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All Journal HAYATI Journal of Biosciences Jurnal Teknosains Indonesian Journal of Cancer Chemoprevention ANNALES BOGORIENSES JFIOnline Annales Bogorienses
Endah Puji Septisetyani, Endah Puji
Research Center for Biotechnology Indonesian Institute of Sciences Jl. Raya Bogor KM 46, Cibinong, Bogor, Indonesia 16911
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Sequential Adaptation in Mammalian CHO-K1 Cells Producing Human Erythropoietin Wisnuwardhani, Popi Hadi; Septisetyani, Endah Puji; Santoso, Adi
ANNALES BOGORIENSES Vol 21, No 1 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (289.139 KB) | DOI: 10.14203/ab.v21i1.282

Abstract

The production of recombinant proteins for clinical applications using mammalian cell technology has become a prevalent system because of its capacity in assembling functional proteins.  One of the main problems with CHO-K1 cells is that this cell has to grow in the presence of serum. However, the presence of serum will complicate the downstream step for protein production. Thus, protein produced in media without serum, theoretically, would be easier to purify.  Technically, this type of cell can be produced by growing the CHO-K1 cells in serum-free media by using adaptation method in suspension condition. This research showed that through sequential adaptation using conditioned media, the CHO-K1 cell line that produces the human erythropoietin gene (hEPO) was able to grow in suspension culture using serum-free media.  Based on Western blot analysis, it showed that the protein (hEPO) was able to be expressed in suspension culture with molecular mass of about 47 kDa.
Optimizaton of Cationic Lipid Mediated Transfection of pEGFP-c1 and pJ-EPO Plasmids in Chinese Hamster Ovary (CHO) Cells Attached Culture for Transient and Stable Recombinant Human Erythropoietin (rhEPO) Expression Septisetyani, Endah Puji; Kusumawati, Arizah; Santoso, Adi
ANNALES BOGORIENSES Vol 19, No 1 (2015): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ab.v19i1.84

Abstract

Cationic lipid is one of transfection agents which show high efficiency and low cytotoxicity. The transfection efficiencies may depend upon the type or amount of cationic lipids, the cell line or DNA plasmid being used for transfection. The purpose of this study was to find optimal condition for transfection of CHO-K1 and CHO-S cells with pJ-EPO plasmid (contain human epo gene) compared with pEGFP-c1 plasmid (contain gfp gene) by cationic lipid Lipofectamin 2000TM to generate stable transfectant expressing recombinant human erythropoietin (rhEPO). Optimization was carried out regarding the amount of lipofectamin, DNA concentration, and concentration of antibiotic Geneticin (G418) for selection of stable transfectants. Using standard amount of lipofectamin (10µl/well) in 6-well plate, highest expression level of green fluorescent protein (GFP) was shown after transfection of CHO-K1 cells with 4µg/well pEGFP-c1 while highest expression level of rhEPO was observed after transfection of CHO-K1 cells with 6, 8, and 10µg/well pJ-EPO plasmids. The data also indicated that optimal transfection conditions of CHO-K1 and CHO-S cells with pJ-EPO were shown with the use of 4µg/well DNA and 15µl lipofectamin, respectively. Concentration of G418 affected the expression where strongest rhEPO expression was shown at 1,000 ng/µl G418 concentration. 
8-HIDROKSIISOKAPNOLAKTON-2',3'-DIOL, KUMARIN BIOAKTIF DARI Micromelum minutum Susidarti, Ratna Asmah; Rahmani, Mawardi; Sukari, Mohd. Aspollah; Ali, Abdul Manaf; Mustofa, .; Yasmina, Alfi; Handayani, Sri; Mintarsih, Betty; Ikawati, Muthi’; Septisetyani, Endah Puji
JFIOnline | Print ISSN 1412-1107 | e-ISSN 2355-696X Vol 4, No 3 (2009)
Publisher : Indonesian Research Gateway

