Puromycin Research Articles

Escherichia coli resistance mechanism AcrAB-TolC efflux pump interactions with commonly used antibiotics: a molecular dynamics study

While antibiotic resistance poses a threat from both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB), GNB pose a more imminent public health hazard globally. GNB are a threat to growing antibiotic resistance because of the complex makeup of the membrane. The AcrAB-TolC efflux pump is a known resistance mechanism of Escherichia coli (E. coli) cells. This study utilized molecular dynamics modeling to visualize some of the changes occurring at a molecular level when airborne bacteria are exposed to stress and antibiotics. This study was conducted to build upon previous experimental research showing that there is an increase in antibiotic resistance and efflux pump activity when exposed to aerosolization. AcrB and AcrAB-TolC proteins were simulated under standard and increased pressure to compare the effect of aerosolization on the binding to the three different antibiotics (puromycin (PUY), ampicillin (AMP) and sulfamethoxazole-trimethoprim (SXT)) to the AcrB binding site. Analysis such as root-mean-square deviation of atomic positions and root-mean-square fluctuation, the opening of TolC, and the significant molecular mechanics with generalized Born and surface area solvation (MM-GBSA) scores associated with specific ligands were recorded. Resistance in experimental data indicated a relationship between the docking scores and some ligand–protein interactions. Results showed that there was more flexibility in the proteins within simulations conducted under standard pressure for the AcrB protein and the full tripartite complex AcrAB-TolC, showing that increased pressure causes more rigidity. MM-GBSA scores, used to calculate the free energy of ligand–protein binding, did not show a significant change, but interestingly, the strongest MM-GBSA scores were for ligands that moved to another binding pocket and did not result in resistance or opening of the efflux pump. However, the ligand moved from the binding site and did not cause the opening of TolC to increase significantly, whereas PUY and AMP were bound to the binding site for the duration of all simulations. AMP ligands under increased pressure showed the largest change in opening of the TolC efflux pump and aligns with experimental data showing E. coli cells had the most resistance to AMP after aerosolization. These results, in addition to other real-time changes such as OM proteins and mutations of targets within the cell, could be used to delineate and mitigate antibiotic resistance mechanisms.

Low-frequency electromagnetic fields influence the expression of calcium metabolism related proteins in leukocytic cell lines

Our study aimed to verify the hypothesis concerning low-frequency magnetic fields (LF-MFs)-related changes in cell viability through the biomechanism(s) based on calcineurin (CaN)-mediated signaling pathways triggered via ROS-like molecules. For experiments, Mono Mac 6 and U937 leukocytic cell lines were chosen and exposed to various LF-MFs and/or puromycin (PMC). The protein expression level of key regulatory proteins of calcium metabolism was examined by Western Blot analysis. In turn, the reactive oxygen species (ROS) and cell viability parameters were evaluated by cytochrome C reduction assay and flow cytometry, respectively. The simultaneous action of applied MF and PMC influenced cell viability in a MF-dependent manner. The changes in cell viability were correlated with protein expression and ROS levels. It was verified experimentally that applied stress stimuli influence cell susceptibility to undergo cell death. Moreover, the evoked bioeffects might be recognized as specific to both types of leukocyte populations.

Kinetics of the thapsigargin-induced Ca2+ mobilisation: A quantitative analysis in the HEK-293 cell line.

