Cell line and culture
HL60 and MOLM13 cell lines were cultured using RPMI 1640 medium (Gibco, USA), while the 293T cell line was cultured using DMEM medium. The culture media were supplemented with 10% fetal bovine serum (VivaCell, China) and 1% penicillin-streptomycin (Abiowell, China) to provide optimal conditions for cell growth and viability. The prepared medium was stored at 4°C to maintain its freshness and prevent contamination.The cells were cultivated in a sterile culture dish under controlled conditions. The cultivation environment included a temperature of 37 degrees Celsius and a carbon dioxide concentration of 5%, which mimicked the physiological conditions suitable for cell growth and proliferation. To ensure the integrity and purity of the cell cultures, all procedures were carried out in an aseptic environment within a dedicated cell culture facility. The cells were handled with utmost care to minimize any potential contamination. Regular checks for mycoplasma contamination were performed, and the results were consistently negative, affirming the high-quality of the cell lines used. During the experimental process, the appropriate size of culture dishes was selected for plating the cells. The cell density in each dish was carefully controlled by counting the cells using a specialized cell counting board, allowing for consistent and reproducible results across experiments. For drug treatments, the drugs were prepared using dimethyl sulfoxide (DMSO). To ensure minimal impact on the cells, the concentration of DMSO in the experiments was maintained below 0.01%. If the concentration exceeded this threshold, the control group received the corresponding volume of DMSO to maintain consistency in the experimental conditions. In cases where drugs were prepared using other solvents, the control group received the equivalent volume of the solvent to ensure accuracy and reliability in the experimental design. After plating, the culture environment was meticulously maintained to mirror the standard cell culture conditions. This ensured that the cells experienced a consistent and optimal growth environment, allowing for reliable and meaningful observations throughout the course of the experiments.
Animal models
Male nude mice aged 6–8 weeks were selected for our study. These mice were procured from Hunan SJA Laboratory Animal Co., Ltd(China), a reputable supplier known for providing high-quality laboratory animals. To maintain a controlled environment conducive to the mice's well-being, they were housed in a specific pathogen-free facility with appropriate temperature and humidity levels. A 12-hour light-dark cycle was implemented to mimic natural day-night conditions. To ensure the hygiene and safety of the mice, sterilized feed and water were provided throughout the experiment. The growth density of the mice adhered to established hygiene standards, and their growth status was closely monitored on a daily basis. This allowed us to promptly identify any signs of illness or distress and take appropriate action, ensuring the mice remained healthy and robust. During the tumor modeling process, confounding factors such as gender, age, and weight were carefully considered and balanced. Random assignment of mice into experimental groups was performed to minimize bias and ensure the reliability of our results. Subcutaneous tumor induction in mice was achieved using HL60 cells at a density of (2 × 106)/100 µl, suspended in phosphate-buffered saline. The injection site on the back of the mice was chosen to avoid hindering their movement or causing discomfort. After approximately one week, we confirmed the successful tumor formation in the mice, meeting the requirements of a tumor volume between 90-110mm3. Throughout the entire experimental process, we strictly adhered to ethical standards, ensuring that the tumor volume did not exceed 2000mm3, thereby preventing unnecessary harm to the animals. The drugs used in the animal experiments were sourced from Selleck, a reputable supplier known for providing high-quality compounds for research purposes. The preparation and administration of the drugs followed the specific standards provided by each drug's manufacturer, ensuring accuracy and consistency. For intraperitoneal injections, a stock solution was prepared at a concentration of 125 mg/ml using a mixture of 5% DMSO, 40% PEG300, 5% Tween80, and 50% ddH2O. The working solution for injection had a concentration of 6.25 mg/ml. In the case of oral administration, the solvent was prepared fresh and immediately before use, with the components (4% DMSO, 30% PEG300, 5% Tween80, and ddH2O) added sequentially to the product from left to right. To monitor the mice's health and response to the experimental treatments, their body weight was periodically measured and recorded as per the requirements of the study. This allowed us to evaluate any potential effects of the drugs on the mice's overall well-being. At the conclusion of the experiment, the mice were euthanized in a humane manner, following approved protocols. Organs and tissues relevant to the study were collected for further analysis and experimentation, contributing valuable insights to our research endeavor.
