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Repair of Radiation Damage
Published in Kedar N. Prasad, Handbook of RADIOBIOLOGY, 2020
A concentration of puromycin (1–6 μg/ml), which inhibits protein synthesis by about 60% of the controls, does not affect the repair process. One can argue that the remainder of protein synthesis is enough for the repair process. Because of high toxicity, it is not feasible to use a puromycin concentration that would inhibit protein synthesis 100%. Nevertheless, some authors10 have concluded that the repair of sublethal damage is independent of protein synthesis.
A Transcriptomic Analysis and shRNA Screen for Intracellular Ion Channels and Transporters Regulating Pigmentation
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Donald C. Koroma, Salwa Y. Hafez, Elena Oancea
For improved viral transduction efficiency, especially when viral titre was low, the antibiotic selection could start 48 h post viral transduction. Cell media was changed every 2–3 days with fresh media containing 1 μg/ml puromycin. The cells were kept under puromycin selection even after the cells that were not transduced were eliminated. This method allows for the successful selection of the MNT1 cells within 5–6 days post viral transduction.
In Vitro Cellular Aging and Immortalization
Published in George E. Milo, Bruce C. Casto, Charles F. Shuler, Transformation of Human Epithelial Cells: Molecular and Oncogenetic Mechanisms, 2017
Further evidence for this conclusion was obtained by preparing membrane-enriched fractions from senescent cells and adding them to young-cell cultures. These membrane-enriched fractions were very effective in inhibiting the initiation of DNA synthesis in young-cell cultures.31,32 Furthermore, proteins extracted from the membrane preparations and added directly to cultures of young cells were also effective in inhibiting the initiation of DNA synthesis.31 We next examined the role of protein synthesis in the production of this inhibitor by treating the cells with cyclohexamine or puromycin at concentrations which would inhibit protein synthesis by at least 90%. We found that a relatively short treatment, approximately 2 h, with cyclohexamine or puromycin was sufficient to eliminate the inhibitory activity from senescent cells. Upon removal of cyclohexamine from the culture and incubation of the cytoplasts in the absence of cyclohexamine, inhibitory activity was regained in about 4 h,31 indicating that cytoplasts were still active and able to synthesize DNA. Further, this indicated that the messenger RNA coding for the inhibitor was relatively long-lived.
Targeting GPC3high cancer-associated fibroblasts sensitizing the PD-1 blockage therapy in gastric cancer
Published in Annals of Medicine, 2023
Dinuo Li, Yu Wang, Ce Shi, Shuai Fu, Yi-Fei Sun, Chen Li
Mouse GC cell line (YTN16) was described previously. Mice L929 cell line (mouse fibroblast) purchased from the ATCC. Human skin fibroblasts and HGC-27 gastric cancer cell line were purchased from IMMOCELL company. These four cell lines were cultured in 90% Roswell Park Memorial Institute (RPMI) 1640 medium, with 10% foetal bovine serum (FBS) and 1% streptomycin and penicillin at 37 °C in a 5% CO2 cell incubator. Mouse and human GPC3-overexpressing (GPC3-OE) lentivirus was purchased from Invabio (Shanghai, China). GPC3-overexpressing lentivirus (1 × 108 TU/ml) and polybrene (8 μg/ml) were added into L929 cells and cultured for 72 h. The transfected cells were screened using puromycin (1 μg/ml). L929 cells successfully overexpressing GPC3 were detected by immunofluorescence. The control group was transfected by lentivirus with empty vector.
High SPINK1 Expression Predicts Poor Prognosis and Promotes Cell Proliferation and Metastasis of Hepatocellular Carcinoma
Published in Journal of Investigative Surgery, 2021
Kaijun Huang, Wenxuan Xie, Shutong Wang, Qiao Li, Xiangling Wei, Bin Chen, Yunpeng Hua, Shaoqiang Li, Baogang Peng, Shunli Shen
HCC cells (MHCC97H, MHCC97L, SMMC-7721, HepG2, Huh7, BEL7402, and HCCLM6, CRL-8024-PLC, SK-hep-1) and the normal human liver cell line LO2 were purchased from Cell Bank of Typical Culture Preservation Committee of Chinese Academy of Science (Shanghai, China). Lentiviral vector was used to up or down-regulate SPINK1 expression in HCC cells. Lentiviral vector carrying SPINK1 gene and SPINK1 RNAi was constructed in GeneChem (GeneChem, Shanghai, China). Lentivirus infection was performed following the instruction supplied by the company. HCC cells were infected for 16 h with a lentiviral vector in enhanced infection solution and subsequently placed in fresh medium. As the infected cells will express green fluorescence, the fluorescence of infected cells was measured 72 h after infected. As the infected cell lines were also puromycin resistant, they were screened with 0.5 μg/ml puromycin for 10 days after infection. Then, the expression of SPINK1 in stable infected cell lines will be examined by western blot.
Depletion of gut microbiota induces skeletal muscle atrophy by FXR-FGF15/19 signalling
Published in Annals of Medicine, 2021
Yixuan Qiu, Jiaming Yu, Yi Li, Fan Yang, Huiyuan Yu, Mengjuan Xue, Fan Zhang, Xin Jiang, Xueying Ji, Zhijun Bao
To further verify whether skeletal muscle atrophy in Abx mice was caused by decreased FGF15, another subset of mice were given the same antibiotic treatment with recombinant FGF19 injected subcutaneously for 4 weeks. The concentration of plasma FGF19 was undetectable in both vehicle- and PBS-treated mice (VP mice) and antibiotic- and PBS-treated mice (AP mice), but increased in antibiotic- and FGF19-treated mice (AF) mice (Figure S4). We used surface sensing of translation (SUnSET) method in vivo to estimate MPS rate in the three groups of mice [40–42]. Puromycin acts as a protein synthesis inhibitor that blocks protein translation through premature chain termination in the ribosome, so it becomes a biomarker to quantify MPS rate. Thirty minutes after puromycin injection, we detected puromycin in gastrocnemius muscle by western blotting using anti-puromycin antibody. Compared with VP mice, MPS rate, which was shown by puromycin-labelled peptides, was obviously decreased in AP mice, while MPS rate was significantly reversed in AF mice (Figure 5(E,F)). We also noted the phosphorylation of ERK1/2, p90RSK and RPS6 were reversed in AF mice compared with AP mice (Figure 5(G,H)). In line with these results, quadriceps, gastrocnemius and TA muscle mass and grip strength were increased in AF mice compared with AP mice (Figure 5(I,J)), but body weight was comparable among three groups (Figure S5). Collectively, we suggest that depleting gut microbiota disturbed gut microbiota-BA-FXR-FGF15/19-ERK signalling axis, which in turn decreased MPS, thereby inducing host skeletal muscle atrophy.