Thapsigargin (TG) inhibits the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump and, when applied acutely, it initiates a Ca2+ mobilisation that begins with the loss of Ca2+ from the endoplasmic reticulum (ER) and culminates with store-operated Ca2+ entry (SOCE) from the extracellular space. Using the popular model cell line HEK-293, we quantified TG-induced changes in cytosolic and ER Ca2+ levels using FURA-2 and the FRET-based ER Ca2+ sensor D1ER, respectively. Our analysis predicts an ER Ca2+ leak of 5-6µM⋅s-1 for the typical basal ER Ca2+ level of 335-407µM in HEK-293 cells. The resulting cytosolic Ca2+ transients reached peak amplitudes of 0.6-1.0µM in the absence of external Ca2+ and were amplified by SOCE that amounted to 28-30nM⋅s-1 in 1mM external Ca2+. Additionally, cytosolic Ca2+ transients were shaped by a Ca2+ clearance of 10-13nM⋅s-1. Using puromycin (PURO), which enhances the ER Ca2+ leak, we show that TG-induced cytosolic Ca2+ transients are directly related to ER Ca2+ levels and to the ER Ca2+ leak. A one-compartment model incorporating ER Ca2+ leak and cytosolic Ca2+ clearance accounted satisfactorily for the basic features of TG-induced Ca2+ transients and underpinned the rule that an increase in amplitude associated with shortening of TG-induced cytosolic Ca2+ transients most likely reflects an increase in ER Ca2+ leak.

Application of Fucoidan in Caco-2 Model Establishment.

The Caco-2 model is a common cell model for material intestinal absorption in vitro, which usually takes 21 days to establish. Although some studies have shown that adding puromycin (PM) can shorten the model establishment period to 7 days, this still requires a long modeling time. Therefore, exploring a shorter modeling method can reduce the experimental costs and promote the development and application of the model. Fucoidan is an acidic polysaccharide with various biological activities. Our study showed that the transepithelial electrical resistance (TEER) value could reach 600 Ω·cm2 on the fourth day after the addition of fucoidan and puromycin, which met the applicable standards of the model (>500 Ω). Moreover, the alkaline phosphatase (AKP) activity, fluorescein sodium transmittance, and cell morphology of this model all met the requirements of model establishment. Fucoidan did not affect the absorption of macromolecular proteins and drugs. The results indicate that fucoidan can be applied to establish the Caco-2 model and can shorten the model establishment period to 5 days.

CRISPR Guide RNA Library Screens in Human Induced Pluripotent Stem Cells.

High-throughput CRISPR guide RNA (gRNA) library screen, that is, CRISPR/Cas9 screen, enables the unbiased identification of gene functions in a variety of biological processes. Typical pooled CRISPR/Cas9 screen couples a gRNA library and a guided Cas9 or dCas9 endonuclease to target specific gene loci, and then systematically uncover the causal link between candidate genes and observed cellular phenotypes via gRNA depletion or enrichment in screens. Here, we describe a detailed method of puromycin (PURO) concentration titration and lentiviral CRISPR gRNA library titration in Cas9 expressing monoclonal human iPSC line (Cas9+MNhiPSC) prior to performing the screens, conducting pooled CRISPR gRNA library screens in Cas9+MNhiPSC, genomic DNA extraction from the selected cell subpopulation and sequencing library preparation as well as next generation sequencing (NGS) to generate gRNA read counts. In CRISPR/Cas9 screen, we aim for 30% transduction efficiency (i.e., multiplicity of infection=0.3) to ensure most of infected cells receive only one gRNA. The principles in this method can be applied to CRISPR perturbation (knockout, activation, repression or base editing) screens with other CRISPR gRNA libraries across many other cell models and other species.

Cytoplasmic Expression of the ALS/FTD-Related Protein TDP-43 Decreases Global Translation Both in vitro and in vivo.

TDP-43 is a major component of cytoplasmic inclusions observed in neurodegenerative diseases like frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To further understand the role of TDP-43 in mRNA/protein metabolism and proteostasis, we used a combined approach with cellular and animal models overexpressing a cytoplasmic form of human TDP-43 (TDP-43-ΔNLS), recapitulating ALS/FTD features. We applied in HEK293 cells a method for labeling de novo translation, surface sensing of translation (SUnSET), based on puromycin (PURO) incorporation. While control cells displayed robust puromycilation, TDP-43-ΔNLS transfected cells exhibited reduced ongoing protein synthesis. Next, by using a transgenic mouse overexpressing cytoplasmic TDP-43 in the forebrain (TDP-43-ΔNLS mice) we assessed whether cytoplasmic TDP-43 regulates global translation in vivo. Polysome profiling of brain cortices from transgenic mice showed a shift toward non-polysomal fractions as compared to wild-type littermates, indicating a decrease in global translation. Lastly, cellular level translational assessment by SUNSET was performed in TDP-43-ΔNLS mice brain slices. Control mice slices incubated with PURO exhibited robust cytoplasmic PURO signal in layer 5 neurons from motor cortex, and normal nuclear TDP-43 staining. Neurons in TDP-43-ΔNLS mice slices incubated with PURO exhibited high cytoplasmic expression of TDP-43 and reduced puromycilation respect to control mice. These in vitro and in vivo results indicate that cytoplasmic TDP-43 decreases global translation and potentially cause functional/cytotoxic effects as observed in ALS/FTD. Our study provide in vivo evidence (by two independent and complementary methods) for a role of mislocalized TDP-43 in the regulation of global mRNA translation, with implications for TDP-43 proteinopathies.

