quantitative estimation of apoptotic cells was carried out using flow cytometry with annexin V-FITC and PI. Our data showed 6.77 % of apoptosis (early apoptotic + late apoptotic population) in CSNK2β siRNA knockdown cells compared to 3.52 % (early apoptotic + late apoptotic population) in Scramble siRNA cells which comes out to be significant with p < 0.01 as shown in (Figure 2E, F(Fig. 2)). To unravel the cell death mechanism induced after the knockdown of CSNK2β, western blot analysis was performed to determine the level of proteins related to apoptosis. We found an increased expression of BAX and cleaved caspase 3 along with a decreased level of Bcl-xL expression (Figure 2G(Fig. 2)). Altogether these results showed that the silencing of CSNK2β drives cell death via apoptosis. Moreover, we also analyzed the expression of two vital autophagy markers, Beclin-1 and LC3, and found both markers were upregulated in CSNK2β siRNA transfected samples than Scramble siRNA (Figure 2G(Fig. 2)). These findings together showed that targeting CSNK2β triggers the cells towards both apoptosis and autophagy cell death pathway.

Next, we questioned whether the depletion of CSNK2β could alter the migration and invasion potential of MDA-MB-231 cells. In wound healing assay, migratory capacity was observed for each sample at different time points (0, 6, and 12 h). After 12 hours, the area of a wound was significantly wide in CSNK2β transfected wells as compared with Scramble suggesting the role of CSNK2β gene in the migration of MDA-MB-231 cells in vitro (p < 0.001) (Figure 3A, B(Fig. 3)). Next, we also checked the invasive potential of MDA-MB-231 following CSNK2β knockdown by transwell invasion assay. We found that cells transfected with CSNK2β siRNA were unable to invade the matrigel layer as efficient as the Scramble siRNA transfected cells (Figure 3C, D(Fig. 3)). Some critical markers associated with cell migration were also checked by western blotting and it was found that knockdown of CSNK2β caused inhibition of MMP-9, Vimentin, β Integrin1, and β-catenin along with upregulation of E-cadherin (Figure 3E(Fig. 3)).

To elucidate the role of CSNK2β in MDA-MB-231 cell proliferation, survival, and apoptosis, we examined the expression of some selected genes related to these phenomena at mRNA and protein level by real time PCR and western blotting respectively. Knockdown of CSNK2β led to downregulation of PCNA, E2F1, c-Myc, NF-κB, p-ERK, and p-38α protein level, as shown in (Figure 4A(Fig. 4)). In 72 hours transfected samples, we found that the mRNA level of p21, a cell cycle inhibitor, was increased up to about 2.7 fold, while the expressions of survivin (anti-apoptotic and metastatic marker), Nanog, SLUG, OCT4, SOX2 (transcription factor for stemness) and cyclin E1, Cyclin A1, cyclin B1 (cell cycle cyclins) and E2F1, mTOR, PCNA proliferative marker gene expression were decreased following the knockdown of CSNK2β (Figure 4B(Fig. 4)). These results showed the critical role of CSNK2β in the expression of genes associated with cell cycle, cancer stemness, and metastasis.