Flow Cytometric Analysis of Cell Cycle

The effects of different doses of SDEA on the cell cycle of HT29 and HCT116 cells were investigated. The cells were treated as follows: one group was intervened for 48 h with different SDEA concentrations (0, 20, 60, and 100 μg/mL), and the other group was treated with 100 μg/mL at different times (0, 12, 24, and 48 h). Then, the cells were digested, washed with 75% alcohol, and pre-cooled at 4°C. The cells were slowly added to the cell precipitation and fixed overnight at 4°C. The fixed cells were washed, and the residual fixative was removed. PI/RNase staining buffer (0.5 mL) was added, followed by incubation at room temperature and under light for 15 min. Afterward, the cells were stored in ice in the dark and detected using flow cytometry within 1 h (Jung et al., 2020).

The autophagy- and apoptosis-related pathways were detected to reveal the mechanism of inhibition in colorectal carcinoma HT29 and HCT116 cells interfered by SDEA. After the cells were treated with the extract under different conditions, they were treated for protein extraction, which was performed using direct lysis with 1× RIPA cell lysis buffer (Beyotime Biotechnology, no. P0013K), and 1× protease and phosphatase inhibitor (Beyotime Biotechnology, cocktail for general use, MS-safe, 50×, no. P1048) were added immediately before use. The samples were boiled with added 5× SDS sample buffer for 10 min at 100°C and resolved using SDS–PAGE (Zhao et al., 2020).

The following antibodies were used: anti-ULK1 (1:1000, no. 8054S), Beclin 1 (1:1000, no. 3495S), SQSTM1/P62 (1:500, no. 8025S), LC3 A/B (1:1000, no. 4108S), p-Akt (1:1000, no. 4060S), cleaved-Caspase-9 (1:1000, no. 7237S), Caspase-3 (1:1000, no. 9665S), Bax (1:1000, no. 2774S), Bcl-2 (1:1000, no. 2870S), and Rabbit IgG (1:1000, no. 7074P2). All antibodies were purchased from Cell Signaling Technology.

For qRT-PCR, the total RNAs from the harvested cells were prepared using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. RNA concentrations were determined using a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA). The quality of the RNA samples was ensured to have an A260/A230 ratio > 1.7 and A260/A280 ratio between 1.8 and 2.0. Approximately 1 μg of total RNA was subjected to RT-PCR using Revert Aid First Strand complementary DNA (cDNA) synthesis kit (Thermo Scientific, no. K1622). The messenger RNA (mRNA) expression levels of Caspase-3, Bax, Bcl-2, LC3, P62, Beclin 1, and β-actin were measured using RT-PCR. The primers used in the PCR were as follows:

β-actin forward: 5′- CACCCAGCACAATGAAGATCAAGAT -3′, reverse: 5′-CCAGTTTTTAAATCCTGAGTCAAGC -3′, Caspase-3 forward: 5′-TGGAAGCGAATCAATGGACTCT -3′, reverse: 5′- TGAATGTTTCCCTGAGGTTTGC -3′, Bax forward: 5′- TTTTGCTTCAGGGTTTCATCCA -3′, reverse: 5′- TGCCACTCGGAAAAAGACCTC -3′, Bcl-2 forward: 5′- ATCGCCCTGTGGATGACTGA -3′, reverse: 5′- GAGACAGCCAGGAGAAATCAAAC -3′, LC3 forward: 5′- AGCAAAATCCCGGTGATCATC -3′, reverse: 5′- GCCGGATGATCTTGACCAACT -3′, P62 forward: 5′- CAGTCCCTACAGATGCCAGAAT -3′, reverse: 5′- GCCGCTCCGATGTCATAGTT -3′, Beclin 1 forward: 5′- GAGCCATTTATTGAAACTCCTCG -3′, and reverse: 5′ - CCCAGTGACCTTCAGTCTTCG -3′.

The annealing temperatures and the thermal cycles for β-actin, Caspase-3, Bax, Bcl-2, LC3, P62, and Beclin 1 were 60°C and 40 cycles. For each cDNA, the mRNA level of the target gene was normalized to β-actin mRNA level. The results were expressed as the ratio of the normalized mRNA level of the target gene in cells treated with the SDEA to that in the control group. The experiments were performed in triplicate.