Cytoplasmic expression of TDP‐43 decreases global translation both in vitro and in vivo

AbstractBackgroundTDP‐43 is a major component of cytoplasmic inclusions observed in neurodegenerative diseases like frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To further understand the role of this predominantly nuclear RNA‐binding protein in disease‐related mRNA/protein metabolism and proteostasis, we used a combined approach with cellular and animal models that overexpress a cytoplasmic form of human TDP‐43 (TDP‐43‐ΔNLS), recapitulating ALS/FTD features.MethodHEK293 cells were transfected with empty vector or hTDP‐43‐ΔNLS followed by a method for labeling de novo translation, surface sensing of translation (SUnSET), based on puromycin (PURO) incorporation. We also assessed whether cytoplasmic TDP‐43 regulates global translation in vivo using a transgenic TDP‐43‐ΔNLS mouse model. Brain cortices from control and transgenic mice were subjected to polysome profiling and western blotting. Lastly, cellular level analysis of ongoing protein synthesis was performed by applying the SUnSET method in acute thick brain slices from control and TDP‐43‐ΔNLS mice. Immunofluorescence for TDP‐43 and PURO was performed and signal intensity was measured.ResultOur in vitro approach showed that, while control cells displayed robust puromycilation, TDP‐43‐ΔNLS positive cells exhibited reduced ongoing protein synthesis when compared to control groups. In vivo results from polysome profiling revealed a shift towards light (non‐polysomal) fractions as compared to wild‐type littermates, indicating a decrease in global mRNA translation. Cellular level analysis of ongoing protein synthesis showed that slices from control mice incubated with PURO exhibited robust cytoplasmic PURO signal in layer 5 neurons from motor cortex, and normal nuclear TDP‐43 levels similar to control (‐PURO) neurons. Slices from TDP‐43‐ΔNLS mice incubated with PURO exhibited high cytoplasmic expression of TDP‐43 and a reduction in puromycilation respect to control mice. Quantification of neuronal staining intensity revealed a significant decrease in PURO incorporation in transgenic vs. control mice.Conclusion In vitro and in vivo results indicate that cytoplasmic TDP‐43 can decrease global translation and potentially cause functional/cytotoxic effects as observed in ALS/FTD. We provide in vivo evidence (by two independent and complementary methods) for a role of mislocalized TDP‐43 in the regulation of global protein synthesis, with implications for understanding the molecular changes underlying the clinic‐pathological manifestations of TDP‐43 proteinopathies.

The mechanism of human epidermal growth factor receptor 2 insertion mutation leading to lapatinib resistance