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Abstract

Separation of leaves chloroform extract of Micromelum minutum (Rutaceae) yielded a new coumarin, 8-hydroxyisocapnolactone-2¢,3¢-diol. The structure of this compound was characterized by UV, IR, MS and NMR spectroscopic methods, including 1H, 13C, HSQC, COSY, HMBC dan NOESY experiments. This compound is significantly toksisk towards several cancer cell lines (CEM-SS, HL60, HeLa, HepG2, MCF7, T47D and NS1), active against chloroquin sensitive (D10) and resistance (FCR3) Plasmodium falciparum and showed strong antibacterial activity against Bacillus Subtilis mutan, Bacillus. Subtilis wild type, Pseudomonas Aeruginosa dan Staphylococcus aureus resisten meticilin).  ABSTRAK Pemisahan ekstrak kloroform daun Micromelum minutum (Rutaceae) menghasilkan suatu kumarin baru, 8-hidroksiisokapnolakton-2΄,3΄-diol yang strukturnya diidentifikasi secara spektroskopi UV, IR, MS dan NMR termasuk 1H, 13C, HSQC, COSY, HMBC dan NOESY. Senyawa tersebut secara signifikan toksik terhadap beberapa sel kanker (CEM-SS, HL60, HeLa, HepG2, MCF7, T47D dan NS1), aktif terhadap Plasmodium falciparum yang sensitif (D10) maupun resisten (FCR3) kloroquin dan mempunyai aktivitas antibakteri yang kuat terhadap Bacillus Subtilis mutan, Bacillus. Subtilis wild type, Pseudomonas Aeruginosa dan Staphylococcus aureus resisten meticilin).
8-HIDROKSIISOKAPNOLAKTON-2',3'-DIOL, KUMARIN BIOAKTIF DARI Micromelum minutum Susidarti, Ratna Asmah; Rahmani, Mawardi; Sukari, Mohd. Aspollah; Ali, Abdul Manaf; Mustofa, .; Yasmina, Alfi; Handayani, Sri; Mintarsih, Betty; Ikawati, Muthiâ??; Septisetyani, Endah Puji
Jurnal Farmasi Indonesia Vol 4, No 3 (2009)
Publisher : Jurnal Farmasi Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.35617/jfi.v4i3.18

Abstract

Separation of leaves chloroform extract of Micromelum minutum (Rutaceae) yielded a new coumarin, 8-hydroxyisocapnolactone-2¢,3¢-diol. The structure of this compound was characterized by UV, IR, MS and NMR spectroscopic methods, including 1H, 13C, HSQC, COSY, HMBC dan NOESY experiments. This compound is significantly toksisk towards several cancer cell lines (CEM-SS, HL60, HeLa, HepG2, MCF7, T47D and NS1), active against chloroquin sensitive (D10) and resistance (FCR3) Plasmodium falciparum and showed strong antibacterial activity against Bacillus Subtilis mutan, Bacillus. Subtilis wild type, Pseudomonas Aeruginosa dan Staphylococcus aureus resisten meticilin).  ABSTRAK Pemisahan ekstrak kloroform daun Micromelum minutum (Rutaceae) menghasilkan suatu kumarin baru, 8-hidroksiisokapnolakton-2Î?,3Î?-diol yang strukturnya diidentifikasi secara spektroskopi UV, IR, MS dan NMR termasuk 1H, 13C, HSQC, COSY, HMBC dan NOESY. Senyawa tersebut secara signifikan toksik terhadap beberapa sel kanker (CEM-SS, HL60, HeLa, HepG2, MCF7, T47D dan NS1), aktif terhadap Plasmodium falciparum yang sensitif (D10) maupun resisten (FCR3) kloroquin dan mempunyai aktivitas antibakteri yang kuat terhadap Bacillus Subtilis mutan, Bacillus. Subtilis wild type, Pseudomonas Aeruginosa dan Staphylococcus aureus resisten meticilin).
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.
Viability of bhk-21 fibroblast cells toward acrylic denture bases after reinforced by natural fibers Prawesthi, Endang; Tetelepta, Marzia Magdalena; Heldayani, Heldayani; Kastian, Ria Fajarwati; Septisetyani, Endah Puji
Jurnal Teknosains Vol 14, No 1 (2024): December
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/teknosains.90278

