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All Journal HAYATI Journal of Biosciences Jurnal Teknosains Indonesian Journal of Cancer Chemoprevention
Prasetyaningrum, Pekik Wiji
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Civil Engineering, Building, Construction & Architecture Earth & Planetary Sciences Industrial & Manufacturing Engineering Mechanical Engineering Medicine & Pharmacology

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Optimized condition for pei-based transient transfection of lifeact-gfp/nls-mcherry expressing plasmid used as cell barcode for syncytia live cell imaging Kumara, Dennaya; Harsan, Hayfa Salsabila; Novianti, Metta; Lestari, Dinda; Septisetyani, Endah Puji; Prasetyaningrum, Pekik Wiji; Paramitasari, Komang Alit; Meiyanto, Edy
Jurnal Teknosains Vol 13, No 1 (2023): December
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/teknosains.89479

Abstract

The transfection efficiency positively affects the successful plasmid DNA transfer into cells, with the highlight on the amount of plasmid DNA and its ratio to the transfection reagent. Polyethyleneimine (PEI) is a cost-effective transfection reagent that facilitates DNA transfer by forming positively charged DNA complexes. It allows DNA to interact with negatively charged cell surfaces and enter the cells by endocytosis. In this study, we optimized the condition for transient transfection of life act-GFP/NLS-mCherry-expressing plasmid in BHK-21 and 293T cells using PEI. This plasmid is helpful as a biosensor of the cytoskeleton and nucleus that enables live imaging observation using a fluorescence microscope, for instance, in the observation of syncytium. Here, we optimized two independent variables: the amount of DNA (0.5 and 1 µg) and the ratio of DNA-PEI (1:3 and 1:4). GFP and mCherry expressions were observed at 24, 48, and 72 h post-transfection. As a result, transfection efficiency achieved by using PEI in 293T cells is higher than in BHK-21 cells, which are ~90% and ~50%, respectively. Moreover, amongst four different transfection conditions, in both cell lines, 1 µg of plasmid DNA with a 1:3 DNA-PEI ratio yields the most efficiency with the least amount of toxicity. We used this condition for the syncytia observation in 293T cells as a model of the cell-to-cell transmission of SARS-CoV-2. Syncytia formation was successfully observed by detecting the giant cells expressing GFP/mCherry with multiple nuclei.
Naringin Effect on SARS-CoV-2 Pseudovirus Entry and Spike Mediated Syncytia Formation in hACE2-overexpressing Cells Septisetyani, Endah Puji; Prasetyaningrum, Pekik Wiji; Paramitasari, Komang Alit; Suyoko, Ahmad; Himawan, Alayna Lillahida Indri; Azzahra, Salsabila; Wisnuwardhani, Popi Hadi; Anam, Khairul; Ramadani, Ratna Dwi; Santoso, Adi; Ningrum, Ratih Asmana; Herawati, Neng; Rubiyana, Yana
HAYATI Journal of Biosciences Vol. 31 No. 2 (2024): March 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.2.336-347

Abstract

A molecular docking study demonstrates the interaction between naringin, a citrus flavonoid, with SARS-CoV-2 spike RBD. Nevertheless, in vitro investigation of the inhibitory effect of naringin on SARS-CoV-2 entry and syncytia models has yet to be carried out. We synthesized VSV∆G-GFP/Spike* pseudovirus (PSV) as a SARS-CoV-2 model by pseudotyping VSV∆G-GFP/S* in BHK-21 cells overexpressing the SARS-CoV-2 spike glycoprotein. In the SARS-CoV-2 PSV entry assay, we utilized CHO-K1 cells transfected with hACE2 plasmid, which were then treated with naringin and SARS-CoV-2 PSV/naringin. After 16-18 h incubation, PSV internalization represented by the GFP signal was observed under a fluorescence microscope. Immunofluorescence staining was also performed to probe the SARS-CoV-2 spike and confirm the PSV entry. We performed a syncytia assay using 293T cells co-transfected with SARS-CoV-2 spike/hACE2. Six hours after transfection, the cells were treated with naringin and incubated for another 16-18 hours. Then, we observed syncytia using a phase contrast microscope. Based on fluorescence foci quantification, the results indicated that naringin might inhibit SARS-CoV-2 PSV entry at a concentration of 100 µM (P<0.05). However, naringin did not prevent syncytia formation compared to solvent control. These PSV entry and syncytia assay results suggested that naringin potentially inhibited SARS-CoV-2 viral infection but not cell-to-cell viral transmission.
Anti-SARS-CoV-2 Activity of Andrographis paniculata (Burm.f.) Nees Extract via Inhibition of Spike-mediated Syncytia Formation in HEK293T Cell Model Prasetyaningrum, Pekik Wiji; Kastian, Ria Fajarwati; Novianti, Metta; Santoso, Adi; Septisetyani, Endah Puji
HAYATI Journal of Biosciences Vol. 31 No. 5 (2024): September 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.5.996-1006