Objective The insertion mutation of human epidermal growth factor receptor 2 (ERBB2) (p. Pro780_Tyr781insGSP)leads to the emergence of rapatinib resistance through activating protein kinase B (Akt) signaling pathway, in order to explore the potential mechanism of the mutation. Methods Polymerase chain reaction (PCR), restriction enzyme and ligation were used to construct control group pHelper. CopGFP, wild group pHelper. GV358-ERBB2, mutant group pHelper. GV492-ERBB2 plasmid to transfect 293T cells, collect supernatant and package lentivirus. The titer was 108, transfected in MCF-7 cells with a fusion rate of 70%-80%, and the drug was sieved in 1 mg/L puromycin to construct a stable strain, by fluorescence microscopy and western blotting to detect transfection efficiency. The experiment was divided into three groups: ERBB2-NC group, ERBB2-WT group and ERBB2-MT group. The half maximal inhibitory concentration (IC50) of MCF-7 cells treated with lapatinib for 48 h was detected by cell proliferation cell counting kit-8 (CCK-8) method. The expression of ERBB2 related signaling pathway protein was detected by Western blot after lapatinib treatment. The effect of lapatinib on cell cycle and apoptosis was determined. Results The vector was successfully constructed and sequenced correctly. The wild type ERBB2 (ERBB2-WT) and mutant ERBB2 (ERBB2-MT) stable transformants were successfully constructed. The cell proliferation inhibition experiment showed that the IC50 in the ERBB2-MT group was (90.41±7.47) μmol/L, which was significantly higher than that of the ERBB2-WT group (43.94±9.84) μmol/L. Compared with the other two groups, the ERBB2-MT group showed significant resistance to lapatinib (F=55.530, P<0.01). Cell growth was promoted by increasing the phosphorylation level of Akt, The ERBB2-MT group (326.00±36.16) cell clones increased compared with the ERBB2-WT group (201.00±10.03) (t=7.478, P<0.05). And advanced Apoptosis (WT: 22.29±1.53, MT: 10.89±2.42, t=6.905, P<0.01) and enhanced cell proliferation (WT: 18.95±0.73, MT: 33.05±2.69, t=8.774, P<0.01)developed resistance to lapatinib. Conclusion The results indicate that the insertion mutation of ERBB2-MT is a cancer-promoting mutation that is resistant to the treatment of lapatinib by activating phosphorylation of the Akt signaling pathway. Key words: Breast cancer; Insertion mutation; Resistance

Construction and identification of recombinant lentiviral plasmid of receptor for advanced glycation end products

Objective To construct a recombinant lentiviral plasmid of receptor for advanced glycation end products (RAGE) and establish RAGE stable expressing hepatocellular carcinoma cell models in order to explore the pathogenesis of RAGE in the occurrence and development of hepatocellular carcinoma. Methods RAGE gene fragments were amplified by PCR and cloned into the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro. Then the recombinant lentiviral plasmid and packaging plasmid were used to package lentivirus in HEK293T cells by calcium phosphate transfection. Lentivirus supernatant was collected to infect hepatoma cells HepG2 and Huh7. After that cells were screened by puromycin. Finally, RAGE expression was detected by real-time PCR and Western blot. Results RAGE gene fragment was successfully amplified and inserted into lentiviral expression vector. After packaging the lentivirus-infected liver cancer cells, real-time quantitative PCR and Western blot results showed that the mRNA (P<0.05) and protein expression of RAGE cells stably expressing RAGE were significantly higher than that of the control cells. Conclusions The RAGE overexpressing hepatocarcinoma cell line was successfully constructed by using lentiviral expression vector, which laid a good foundation for further study on the pathogenesis of RAGE. Key words: Liver neoplasms; Lentiviral expression vector; Receptor for advanced glycation end products

Data for: Enabling HEK293 cells for antibiotic-free media bioprocessing through CRISPR/Cas9 gene editing

Abstract Unlike other cell lines used in industry, HEK293 have not well-established metabolic markers yet for the selection of positive transfected cells expressing recombinant proteins. Three different metabolic markers were explored and assessed in HEK293 to avoid antibiotic-based selection methods. Firstly, PAH gene (Phenylalanine Hydroxylase) was tested as selection marker in tyrosine free medium exploring the potential of the natural tyrosine auxotrophy found in HEK cells. Secondly, two HEK293 mutant cell lines defectives in Thymidylate Synthase (TYMS) and Glutamine Synthetase (GS) were generated using CRISPR/Cas9, and the knocked-out genes were used as selectable markers. Efficiency of selection and eGFP production were evaluated for each selection marker by FACS and compared with the performance based on puromycin selection. Selection efficiencies of 98.60% and 99.95% eGFP + cells were found for PAH and GS selection markers respectively, comparable to puromycin-based selection (99.86% eGFP+). TYMS-based selection system achieved 87.40% eGFP + cells in transfected cultures after one week. Regarding eGFP expression levels, PAH system showed the highest specific production (4,7 × 10 -4 rfu/cell) followed by GS and puromycin (about 2.0 × 10 -4 rfu/cell and 2.7 × 10 -4 rfu/cell, respectively). Inhibitors were then used to increase selection astringency to enhance productivity in selected pools. Eventually, the GS system was used to produce human interferon gamma (hINFγ) as an additional proof of concept. Growth kinetics, advantages and intrinsic limitations of each strategy are assessed and discussed.

