Cells
MCF-7 cells were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China). They were cultured in minimum essential medium containing 10% fetal calf serum (MilliporeSigma, Burlington, MA, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL). Cell culture was undertaken in an atmosphere of 5% CO2 at 37°C.
Assays to measure the viability and proliferation of cells
MCF-7 cells were plated at 2×103 cells/well on 96-well plates and cultivated overnight to allow attachment. Cells were cultured with ATO (0, 0.5, 1, 2, 4, 8, or 16 µmol/L; MilliporeSigma) and RSV (0, 5, 10, 20, 40, 60, or 100 µmol/L; Aladdin, Shanghai, China). The number of viable cells was determined by Cell Counting Kit-8 (CCK-8; Dojindo, Tabaru, Japan) according to manufacturer instructions at 24, 48, or 72 h. Six replicates of each experimental condition were carried out.
The half-maximal inhibitory concentration (IC50) of ATO and RSV was calculated using Prism 8.4 (GraphPad, San Diego, CA, USA), so we obtained three IC50 values for ATO and RSV, respectively (Fig. 1). When considering the serious adverse reactions of high-dose ATO and the efficacy of low-dose ATO [24–26], we chose ATO at 3 μM for 48 h and RSV at 53 μM for 48 h.
Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay
TUNEL staining was undertaken to assess the death of MCF-7 cells. The assay was undertaken in accordance with manufacturer (Invitrogen, Carlsbad, CA, USA) instructions. TUNEL-positive green fluorescence images were captured using a microscope. The number and percentage of positive cells were counted, and data presented as the mean percentage of positive cells.
Western blotting
MCF-7 cells were treated with ATO (3 μM) plus RSV (53 μM), respectively, for 48 h. Phosphate-buffered was administered under identical conditions and used as the control. Cell lysates were collected and western blotting undertaken according to manufacturer instructions.
The antibodies we used (all at 1:2000 dilution and obtained from Abcam, Cambridge, UK) were: rabbit monoclonal (E17) to B-cell lymphoma (Bcl)-2 (catalog number: ab32124), rabbit monoclonal (E63) to Bax (ab32503), mouse monoclonal (6C5) to GAPDH (loading control; ab8245), rabbit monoclonal (EPR22840-25) to cleaved caspase-7 (ab256469), and rabbit monoclonal (E83-77) to cleaved caspase-3 (ab32042).
Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA from the cultured cells of each group was extracted using TRIzol® Reagent (Invitrogen). Complementary (c)DNA was synthesized from 2 μg of total RNA using the First Strand cDNA Synthesis kit (Vazyme Biotech, Nanjing, China), Then, cDNA was analyzed by the SYBR™ Green Realtime PCR kit (Vazyme Biotech). RT-qPCR was carried out using the LightCycler® 96 system (Roche, Basel, Switzerland) in 96-well plates with each reaction run in triplicate. Finally, analysis of melting curves was done after amplification to verify PCR specificity. All kits were used according to manufacturer instructions. The cDNAs encoding Bax, Bcl-2, caspase-3, caspase-7, and β-actin were amplified using the primer pairs detailed in Table 1.
Table 1 Sequences for primers
Primer
|
Sequence
|
Length (bp)
|
Bax
|
Sense 5'- CCCGAGAGGTCTTTTTCCGAG -3'
|
155
|
|
Antisense 5'- CCAGCCCATGATGGTTCTGAT -3'
|
|
Bcl-2
|
Sense 5'- GGTGGGGTCATGTGTGTGG -3'
|
89
|
|
Antisense 5'- CGGTTCAGGTACTCAGTCATCC -3'
|
|
Caspase-3
|
Sense 5'- AGAGGGGATCGTTGTAGAAGTC -3'
|
81
|
|
Antisense 5'- ACAGTCCAGTTCTGTACCACG -3
|
|
Caspase-7
|
Sense 5'- AGGGACCGAGCTTGATGATG -3'
|
85
|
|
Antisense 5'- CACTGGGATCTTGTATCGAGGA -3'
|
|
β-actin
|
Sense 5'- CATGTACGTTGCTATCCAGGC -3'
|
250
|
|
Antisense 5'- CTCCTTAATGTCACGCACGAT -3'
|
|
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was carried out using a ChIP kit (Beyotime Institute of Biotechnology, Shanghai, China) following manufacturer instructions. Cells were fixed with 1% formaldehyde for 10 min at room temperature. Then, the crosslinking reaction was quenched by addition glycine solution (0.125 M) for 5 min. Chromatin was sheared to 200–1000 bp by sonication with 10 pulses of duration 15 s at 150 W each time (Xinzhi, Ningbo, China). Fragmented chromatin was immunoprecipitated with acetyl-histone H3 (acetyl K27) antibody (ab4729; Abcam) and human immunoglobulin-G (A7001; Beyotime Institute of Biotechnology). Then, the acetyl-histone H3-bound DNA was precipitated, and DNA fragments were purified. Enriched DNA fragments were analyzed by RT-qPCR. Except for precipitated samples, the 10% chromatin inputted for each sample was de-crosslinked, processed, and analyzed by RT-qPCR in the same way as described above. For quantitation, inputted samples were used to normalize each sample. The amount of immunoprecipitated fragments was calculated as a percentage of the amount inputted. The sequences of the primers used for amplification are given in Table 2.
Statistical analyses
Statistical analyses of data were carried out using SPSS 18.0 (IBM, Armonk, NY, USA). Data are the mean ± SD. One-way analysis of variance or the Student’s t-test was used to identify significant differences between individual groups. P < 0.05 was considered significant.