Abstract

The resurgence of COVID-19 endemic cases at the end of 2023 has underscored the need for effective treatments. Some severe cases of COVID-19 are often characterized by the formation of multinucleated syncytial pneumocytes in the lungs. Therefore, our study aimed to explore the potential of Andrographis paniculata (Burm. f) Nees as an antivirus against SARS-CoV-2, which involves syncitia formation. We utilized the non-toxic concentrations of A. paniculata extract on HEK293T cells determined by MTT assay, which were 1 μg/ml (cell viability 97.96%) and 10 μg/ml (cell viability 95.24%) for further assays. First, we conducted a pseudovirus cellular entry assay as a model of SARS-CoV-2 infection in HEK293T cells expressing hACE2/TMPRSS2. The HEK293T cells were co-transfected with plasmids expressing hACE2 and TMPRSS2, then infected with pseudotyped spike*∆G-GFP rVSV with or without A. paniculata extract. The internalized pseudovirus would trigger GFP expression as a reporter of the infected cells. Next, we performed a syncytia assay by transfecting HEK293T cells with hACE2, TMPRSS2, and SARS-CoV-2 spike expression vectors to induce syncytia formation as a model of intercellular viral transmission. As the results, 10 μg/mL of the extract significantly lowered the number of SARS-CoV-2 pseudovirus-infected cells by 54.69% (P = 0.02) and spike-mediated syncytia formation by 42.39% (P<0.001). In conclusion, our results suggested that A. paniculata has a potential antiviral activity against SARS-CoV-2 by hindering virus infection and cell-to-cell transmission.
Simple Procedure for the Isolation of Mesenchymal Stem Cells from Different Parts of the Human Umbilical Cord Suyoko, Ahmad; Prasetyaningrum, Pekik Wiji; Septisetyani, Endah Puji
Indonesian Journal of Cancer Chemoprevention Vol 13, No 2 (2022)
Publisher : Indonesian Society for Cancer Chemoprevention

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14499/indonesianjcanchemoprev13iss2pp104-113

Abstract

The umbilical cord and placenta are both sources of mesenchymal stem cells (MSCs) that are promising for cell-based therapy. Furthermore, compared to other MSCs sources, they are easy to obtain with no invasive procedures. This study presents an adapted method for stem cell isolation from three different parts of the human umbilical cord, including Wharton’s jelly (WJ), cord lining (CL), and cord-placenta junction (CPJ). The isolation consists of sample preparation, tissue dissection into distinct anatomical regions, mincing and enzyme digestion, and explant culturing. In addition, we monitored when the cells migrated from the explant to the bottom of the cell culture dish and passed the cells after they became confluent. This study found that WJ cells were the first to reach confluence at Passage 0 (P0). In contrast, CL cells needed the longest time to get confluence at P0 but displayed faster cell growth after subsequent passages (P1-P2). In addition, CPJ cells showed growth retardation after P1 and P2. Altogether, we could extract the MSCs from umbilical cord tissue explants by using DMEM supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin as general cell culture medium and omitting the use of gentamicin. However, the MSCs may need a more complex specified medium for optimum cell regeneration for further cell expansion.Keywords: mesenchymal stem cells, umbilical cord, Wharton’s jelly, cord lining, cord-placenta junction.
Recloning and Characterization of C2C12 Myoblast and Its Clonal Derivatives Prasetyaningrum, Pekik Wiji; Septisetyani, Endah Puji; Suyoko, Ahmad; Santoso, Adi
Indonesian Journal of Cancer Chemoprevention Vol 12, No 2 (2021)
Publisher : Indonesian Society for Cancer Chemoprevention

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14499/indonesianjcanchemoprev12iss2pp99-105

Abstract

The C2C12 myoblasts are adult murine muscle stem cells which isolated after injury to induce muscle regeneration. The cells are widely used in pharmaceutical and biological researches to represent skeletal muscle cells. In our laboratory, we utilize the cells for glucose uptake assay after insulin treatment and studying the muscle regeneration. In this study we conducted recloning of C2C12 cells by limiting dilution cloning (LDC) and investigated the biological properties incuding cell proliferation, adhesion and differentiation of the clonal cells in comparison to the parental cells. Cell proliferation rate had been determined by WST assay, cell adhesion had been observed after cell detachment by EDTA and cell differentiation into multinucleated myotube had been investigated after induction and incubation with horse serum. As results, two clonal derivatives of C2C12 myoblast cells had been retrieved by LDC and used for cell assays. Moreover, the results indicated that parental cells showed faster proliferation rate and better differentiation ability than that of clonal cells. In the contrary the parental cells exhibited weaker adhesion rate than clonal cells. To conclude, C2C12 parental cells are better for performing the glucose uptake or muscle regeneration assays since they showed better differentiation capability.Keywords: C2C12 cells, cells differentiation, myoblast, myotube, recloning.
Co-Authors Adi Santoso Anam, M. Khairul Azzahra, Salsabila Edy Meiyanto Endah Puji Septisetyani, Endah Puji Harsan, Hayfa Salsabila Himawan, Alayna Lillahida Indri Kastian, Ria Fajarwati Komang Alit Paramitasari Kumara, Dennaya Lestari, Dinda Neng Herawati, Neng Novianti, Metta Popi Hadi Wisnuwardhani, Popi Hadi Ramadani, Ratna Dwi RATIH ASMANA NINGRUM Suyoko, Ahmad Yana Rubiyana, Yana