Construction of lentivirus-mediated mito-OGG1 gene overexpression and OGG1 gene knockdown model in 661W cells

Objective To construct and authenticate the lentiviral-mediated overexpression of mouse mitochondrial-targeted-8-oxoguanine DNA-glycosylase 1 (mito-OGG1) gene and the lentiviral-mediated short hairpin RNA (shRNA) down-regulation of OGG1 gene expression model in 661W cells. Methods Constructed the target plasmids, including pLenti-EF1a-EGFP-P2A-Puro-CMV-Mito-OGG1-3Flag (pLenti-OGG1-GFP) and pLKD-CMV-G&PR-U6-shRNA (pLKD-shRNA).293T cells were used to obtain green fluorescent protein (GFP)-tagged lentiviral vector of interest by using a second generation lentivirus packaging system.293T cells were also used for the virus titer estimation.The multiplicity of infection (MOI) of 661W cells was detected by fluorescence microscopy.A stable transfected cell line was screened by puromycin.Immunofluorescence was used to detect transfection efficiency and cytochrome C oxidase Ⅳ(COXⅣ)-OGG1 co-localization.OGG1 mRNA and protein expression levels were detected by real-time qantitative PCR (QPCR) and Western blot. Results Sequencing results showed that the inserted sequence in the over-expression plasmid was consistent with the mouse OGG1 (NM_010957.4) gene sequence in the gene library.The original lentiviral titer after packaging and purification was between 2.0×107 to 6.0×107 TU/ml.The optimal MOI of 661W cells was 40, and puromycin with a concentration of 4.0 μg/ml successfully screened stable transformation.The transfection efficiency was up to 100% after screening.Immunofluorescence demonstrated successful co-localization of OGG1 and COXⅣ.The relative expression levels of OGG1 mRNA in the blank control group, OGG1 group, overexpression control group, shRNA group and low expression control group were 1.000±0.000, 41.581±12.206, 0.888±0.056, 0.239±0.121 and 1.081±0.083, and the relative expression levels of OGG1 protein were 1.029±0.153, 1.657±0.237, 0.752±0.143, 0.471±0.149 and 1.036±0.185, respectively, with significant differences between them (F=44.654, 30.948; both at P<0.05), the relative expression levels of OGG1 mRNA and protein in the OGG1 group were significantly higher than those in the overexpression control group, the relative expression levels of OGG1 mRNA and protein in the shRNA group were significantly lower than those in the lower expression control group, with significant differences between them (all at P<0.05). Conclusions The mito-OGG1 overexpression and OGG1 knockdown models of 661W cells are successfully constructed, which provides the preliminary experimental basis for follow-up study. Key words: Lentivirus vector; Overexpression; Short hairpin RNA; OGG1 gene; 661W cell; Mitochondrial targeting sequence

