For obtaining Mef cells, the 8-wk-old CD-1 pregnant mice with E13.5 fetuses from Charles River Laboratories were used in this study. Animal studies were approved by the Institutional Animal Care and Use Committee of Peking University (approval No. Urban-HuJY-1). The mice were anesthetized by sodium pentobarbital via intramuscular injection and euthanized via cervical dislocation. The embryos were carefully stripped, and the bodies were fully cut into pieces, and the pieces were mixed with Trypsin-EDTA (T4090; Sigma). After digestion for 5 min at 37°C and standing for 1 min, the supernatant was removed, and FBS (10091148; Thermo Fisher Scientific) was added to the supernatant. The digestion was repeated twice. After mixing the supernatants and filtration by cell strainer (251100; Abcam), the obtained cells were placed on cell culture dishes at the density of and cultured with DMEM (C11965500BT; Thermo Fisher Scientific) and 10% FBS (10091148; Thermo Fisher Scientific). When the cells reached , 0.25% Trypsin-EDTA was used for digestion and expanded culture. Cells were then incubated in an atmosphere of 5% at 37°C and were passaged at a split ratio of 1:3 to 1:5 using 0.05% Trypsin-EDTA (T4090; Sigma).
C11965500bt
Manufactured by Thermo Fisher Scientific
Sourced in United States
The C11965500BT is a laboratory centrifuge designed for general benchtop use. It has a maximum speed of 6,000 RPM and a maximum capacity of 6 x 50 mL tubes. The centrifuge features a brushless motor and a microprocessor-controlled digital display for setting time, speed, and other parameters.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
T4090, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Btx ecm 830, by Harvard Apparatus (1 mentions)
A3160802, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Lv sh nc, by GenePharma (1 mentions)
A3840001, by Thermo Fisher Scientific (1 mentions)
Anti α actinin antibody, by Merck Group (1 mentions)
B5002, by Merck Group (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Rwpe 1, by iCell Bioscience (1 mentions)
Htb 81, by American Type Culture Collection (1 mentions)
Sodium lactate, by Merck Group (1 mentions)
Crl 1772, by Thermo Fisher Scientific (1 mentions)
C0222, by Beyotime (1 mentions)
A019 2 1, by Nanjing Jiancheng (1 mentions)
12 protocols using c11965500bt
1
Isolation and Culture of Murine Embryonic Fibroblasts
2
Transfection and Electroporation of N2a Cells
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The N2a cell line was a gift from Dr. Yichang Jia (Tsinghua University), and was maintained in normal DMEM (C11965500BT, Thermo Fisher) containing 15% fetal bovine serum (AB-FBS-0500s, ABW) and 100 U/ml penicillin-streptomycin (15140148, Thermo Fisher). The N2a cells were seeded in 48-well plate in ~70% confluence at 24 hr before the transfection. The plasmids were transfected with Lipofectamine 2000 (11668019, Thermo Fisher) following manufacturer's instructions. Normally, N1-EGFP was co-transfected to guarantee successful transfection.
Cochlear culture and injectoporation were as previously described 21 (link) . In this study, the organ of Corti was dissected out from mouse pups and was attached on the inner side of a 35-mm culture dish lid cultured with 2ml DMEM/F12 medium (21041-025, Thermo Fisher) with ampicillin (1.5 µg/ml, GG101, TransGen Biotech) addition. For electroporation, plasmids (1 µg/µl in 1x HBSS) were injected into gaps between hair cells via a glass electrode with an open tip of 2 µm diameter. A series of 3 electrical pulses (80-V amplitude, 20-ms duration, 1-s interval) were immediately applied by an electroporator (BTX ECM830, Harvard Apparatus). After electroporation, the tissues were cultured in a humid incubator (37°C, 5% CO2).
Cochlear culture and injectoporation were as previously described 21 (link) . In this study, the organ of Corti was dissected out from mouse pups and was attached on the inner side of a 35-mm culture dish lid cultured with 2ml DMEM/F12 medium (21041-025, Thermo Fisher) with ampicillin (1.5 µg/ml, GG101, TransGen Biotech) addition. For electroporation, plasmids (1 µg/µl in 1x HBSS) were injected into gaps between hair cells via a glass electrode with an open tip of 2 µm diameter. A series of 3 electrical pulses (80-V amplitude, 20-ms duration, 1-s interval) were immediately applied by an electroporator (BTX ECM830, Harvard Apparatus). After electroporation, the tissues were cultured in a humid incubator (37°C, 5% CO2).
3
SNRPB Knockdown in Cell Culture
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Cells were grown at 37° C with 5% CO2 using DMEM (Gibco, C11965500BT) with 10% FBS (Gibco, A3160802) and 1% Penicillin/Streptomycin (Gibco, 15140122). SNRPB knockdown lentivirus (Lv-shSNRPB1/Lv-shSNRPB2) and the negative control (Lv-shNC) were purchased from GenePharma Co. Ltd. Cells were transfected and screened using puromycin (Sigma-Aldrich, P9620) for 4 days. Target sequences were listed below, Lv-shSNRPB1: ccACAAGGAAGAGGTACTGTT, Lv-shSNRPB2: ccTCCCAAAGATACTGGTATT, Lv-shNC: TTCTCCGAACGTGTCACGT.