Immunoblotting
The cells were collected using a centrifugation speed of 2000r/min for 3 minutes to ensure the efficient collection of all cells. To remove any residual contaminants, the cells were washed once with pre-chilled PBS. Following this, the cells were centrifuged again to collect them, and then lysed using a prepared RIPA cell lysis buffer. The RIPA lysis buffer was supplemented with 1X proteinase inhibitor and phosphatase inhibitor to maintain the integrity of the proteins during the lysis process.To prevent protein degradation, the entire process of cell lysis was conducted on ice, maintaining a low temperature throughout. After cell lysis, the cells were sonicated to further disrupt the cellular structures and release the proteins of interest. The sonication conditions were carefully set as follows: 30 watts of power, 3 seconds of sonication followed by a 10-second rest period, repeated 5 times. This ensured effective disruption without excessive heat generation. To quantify the protein concentration in the cell lysates, the BCA assay kit (Thermo Fisher Scientific) was used. This assay allowed for accurate measurement of protein content based on the colorimetric detection of the reaction between proteins and the BCA reagent. Following the quantification, the cell lysates were mixed with the loading buffer and boiled for 10 minutes. This step denatured the proteins and prepared them for subsequent gel electrophoresis. For gel electrophoresis, each protein sample, approximately 30 micrograms in mass, was loaded onto an SDS-PAGE gel. The electrophoresis conditions were set initially at 70 volts for 30 minutes to allow for efficient sample migration through the stacking gel. Subsequently, the voltage was adjusted to 120 volts, with the time adjusted based on the desired resolution and separation of the target protein bands. After electrophoresis, the proteins were transferred from the SDS-PAGE gel to a PVDF membrane using a wet transfer system. The transfer conditions involved a constant current of 290mA for 90 minutes, ensuring efficient and complete transfer of the proteins to the membrane. To prevent non-specific binding and enhance the specificity of antibody binding, the PVDF membrane was blocked at 4 degrees Celsius for 2 hours. The blocking buffer consisted of 5% milk in TBST, which effectively blocked any remaining unoccupied binding sites on the membrane. Following the blocking step, the primary antibody, specific to the protein of interest, was incubated with the membrane overnight at 4 degrees Celsius. The antibody dilutions were prepared according to the recommendations provided by the antibody manufacturers, ensuring optimal antibody-antigen interaction and detection. The choice of primary antibodies relied on the specific target proteins being investigated in the experiment. These antibodies were carefully selected based on their known specificity and affinity towards the proteins of interest. The antibodies used were as follows:
ACTB: AC026, 1:5000 dilution, Abclonal.
BMAL1: 14040, 1:500 dilution, Cell Signaling Technology.
GPX4: 125066, 1:1000 dilution, Abcam.
P62: A19700, 1:1000 dilution, Abclonal.
HMGB1:3935, 1:3000, Cell Signaling Technology.
LC3A/B: 4108, 1:1000 dilution, Cell Signaling Technology.
The secondary antibody, diluted at a ratio of 1:10000, was carefully incubated with the membrane for 2 hours at 4 degrees Celsius to ensure specific binding to the primary antibody-antigen complex. Following this incubation, the membrane was subjected to a thorough washing process using 0.1% TBST, with each wash lasting 7 minutes and a total of 3 washes being performed. This rigorous washing step effectively removed any unbound or non-specifically bound antibodies, minimizing background noise and ensuring the specificity of signal detection.To further ensure the removal of residual washing buffer, the membrane was then washed once with TBS, providing a clean and neutral environment for subsequent chemiluminescent detection.For the final step of signal detection, either the highly sensitive SuperBright Subpico ECL (SUDGEN, catalog no. 31060) or the Super-sensitive ECL chemiluminescent substrate (Biosharp, catalog no. BL520B) was employed. These substrates offered exceptional sensitivity and signal strength, allowing for the precise visualization and detection of the target proteins on the membrane. The choice between the two substrates depended on the specific experimental requirements and the desired balance between sensitivity and signal duration.