Construction of human ubiquitin specific peptidase 53 overexpressing cell line

Objective To construct an Esophageal cancer Eca109 cell line which can stably overexpressing the ubiquitin specific peptidase 53 (USP53). Methods The USP53 full-length gene was amplified by Polymerase Chain Reaction (PCR) using a plasmid containing the full-length sequence of USP53 purchased by GE. The constructed USP53 lentiviral plasmid and packaging plasmid were co-transfected into 293T cells to obtain concentrated lentiviral particles, and then the lentiviral particles were infected with Eca109 cells. The medium containing puromycin was selected for stable transfected cell lines. Results The mRNA expression of USP53 in overexpressing cell lines (45.070±0.942) was significantly higher than that in the control group (1.091±0.075) and the normal group (1.000±0.023). The difference was statistically significant (P<0.05), Western blotting showed that the expression of the tagged protein HA was significant expressed in the overexpressed cell line, and no tagged protein expression was observed in the control group and the normal group. Conclusion Human ubiquitin specific peptidase 53 overexpressing cell line was successfully constructed. Key words: Esophageal cancer; Eca109; Ubiquitin specific peptidase 53; Lentivirus

An Improved Photocaged Puromycin

An Improved Photocaged Puromycin

Immortalization of human umbilical vein endothelial cells and the effect of immortalization on its biological characteristics

Objective To establish the immortalized human umbilical vein endothelial cell lines and investigate the effect of immortalization on biological characteristics of proliferation, apoptosis and tumorigenesis of the cells. Methods Plasmid containing simian virus 40 large T antigen (SV40LT) gene or human telomerase reverse transcriptase (hTERT) gene was amplified and extracted; the target genes marked by puromycin resistance were packed by human embryo kidney cells (293T) to get the retroviral vectors and used to infect human umbilical vein endothelial cells (HUVECs); then the infected cells were screened by culture medium containing 2 μg/ml puromycin for two weeks; and polymerase chain reaction (PCR) was used to identify whether the target genes were successfully transferred into HUVECs; cell counting method, flow cytometry and tumorigenesis experiments in nude mice were used to detect the biological characteristics of proliferation, apoptosis and malignant transformation of immortalized cells respectively. Results The human umbilical vein endothelial cells were successfully immortalized by retrovirus-mediated SV40LT and hTERT; On the 5th to the 7th day, there were significant differences in cells number between the SV40LT immortalized group and HUVECs group (P<0.05) and between the hTERT immortalized group and HUVECs group (P<0.05). The apoptosis rate of HUVECs group was (11.2±0.9)%, the apoptosis rate of HUVECs immortalized by SV40LT was (3.4±0.4)%, the apoptosis rate of HUVECs immortalized by hTERT was (2.8±0.4)%. The apoptotic rates of the two immortalized group were lower than that of HUVECs group (both P<0.01). These immortalized HUVECs had no tumorigenicity. Conclusion Retrovirus-mediated SV40LT and hTERT can successfully immortalize HUVECs; the proliferative capacity of immortalized HUVECs is greatly enhanced; the apoptosis rate is decreased, and no malignant transformation occurs. Key words: Simian virus 40 large T antigen; Human telomerase reverse transcriptase; Retroviral vector; Human umbilical vein endothelial cell; Immortalization

Construction of lung tumor cells using RFP and Fluc dual-labeled system

Objective To establish a human lung cancer cell line that can stably express firefly luciferase (Fluc) and red fluorescent protein (RFP) gene so as to lay the foundation for the further establishment of a live-imaging lung cancer xenograft model in nude mice and therapeutic research. Methods The lentiviral vector pHBLV-Fluc-RFP containing luciferase and red fluorescent protein was constructed and then transfected into 293T cells for virus packaging. The complete virus was used to infect human lung cancer cell lines A549, H1975 and human B-cell lymphoma cell line K562. The stable cell lines were obtained by puromycin selection. Fluorescence microscopy and quantitative PCR were used to confirm the RFP and Fluc expression. Results The lentiviral vector pHBLV-Fluc-RFP was successfully constructed. Cancer cell line A549, H1975 and K562 stably expressing Fluc and RFP was obtained. The real-time quantitative PCR results showed that the relative expression of Fluc gene in the three stable infected cells was much higher than that in the corresponding wild-type cells, and the differences were statistically significant(all P<0.05). Conclusion The human lung cancer cell line A549, H1975 and human B-cell lymphoma cell line K562 with dual expression of RFP and Fluc were obtained, which provided a new model of fluorescent cells for in vivo imaging of immunodeficient mouse models such as nude mice. Key words: Red fluorescent protein; Firefly luciferase; Lentivirus; Lung cancer cell fluorescence model

