Reagent and antibody
|
Reagent type |
Reagent name |
Company |
|
Antibody |
ACTIN |
Proteintech |
|
|
KDELR3 |
Proteintech |
|
Cell proliferation test reagent |
EDU |
Ribobio |
|
CCK8 |
Selleck |
|
|
Inhibitor |
Sorafenib |
Selleck |
|
Z-VAD-FMK |
Selleck |
|
|
Z-YVAD-FMK |
Selleck |
|
|
Necrostatin-1 |
Selleck |
|
|
Ferrostatin-1 |
Selleck |
|
|
Liproxstatin-1 |
Selleck |
|
|
RNA extraction and reverse transcription |
RNA extraction kit |
Shanghai Feijie Bio-Technology |
|
RNAisoPlus |
TaKaRa |
|
|
RTaseM-MLV |
TaKaRa |
|
|
oligo(dt) |
TaKaRa |
|
|
dNTP |
TaKaRa |
|
|
TBGreen |
TaKaRa |
|
|
Protein extraction and WB
|
10×Cell Lysis Buffer |
CST |
|
5×Protein loading |
Sangon |
|
|
agarose |
Sigma |
|
|
protease inhibitor |
MILLIPORE |
|
|
Other reagents |
FBS FCS |
Gibco |
|
DMEM culture media |
Gibco |
|
|
EDTA-pancreatin |
Gibco |
|
|
DMSO |
Sangon |
|
|
DEPC |
Sangon |
Main instruments and software
|
Instrument type |
Instrument name |
Company |
|
Cell culture |
cell incubator |
Thermo |
|
qPCR |
Quantstudio 7 Flex |
Thermo |
|
Microscope |
optical microscope |
Thermo |
|
Western Blot |
electrophoresis bath |
Biorad |
|
rapid transmembrane instrument |
Genscript |
|
|
toning system |
Tanon |
|
|
FCM |
Foteasa |
BD |
|
Sony 7000 |
Sony |
|
|
Other
|
centrifuge |
Eppendorf |
|
pipette |
Eppendorf |
|
|
dry thermostat |
Eppendorf |
METHODS
Mice
The BALB/c nude mice were purchased from Joint Ventures Sipper BK Experimental Animals (Shanghai, China). Mice were kept and bred in specific-pathogen-free conditions. All animal experiments were undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals with approval of the Scientific Investigation Board of Naval Medical University, Shanghai.
Subcutaneous tumor-bearing model of nude mice: The BALB/c nude mice (6-8 weeks old, 18–20g) were used for subcutaneous tumor xenograft models. 1 × 107 WT or KDELR3 KO HepG2 cells were injected subcutaneously into right flank of each mouse. The tumor was monitored and sorafenib was given when the tumor volume reached 150-200mm3(30mg/kg, oral gavage once every two days). Tumor size was monitored by calipers for every 5 days, and the tumor volumes were calculated using the equation (larger diameter) × (smaller diameter)2/2.
Cell culture
Cell thawing: Removed the cells from the ultra-low temperature refrigerator into 37 °C water. After the suspension was completely frozen, the cells were centrifuged at 1000rpm for 5 minutes and resuspended with fresh medium.
Cell passage: Cells were washed with 1ml of sterile PBS firstly. Appropriate amount of trypsin was added for digestion and cell suspension was centrifuged at 1000rpm for 5 min. After centrifugation, discarding cell supernatant and resuspending cells in fresh medium. Cells we cultured in a sterile chamber with 5% carbon dioxide at a constant temperature of 37°C.
Construction of HepG2 sorafenib-resistant cell line
Sorafenib-resistant cell line were constructed using the hepatocellular carcinoma cell line HepG2. Cells were treated with 2.5 μM sorafenib as the initial concentration, each increase was 0.5μM when the cells tolerated. The final concentration is 7μM and the cells can proliferate normally.
Cell counting kit-8 (CCK-8)
8^103 cells per 100μl DMEM were seeded in each well of 96-well flat-bottomed plate avoiding one circle of the periphery. Cell viability was measured 24 hours after sorafenib treatment.Cells were treated with10 μl CCK8 solution per 100μl DMEM and incubated for 2 hours before measuring the absorptive value at 450nm.
Quantitative Real-Time PCR
Total RNA was extracted from cultured cells with TRIzol reagent according to the manufacturer’s instructions. RNA was reverse transcribed with Oligo (dT) primer for mRNA into cDNA with M-MLV Reverse Transcriptase (TaKaRa). RNA expression was quantified by real-time PCR with SYBR Premix ExTaq Kit (TaKaRa) and normalized by the level of β-actin. A 2−ΔΔCt method was used to calculate relative expression changes. Amplification of cDNA was performed on the ABI-Quant Studio 7 Flex.
RNA interference
The siRNAs for KDELR3 was purchased from Genepharma (Shanghai, China).The HepG2 cells were seeded into 24-well plates at a concentration of 3^104 cells per well. The transfections were performed using INTERFERin transfection reagent (Polyplus) according to the manufacturer's instructions.
5-Ethynyl-2'-deoxyuridine (Edu) assay
Cell proliferation capacity was determined by the EDU assay.10μM EDU solutions were added to the cells for 2 hours before harvest.Adding paraformaldehyde for 15 min and discarding supernatant after adding 2 mg/ml glycine,then the cells were incubated with 0.5%Triton-100 for 10 min and stained with Apollo for 10 min, the dye was washed by adding 0.5%Triton-100 and cells were resuspended in PBS detected by flow cytometry.
Scratch assay
HepG2 cells were seeded in 24-well cell plates. When cells are fully fused,a smooth scratch was created with a sterile 10 μL pipette tip positioned perpendicular to the plate. Cells were cultured for 24hrs and images were acquired to calculate the scratch healing rate.
Flow cytometer
Cell death:HepG2 cells were seeded in 24-well plates and performed RNA interference with the above step. After treated with 10 μg/ml sorafenib for 24 hrs,cells were harvest and resuspended in 500 μl PBS.
Then,cells were incubated with 30 μl Binding buffer, mixed with 0.2 μl Annexin V-APC and 0.1ul PI for 15 min in dark. After the incubation, 200 μl binding buffer was added and transferred to a flow tube for detection.
BODIPY™ 581/591 C11: The cells were seeded to 24-well flat-bottomed plate as above described. After treated with sorafenib, cells were cultured with 2μM BODIPY™ 581/591 C11 (Sigma) for 30min at 37°C and then harvested to detect.
CRISPR/Cas9:
Firstly,KDELR3-Cas9 plasmids (sgRNA sequence:GGTGAAGACGAGAGCAAAC) were constructed and transfected into HepG2 cells. The successfully transfected cells were sorted and continued to be cultured as monoclones for 2 weeks. And then all monoclones were identified for successful knockout. In summary, through the above steps, we used CRISPR/Cas9 technology to construct HepG2 KDELR3 knockout cell lines.
Lipid nanoparticle (LNP) delivery system：
Lipid nanoparticles has been identified as good non-viral drug delivery materials. The preparation of LNP came from the help of a post-doctor in the our research team. KDELR3 siRNA were encapsulated into LNPs and evaluated for their size distribution and encapsulation efficiency.