Abstract

The use of acrylic denture bases in the oral cavity requires biocompatibility. This study investigated the viability of BHK-21 fibroblast cells after treatment with an acrylic denture base modified using natural fibers. Ramie and banana stem fibers were used as cost-effective alternatives to synthetic fibers. The study involved 42 acrylic resin specimens (10 mm diameter, 2 mm thickness) divided into groups: resin without fibers, 0.5%, 1.5%, and 2.5% ramie fibers, and 0.5%, 1.5%, and 2.5% banana stem fibers. The resin was incubated with cell culture media at 37°C for 7 days. Cytotoxicity testing using the MTT method revealed that all treatment groups had cell viability exceeding 70%, meeting ISO 10993-5 standards. No significant differences in cell viability were observed between the treatment groups and the control (media without specimens). Additionally, adding 0.5%, 1.5%, and 2.5% ramie fibers did not affect BHK-21 cell viability compared to the resin-only control, while adding banana stem fibers increased cell viability compared to the control (P = 0.035; P = 0.021; and P = 0.011). In conclusion, increasing the concentration of natural fibers in acrylic denture bases did not negatively impact fibroblast cell growth.
Analysis of SARS-CoV-2 spike-Induced Syncytia with Lifeact-GFP as Biosensor Using High-Content Screening Instrument for Automated Syncytia Counting Fauziah, Dita; Septisetyani, Endah Puji; Yerizel, Eti; Kastian, Ria Fajarwati
Indonesian Journal of Cancer Chemoprevention Vol 15, No 2 (2024)
Publisher : Indonesian Society for Cancer Chemoprevention

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14499/indonesianjcanchemoprev15iss2pp127-136

Abstract

SARS-CoV-2 is believed to cause cytopathic effects in forming multinucleated cells, known as syncytia. Syncytia due to SARS-CoV-2 infection found in lung tissue samples of COVID-19 patients represents a case of COVID-19 with a poor prognosis. Therefore, it is very important to study the mechanism of syncytia formation and to test candidate materials that can inhibit the occurrence of syncytia and potentially be applied in the treatment or prevention of COVID-19. Since syncytia counting and analysis are time-consuming, we utilized a high-content screening (HCS) instrument in this study to automate syncytia analysis. We used 293T cells transfected with plasmids to express the SARS-CoV-2 spike, human angiotensin-converting enzyme-2 (hACE-2), and a plasmid encoding lifeact-GFP as an F-actin biosensor to facilitate syncytia analysis using the HCS instrument. In this study, syncytia analysis was carried out using HCS software. The HCS application categorizes cells as multi-nuclei by counting the number of cell nuclei stained with DAPI in cells that emitted green fluorescence due to lifeact-GFP expression. Syncytia analysis is time-consuming because of the calculation of the number of syncytia formed in a confluent cell monolayer culture. Hopefully, utilizing the HCS platform can accelerate the test of syncytia inhibition after various treatments using test compounds.Keywords: 293T cells, high-content analysis, SARS-CoV-2, spike, syncytia.
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.
Co-Authors Abdul Manaf Ali, Abdul Manaf Adi Santoso Alfi Yasmina Anam, M. Khairul Azzahra, Salsabila Edy Meiyanto Eti Yerizel Fauziah, Dita Harsan, Hayfa Salsabila Heldayani Heldayani, Heldayani Himawan, Alayna Lillahida Indri Kastian, Ria Fajarwati Komang Alit Paramitasari Kumara, Dennaya Kusumawati, Arizah Kusumawati, Arizah Lestari, Dinda Marzia Magdalena Tetelepta Mintarsih, Betty Mintarsih, Betty Mustofa Mustofa Muthi Ikawati Neng Herawati, Neng Novianti, Metta Popi Hadi Wisnuwardhani, Popi Hadi Prasetyaningrum, Pekik Wiji Prawesthi, Endang Rahmani, Mawardi Rahmani, Mawardi Ramadani, Ratna Dwi RATIH ASMANA NINGRUM Ratna Asmah Susidarti Sri Handayani Sukari, Mohd. Aspollah Sukari, Mohd. Aspollah Suyoko, Ahmad Wisnuwardani, Popi Hadi Yana Rubiyana, Yana