Ku80 gene knock out by clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 enhances the sensitivity of HEK293T cells to doxorubicin

Objective 293T cells with Ku80 knockout were constructed using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein9 (CRISPR/Cas9) technique, and the effect of Ku80 deletion on DNA damage repair and doxorubicin resistance of 293T cells was investigated. Methods Firstly, a pair of DNA Oligos targeted Ku80 was designed and the single guide RNA (sgRNA) expression vectors were constructed using pSpCas9 (BB)-2A-Puro (PX459) vector. This vector was transfected into 293T cells. The effectiveness of the designed sgRNA was verified by site-directed Polymerase chain reaction (PCR) and T7E1 enzyme reaction. The 293T cell line was constructed using limited dilution method and puromycin screening. Single cell clones were selected and screened by Western blotting assay and sequencing. Furthermore, DNA damage was induced by doxorubicin treatment, and detected by DNA DSB marker phosphorylated Histone H2AX (γ-H2AX). Cell counting kit-8 (CCK-8) assay was carried out to detect the sensitivity of the cells to doxorubicin. Results The Ku80 gene knockout 293T cells were successfully constructed. After DNA damage induced by doxorubicin treatment and DNA repair, the positive rates of γ-H2AX staining in the control and Ku80 knockout groups were (30.67±2.49)% and (88.00±1.63)%, respectively (P=0.000), indicating that the DNA damage repair ability was significantly inhibited in Ku80 knockout 293T cells. Ku80 knockout increases the sensitivity of 293T cells to doxorubicin. The growth inhibition rates of Ku80 knockout group and control group were (53.99±1.29)% and (32.42±1.20)%, respectively (P=0.000). Conclusion The knockout of Ku80 in 293T cells inhibited the DNA damage repair ability and increased the sensitivity of cells to doxorubicin. Key words: Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 technique; 293T cell; Ku80 gene; Doxorubicin

Tacrolimus inhibits Puromycin induced injury of podocytes by stabilizing the expression and distribution of podocin

Objective To investigate the effect of Puromycin(PAN) and Tacrolimus(FK506) on the expre-ssion of podocin in podocytes, and discuss the mechanism of FK506 in improving proteinuria caused by the damage of glomerular podocytes. Methods Mouse podocyte were cultured and divided into 3 groups, which were control group, PAN group and FK506 group, respectively.The changes of each group after 8 hours, 24 hours and 48 hours of treatment were detected by using phase-contrast microscope, and the expression and distribution of protein and mRNA were tested by adopting Western blot, quantitative real-time PCR and immunofluorescence technique. Results Compared with the control group(8.54±0.25, 8.27±0.07, 7.45±0.13) at each time point(8 hours, 24 hours and 48 hours), the soma size of the PAN group(6.41±0.22, 4.96±0.09, 3.76±0.16) was reduced.But in the FK506 group(7.67±0.06, 6.62±0.04, 5.57±0.27), it was increased at each time point(8 hours, 24 hours and 48 hours) compared with the PAN group.The podocytic process and the intercellular connection disappeared, and the distribution was significantly scattered.The mRNA (0.87±0.15, 0.78±0.15, 0.58±0.12)and protein (0.82±0.02, 0.62±0.03, 0.50±0.02) expressions of podocin increased in FK506 group and mRNA(0.63±0.12, 0.56±0.01, 0.48±0.02), protein(0.71±0.03, 0.46±0.01, 0.34±0.02) were observably reduced in PAN group at each time point(8 hours, 24 hours and 48 hours) and showed abnormal distribution in PAN group, compared with the control group[podocin mRNA(1.22±0.15, 1.18±0.06, 0.87±0.30), protein(0.86±0.03, 0.87±0.03, 0.61±0.07)]. Conclusions PAN attenuates the mRNA and protein expression of podocin by damaging podocytes, inversely, while FK506 protects potocyte injury by stabilizing the mRNA and protein expression of podocin, which maybe involve inhibition of proteinuria.It can be used as a target for the study and treatment of kidney diseases. Key words: Podocytes; Tacrolimus; Puromycin; Podocin

