br Flow cytometry analysis br Translocation was detected bas
2.7. Flow cytometry analysis
Translocation was detected based on the binding of allophyco-cyanin-conjugated Annexin V. The A 83-01 were harvested using Trypsin-EDTA, without causing any harm to the cells. The harvested cells were then mixed with a mixture of Annexin V, phosphatidylinositol reagent, and 1X binding buﬀer from Annexin V-FITC Apoptosis Detection Kit (BioBud, Seoul, Republic of Korea). After 30 min at RT without light, the cells were analyzed using flow cytometry.
2.8. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
TUNEL assay was used to detect apoptosis and was performed using In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany). The cells were first fixed with 3.7% formaldehyde and then permeabilized with 0.5% Triton X-100 for 15 min at RT. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen, CA, USA) for 20 min at 37 °C in 5% CO2 incubator. After washing the cells three times with TBST, the mixture from the assay kit was used to stain dead cells. The cells were then incubated for 1 h at 37 °C in a 5% CO2 incubator. These cells were observed by confocal microscopy.
2.9. RNA interference assay
HCT116 and DLD-1 cells were transfected with Noxa siRNA and CHOP siRNA purchased from Santa Cruz Biotechnology (Santa Cruz).
Prior to siRNA treatment, the opti-minimum essential medium (Opti-MEM; Gibco, Life Technologies, LA, USA) was added to the dish and incubated at 37 °C for 30 min to warm the cells. Then, the mixture containing siRNA, Opti-MEM, and Lipofectamine RNAiMAX (Invitrogen) was incubated for 30 min at RT. The mixture was then added to the warmed cells and incubated for 18 h at 37 °C in a 5% CO2 incubator. The cells were then treated with CBD for subsequent ana-lysis.
2.10. RNA extraction for reverse-transcriptase polymerase chain reaction (RT-PCR) and quantitative real time PCR (qRT-PCR)
TRIzol reagent (TRI reagent, Molecular Research Center, OH, USA) was used for extraction of RNA, according to the manufacturer's in-structions. After lysing the cells, cold chloroform was added and the solution was kept on ice for 30 min and centrifuged for 10 min at 12,000 rpm (14,240×g) at 4 °C. Then, the supernatant was transferred to another tube. Cold isopropanol was added to precipitate the RNA. After gentle mixing, the sample was incubated for 20 min on ice and centrifuged for 10 min at 12,000 rpm (14,240×g). The RNA pellet was then washed with 75% EtOH (diluted with diethyl pyrocarbonate, DEPC). After the pellet was air-dried, it was dissolved in DEPC water and concentrated. RT-PCR assay was performed using the RT-PCR kit (Life Technologies). qRT-PCR was used to determine mRNA levels of Noxa, and GAPDH was used as control. Taqman Probes were purchased from Thermo Fisher Scientific for Noxa and GAPDH.
2.11. Immunocytochemistry assay
HCT116 and DLD-1 cells were seeded on a glass coverslip. The treated cells were fixed with 3.7% formaldehyde for 15 min at RT, and then, 0.5% Triton X-100 was used for permeabilization under same conditions. The cells were blocked with 3% BSA for 1 h, and then in-cubated overnight with primary antibodies, both at 4 °C. DAPI was used to stain the nuclei of cells, followed by the specific secondary antibodies (17 min at 4 °C).
2′,7′-Dichlorodihydrofluorescein Diacetate (DCF-DA, Invitrogen) reagent was used to detect total cell ROS, while mitochondrial super-oxide indicator (MitoSOX, Invitrogen) reagent was used to detect mi-tochondrial ROS. Tetramethylrhodamine, ethyl ester, perchlorate (TMRE, Thermo Fisher Scientific) was used to measure the mitochon-drial transmembrane potential (MMP). For fluorescence staining, the cells, seeded on a coverslip, were fixed and permeabilized with 3.7% formaldehyde and 0.5% Triton X-100 for 15 min at RT. The nuclei were stained with DAPI, followed by treatment with the ROS detection re-agents, namely, DCF-DA and MitoSOX, for 10 min at 37 °C. The treated cells were then observed by confocal microscopy. For flow cytometry analysis, the seeded cells were treated with MitoSOX and TMRE, 30 min, 1 h and 6 h after CBD treatment and incubated for 30 min at 37 °C.
2.13. Chromatin immunoprecipitation (ChIP) assay
For the ChIP assay, 1% formaldehyde was directly added to HCT116 and DLD-1 cells (1.5 × 106 cells per dish) to facilitate the cross-linking of proteins to DNA. After the incubation for 15 min at 37 °C, the cells were harvested with a cell scraper and then centrifuged. Pellets were resuspended in SDS lysis buﬀer containing phenylmethane sulfonyl fluoride (PMSF) and a protease inhibitor and then incubated on ice for 10 min. Cell lysate was sonicated in various conditions to obtain 200 and 1000 base-pair DNA fragments. Lysates were immunoprecipitated with anti-CHOP, anti-ATF3 and anti-ATF4 overnight at 4 °C. The pro-tein-DNA complex was incubated with protein A/salmon sperm DNA