Quantitative PCR analysis for gene expression
RNA extraction was conducted using proper personal protective equipment, including gloves, masks, and protective clothing, to minimize RNA degradation. Initially, cells were centrifuged at 1000 rpm for 3 minutes to form a cell pellet, after which the culture medium was removed. Subsequently, 1 ml of TRIzol was added to each 5–10×10^6 cells and thoroughly pipetted at room temperature for 10 minutes to lyse the cells. Following this, 200 µl of chloroform was added to each 1 ml of TRIzol, and the mixture was vigorously shaken by hand for 15 seconds before being incubated at room temperature for 2–3 minutes. The resulting mixture was then centrifuged at 12000 g for 15 minutes at 4 degrees Celsius, and the aqueous phase was carefully transferred to a new EP tube. Next, 600 µl of isopropanol was added, and the mixture was inverted several times before being placed at -20 degrees Celsius for 30 minutes. After centrifugation at 4 degrees Celsius for 10 minutes at 12000 rpm, the supernatant was discarded. Subsequently, 1 ml of 75% ethanol was used to wash the pellet twice, with the mixture being centrifuged at 4 degrees Celsius for 5 minutes at 7500 g each time. After ethanol washing, the supernatant was removed, and the pellet was air-dried at room temperature for 5–10 minutes. Finally, the RNA was dissolved in DEPC-treated water and stored at -80 degrees Celsius. For RNA integrity assessment, a 1.2% agarose gel was prepared, and the RNA samples were mixed with RNA loading buffer and loaded into the wells for electrophoresis. UV imaging revealed three distinct bands from top to bottom: 28s RNA, 18s RNA, and 5s RNA. The 28s RNA band should exhibit twice the intensity of the 18s RNA band and should not display any smearing, indicating high-quality RNA. Subsequently, RNA reverse transcription was performed using the Takara Reverse Transcription Kit (RR037A) according to the provided manual. Real-time quantitative PCR was then carried out using ChamQ Blue Universal SYBR qPCR Master Mix (Q312-02, Vazyme) in a 10 µl reaction system, incorporating 5 µl of qPCR Master Mix, 0.4 µl of each forward and reverse primer, and the remaining volume filled with DNA and ddH2O. The CFX96 Real-Time System (Bio-Rad) was utilized for the qPCR analysis, with actin serving as the internal reference for data analysis. To ensure experimental accuracy, each experiment was independently repeated three times.
Cell proliferation assays
Cell viability experiments were performed using the highly sensitive CCK-8 assay kit (GK3607-500T, DING GUO PROSPEROUS) for accurate detection and analysis. To establish the experimental setup, a 96-well plate was chosen as the plating platform, with each well initially seeded with a cell density of 5×10^4 cells. Prior to each experiment, precise cell counting was conducted using a specialized cell counting chamber, allowing for the calculation of the required volume of cell suspension based on the desired cell density.Once the cell suspension was prepared, it was thoroughly mixed and dispensed into individual labeled EP tubes. Simultaneously, the drugs or compounds to be tested were appropriately prepared and added to their respective labeled tubes. To ensure comprehensive exposure of the cells to the drugs, a meticulous mixing step was carried out, ensuring proper distribution and interaction between the cells and the test substances. Subsequently, 100 µl of the well-mixed cell suspension was carefully pipetted into each well of the 96-well plate, ensuring uniform distribution across all wells. The plated cells were then placed in a precisely controlled incubator, providing an optimal cultivation environment conducive to cell growth and drug treatment. Following the designated period of drug treatment, 10 µl of the CCK-8 detection reagent was added to each well with utmost care to prevent the formation of bubbles. This reagent is specifically designed to assess cell viability, providing reliable and quantitative results. After an incubation period of 1–2 hours to allow for proper reaction, the absorbance of the samples at 450nm was measured using a high-performance microplate reader.
MDA assay
To ensure the accurate and reliable detection of malondialdehyde (MDA), an important marker of oxidative stress, the MDA assay kit from Beyotime (S0131M) was utilized. First, cells were collected by centrifugation at 1000 rpm for 3 minutes and washed with PBS to remove any extraneous substances. For every 1×10^6 cells, 100 µl of ice-cold lysis buffer was added, and the cells were gently lysed on ice for 15–20 minutes. The lysate was then centrifuged at 12,000 g for 10 minutes, and 100 µl of the supernatant was collected for subsequent measurements.To establish a standard curve, the blank control and standard samples at concentrations of 0, 3.125, 6.25, 12.5, 25, and 50 µM were prepared. In the control, standard, and test samples, 200 µl of the working solution was added. After thorough mixing, the samples were heated at 100°C for 15 minutes to promote the reaction between MDA and the working solution. The samples were then allowed to cool in a water bath and centrifuged at 1000 g for 10 minutes at room temperature. Next, 200 µl of the supernatant was transferred to a 96-well plate, and absorbance was measured at 532 nm using a microplate reader.To account for variations in protein concentration across different samples, the protein content of the test samples was determined using the BCA method. The MDA content in the samples was then calculated as units per unit weight of protein. Standardization was performed using the control group to ensure consistency and accuracy across all experiments.