4
Cardiomyocyte Hypertrophy Induction Protocol
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Mouse hearts were subjected to cardiomyocyte isolation based on a protocol described previously [19 (link), 20 (link)]. The mouse cardiomyocytes were cultured in DMEM (Gibco, #C11965500BT) supplemented with 10% FBS (Gibco, #A3840001). BrdU (Sigma, #B5002) was used to repress fibroblast proliferation. Before hypertrophy induction, the cardiomyocytes were cultured in FBS-free DMEM for 24 h. Cardiomyocyte hypertrophy was induced by treatment with Angiotensin II (Ang II; MCE, #HY-13948) for 48 h in FBS-free DMEM. For KLK11 overexpression or knockdown, cardiomyocytes were infected with adenovirus or transfected with siRNA 24 h before serum starvation and Ang II treatment. Cardiomyocytes were stained with an anti-a-actinin antibody (Sigma, #A7188) for size analysis, and cell size was measured with Image J software. Fifty cardiomyocytes of each group were analyzed, and the mean cardiomyocyte size of independent experiments was used to further statistical analysis. For mTOR inhibition, 100 nM rapamycin (MCE, #HY-10219) was used.
5
Modulation of PAS usage in HEK293T cells
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The HEK293T cells were obtained from the ATCC and cultured in DMEM (Gibco, C11965500BT) with 10% FBS (Gibco, 10270106) and 1% P/S (Gibco, 15070063) with 5% CO2 at 37°C.
For perturbation of PAS usage, HEK293T cells were seeded in 24-well plate and transfected at 70% cell confluence. For transfection, 3 μl PEI (1 μg/μl, Polysciences) was applied with 1 μg total plasmid (0.5 μg dCas13 and 0.5 μg individual gRNA). Forty-eight hours after transfection, cells were harvest for the following assays.
For perturbation of PAS usage, HEK293T cells were seeded in 24-well plate and transfected at 70% cell confluence. For transfection, 3 μl PEI (1 μg/μl, Polysciences) was applied with 1 μg total plasmid (0.5 μg dCas13 and 0.5 μg individual gRNA). Forty-eight hours after transfection, cells were harvest for the following assays.
6
Evaluating miR-155-5p Regulation of SGK3 3' UTR
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The partial wild-type 3′ UTR of Sgk3 (NM_001376043.1) was amplified using PCR and subsequently mutated using site-directed mutagenesis. After being sequenced, the correct recombinant vector was double-digested with restriction enzymes Mlu I and Hind III to release the sequence of interest, which was inserted into the empty luciferase reporting vector (pMIR-REPORTTM Luciferase, 6470bp, Ambion Life Technologies, Carlsbad, CA, USA). HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (C11965500BT, Gibco) containing 10% fetal bovine serum (FBS) (10270-106, Gibco) for 24 h. Then, 0.2 µg Sgk3 WT or MUT recombinant plasmid (Firefly luciferase vector) and 0.01 µg pRL-CMV (Ranilla luciferase vector) were co-transfected with 25 μL of either miR-155-5p mimics or nontargeting control mimics (100 nM) using liposome (Lipofectamine® 2000, Invitrogen Life Technologies). Forty-eight hours later, Firefly and Renilla luciferase activities were quantified using the Dual-Luciferase Reporter Assay System (E1960, Promega, Madison, WI, USA) and normalized to the control group.
7
Prostate Cancer Cell Line Culture
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The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin. The normal human prostate epithelial cell line RWPE-1 was obtained from iCell (Shanghai, China) and grown in keratinocyte serum free medium (SFM; iCell-0019, iCell). Cells were incubated in a humidified, 5% CO2 atmosphere at 37°C. The detailed information of reagents used in this study is shown in the Supplementary Table 1
8
C2C12 Myoblast Differentiation with Lactate
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The C2C12 mouse myoblast cell line (ATCC®, CRL-1772™) was cultured in a growth medium (GM) of Dulbecco’s modified Eagle’s medium (DMEM, C11965500BT, Gibco, USA) with 10% fetal bovine serum (10099-141, Gibco, USA) and 1% penicillin–streptomycin (C0222, Beyotime, China) at 37 °C with 5% CO2. When cell confluence reached 80–90%, the GM was replaced with a differentiation medium (DM) of DMEM containing 2% horse serum (SH30074.02, Hyclone, USA) and 1% penicillin–streptomycin. Cells were fed with fresh DM every other day, and cultures were maintained for 5 d. In some experiments, cells were continuously incubated for 5 d in the DM with sodium lactate (0, 10, or 20 mM) purchased from Sigma-Aldrich. The concentration of lactate was set at 0–20 mM based on previous studies using skeletal muscle cells, and this range corresponds to the physiological range of blood lactate level in humans after an exercise [17 (link)]. Pyruvate or lactate was assessed using a lactic-acid assay kit or pyruvate assay kit (A019-2-1, A081-1-1, Nanjing Jiancheng Bio, China).
9
PANC-1 Cell Culture Conditions
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Cell lines of PANC-1 was purchased from Procell Life Science (Wuhan, China) and cultured in the Dulbecco’s Modified Eagle Medium (DMEM; C11965500BT; Gibco) culture with 10% fetal bovine serum (FBS; cat no.2127186; VivaCell) and 1% penicillin and streptomycin (cat no.15140122; Gibco). The cell was maintained at 37℃ in a humidified 5% CO2 environment.
10
Culturing Human Liver Cancer Cells
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Human HCC (MHCC97H) cell line was purchased from Shanghai Yiyan Biological Technology Co., Ltd. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (C11965500BT; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FSP500; Shanghai ExCell Biology, Inc.,) and 1% penicillin/streptomycin.
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