Construction and identification of LSD1 overexpression and demethylation disfunction plasmids

Objective To construct rat lysine-specific histone demethylase 1(LSD1) overexpression plasmids and LSD1 demethylation fragment disfunction plasmids, and to evaluate their expression levels in HEK293T cells. Methods LSD1 fragments were amplified by PCR, and LSD1 demethylation disfunction fragments were amplified by overlap PCR. Rat-specific LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids were constructed and verified by agarose gel electrophoresis and sequencing. HEK293T cells were infected using the validated recombinant plasmids and blank vectors, and the stably expressed cells were selected by puromycin. The expression of LSD1 in the stably expressed HEK293T was detected by Western Blot and real-time quantitative PCR. Results The results of agarose gel electrophoresis and sequencing showed that LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids were successfully constructed. The Western Blot and real-time quantitative PCR results showed that compared with the blank group, the relative expression of LSD1 and mRNA in the LSD1 overexpression group and the LSD1 demethylation disfunction group were up-regulated, and the differences were statistically significant(all P<0.01). Conclusions The constructed LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids can achieve overexpression of LSD1 gene in HEK293T cells. This paper lays a foundation for further study of the relationship between LSD1 gene and related diseases and demethylation of LSD1. Key words: Lysine-specific histone demethylase 1; Demethylation; Recombinant plasmid

Construction of human ARF4 lentiviral vector and stable expression in ovarian cancer cell line SKOV3

Objective To establish ovarian cancer cell line SKVO3 that can stably express human ADP ribosylation factor-4 (ARF4). Methods A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was constructed and transfected into SKOV3 cells after verifying by DNA sequencing. The expression of ARF4 mRNA was verified by real-time quantitative PCR (qRT-PCR). Then, the recombinant plasmid with lentiviral packaging plasmids were co-transfected into SKOV3 cells for packaging. The recombinant lentiviral particles LV-ARF4 were collected and transfected into SKOV3 cells, and the stable transfected SKOV3 cell line was screening by culture with puromycin. The expression of ARF4 gene was detected by qRT-PCR and Western Blot. Results A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was successfully constructed. The vector could significantly up-regulate the expression of ARF4 mRNA in SKOV3 cells and be successfully packaged into recombinant lentiviral particles in HEK-293T cells. Compared with the control group, the relative expression level of ARF4 mRNA and protein in SKOV3 cells was significantly increased after the infection with LV-ARF4 (all P<0.001). Conclusion The recombinant plasmid pCDH-CMV-MCS-EF1-Puro/ARF4 and lentiviral vector LV-ARF4 were successfully constructed. The establishment of stably infected SKOV3 cell line with LV-ARF4 provides an experimental foundation for further studies on the biological function of ARF4 in ovarian cancer. Key words: Gene cloning; Lentiviral vector; ADP-ribosylation factor 4; SKOV3 cell line

Construction of a stable 4.1R gene knockout cell model in RAW264.7 cells using CRISPR/Cas9 technique

To construct a cell model of 4.1R gene knockout in murine macrophage cell line RAW264.7 using CRISPR/Cas9 technique. Three high?grade small?guide RNAs (sgRNAs) that could specifically identify 4.1R gene were synthesized and inserted into lentiCRISPRv2 plasmid. RAW264.7 cells were infected with sgRNA?Cas9 lentivirus from 293T cells transfected with the recombinant sgRNA?lentiCRISPRv2 plasmid, and the positive cells were screened using puromycin and the monoclonal cells were obtained. The expression of 4.1R protein in the monoclonal cells was measured by Western blotting, and the mutation site was confirmed by sequence analysis. Result A 4.1R gene knockout RAW264.7 cell line was obtained, which showed a 19?bp deletion mutation in the 4.1R gene sequence and obviously enhanced proliferation. We successfully constructed a 4.1R gene knockout macrophage cell line using CRISPR/Cas9 technique, which may facilitate further investigation of the function of 4.1R in macrophages.