Lipid Peroxidation Probe -BDP 581/591 C11-
We used Lipid Peroxidation Probe -BDP 581/591 C11 for lipid peroxidation detection(Dojindo Laboratories, L267). The protocol we used was in accordance with the manufacturer's instructions, and involved following the specific steps outlined below: HL60 and MOLM13 cells were plated in a 12-well plate at a density of 6*10^5 cells/ml, with RSL3 at a concentration of 0.5 µM. After incubating in a cell culture incubator for 24 hours, the culture medium was discarded. The cells were then washed twice with HBSS by centrifuging at 2000 rpm for 3 minutes to collect them. After removing the HBSS, probe working solution was added and the cells were incubated in the cell culture incubator for 30 minutes. The working solution was subsequently removed, and the cells were rinsed twice with HBSS before adding 500 microliters of HBSS per well. Finally, the cells were observed and photographed using a fluorescence microscope.
Cell cycle analysis
To ensure accurate and precise analysis of cell cycle distribution, the following optimized protocol was implemented. First, the processed cells were collected, and to prepare a cell suspension, 300 µl of pre-chilled PBS was added. Subsequently, 900 µl of pre-chilled absolute ethanol was added dropwise while continuously shaking, resulting in a final ethanol concentration of 75%. The samples were then placed in a 4-degree environment for 24 hours to fix the cells.To proceed with cell cycle analysis, the fixed cell suspension was centrifuged at 1000 rpm for 5 minutes, and the supernatant was carefully discarded. Next, 1 ml of pre-chilled PBS was added to resuspend the cells, which were then subjected to another centrifugation step at 1000 rpm for 5 minutes to collect the cells. This washing step was repeated twice to remove any remaining ethanol. Following the removal of ethanol, 200 µl of propidium iodide (PI) working solution was added to the cell suspension, and the cells were stained for 30 minutes in the dark at 4°C. Subsequently, the cells were centrifuged at 1000 rpm for 5 minutes to obtain a cell pellet, which was washed once with PBS to remove excess PI staining. For flow cytometric analysis, the cells were transferred to a flow cytometry tube and analyzed using a flow cytometer. The PI fluorescence was excited by a 488 nm argon ion laser and detected through a 630 nm bandpass filter. To ensure reliable data acquisition, a total of 10,000 cells were collected based on forward scatter (FSC) and side scatter (SSC) scatter plots. Gate technology was employed to exclude adherent cells and debris, ensuring that only viable cells were included in the analysis.
Transmission electron microscope (TEM)
The experiment encompassed various pivotal stages, encompassing fixation, dehydration, infiltration, embedding, sectioning, staining, and imaging. In the fixation phase, cells were initially exposed to a 2.5% glutaraldehyde solution for a duration of 6–12 hours. Subsequently, they were transferred to a PBS buffer solution for 1–6 hours after removing the fixative, followed by fixation using a 1% osmium tetroxide solution for 1–2 hours. Dehydration involved a sequential series of exposure, starting with 30% ethanol for 10 minutes, followed by 50% ethanol for another 10 minutes. The cells underwent an immersion in a 70% uranyl acetate solution for either 3 hours or overnight. This was followed by exposure to 80% ethanol for 10 minutes, 95% ethanol for 15 minutes, and two rounds of 100% ethanol for 50 minutes each. Finally, the cells received a 30-minute treatment with epoxypropane. In the infiltration phase, the cells were immersed in a mixture of epoxy resin and epoxypropane in a 1:1 ratio for a duration of 1–2 hours. They were then soaked in pure epoxy resin for an additional 2–3 hours. For embedding, the cells were fully immersed in pure epoxy resin and incubated in a 40°C oven for 12 hours. This was followed by further incubation in a 60°C oven for 48 hours. Sectioning involved cutting ultra-thin slices from the embedded block, and these slices were retrieved using copper grids. The staining process consisted of electron staining utilizing lead and uranium, while imaging was accomplished by observing the samples through a JEOL JEM1400 transmission electron microscope and capturing images using a Morada G3 digital camera.
Lentivirus infection
To optimize gene overexpression and knockdown experiments, we sourced short hairpin RNAi molecules targeting BMAL1 and HMGB1, as well as the necessary overexpression plasmids, from Genechem CO.LTD (Shanghai, China). Lentiviruses were then prepared by transfecting 293T packaging cells in 10 cm dishes using Lipo8000 (No. C0533, Beyotime) as the transfection reagent. To ensure optimal virus packaging, the following steps were taken: On the first day, 293T cells were seeded in a 10 cm dish with a density controlled at around 70–80% for transfection the next day. On the second day, the constructed plasmids and virus packaging plasmids were co-transfected into 293T cells at a certain ratio. After 48 hours of transfection, the supernatant virus liquid was collected via centrifugation at 1500 rpm for 5 minutes and then filtered using a 0.45 µm filter. The resulting virus liquid can be used for subsequent experimental transfections and is stored at -80 degrees Celsius for long-term preservation.After preparing the lentiviruses, they were introduced into HL60 and MOLM13 cells for transduction. We evaluated multiple hairpin constructs to select those with the highest knockdown efficiency. To ensure optimal infection efficiency based on the different characteristics of virus-infected cells, appropriate volumes of the virus were used to infect the cells. After 24 hours of viral infection, the cells were replenished with fresh cell culture medium, and corresponding antibiotics were used for selection for three days based on the resistance carried by the transfected plasmid. The efficiency of plasmid transfection was determined through RNA or protein level detection.
Chromatin immunoprecipitation (ChIP)
The ChIP assay was conducted following the protocol outlined in the ChIP Kit (No. ab500, Abcam). The steps of the experiment could be divided into cell fixation and collection, cell lysis, immunoprecipitation, and DNA purification. The steps for the cell fixation experiment were as follows: firstly, 288 µL of 37% formaldehyde (final concentration of 1%) was slowly added along the wall of a 10 cm cell culture dish containing 10 mL of medium, and incubated at room temperature on a shaker for 10 minutes. Then, 1 mL of 1.25 M glycine was added to neutralize the unreacted formaldehyde, and the mixture was incubated at room temperature on a shaker for 5 minutes. After washing twice with pre-chilled PBS, the cells were collected using a cell scraper and transferred to a 1.5 mL enzyme-free centrifuge tube containing 1 mL of PBS. The tube was then centrifuged at 4°C, 1300g for 5 minutes using a refrigerated centrifuge, and the supernatant was discarded. The cell sonication conditions were set to obtain desired DNA fragments ranging from 200 to 1000 bp in size. Antibodies against BMAL1 (No.14020s, Cell Signaling Technology), IgG control (No.2729, Cell Signaling Technology), and Flag (F4049, Sigma) were used along with ChIP-Grade Protein G Magnetic Beads for the immunoprecipitation step. DNA purification was performed using DNA purification magnetic beads.The resulting ChIP-enriched chromatin was subjected to qPCR analysis. The final analysis results were expressed as a ratio relative to the input DNA. The specific primers used for PCR amplification are provided below:F-P1: GCTGACGAAAGAGACCTGCT; R-P1: CTTCGGAAGCCCTTCCCTC. F-P2༚CTCCTTTCCTCCCTCCCAGA; R-P2: GGACAGATCGGCTGTTGACT. F-P3༚GCAGTACCTTCCAGTGGGATT; R-P3: AAGCTTCCTCCCTTTAAATCATGT
Luciferase assay
On the first day, 293T cells were seeded into a 24-well plate, with a seeding density of 70–80% confluency targeted for transfection on the following day. On the second day, plasmid transfection was performed with a total of 200 ng of plasmid DNA per well. This included 1 ng of Renilla plasmid, and the transfection ratio of luciferase plasmid to the target plasmid was adjusted according to experimental requirements, with the remaining plasmid volume being made up with empty vector as needed. After transfection, the cells were treated with RSL3 and incubated for 24 hours. Fluorescence quantification was performed 48 hours after transfection completion. The cells were collected, and a Dual-Luciferase reporter system (Promega, USA) was used for fluorescence detection. The 24-well plate was washed once with PBS. After adding 100uL of PLB dilution buffer to each well, it was gently shaken on a shaker at low speed for at least 30 minutes. The lysate was then aspirated into an EP tube and centrifuged at 15000 rpm, 4℃, for 30–60 seconds. One EP tube was taken, and 20uL of LAR II and 4uL of cell lysate supernatant were added. After thorough mixing, the first luminescence reading (experimental value) was measured. Subsequently, 20uL of S&G mixing solution was added to the EP tube and mixed thoroughly, followed by measurement of the second luminescence reading (internal reference value). Each treatment had three replicate wells, and three independent experiments were performed.
Statistical analysis.
A p-value less than 0.05 was considered to indicate significant differences. Student unpaired t-test were used for comparison between two groups, and, one-way or two-way ANOVA was used for comparison among multiple groups. Before conducting any significance tests, all data underwent tests for data applicability. Data analysis was performed using GraphPad Prism software.