GREEN FLUORESCENT PROTEIN (GFP), SUATU SIGNAL PENANDA QUANTITATIF UNTUK MEMONITOR PROLIFERASI SEL

Banyak penelitian sel kultur yang melibatkan sejumlah besar sampel untuk dianalisis, baik yang berhubungan dengan pertumbuhan sel atau toksisitas senyawa terhadap sel. Green Fluorescent Protein (GFP) adalah suatu protein yang secara alami dapat berfluorescence dan banyak digunakan pada berbagai aplikasi seperti penanda untuk ekspresi gena, produksi heterologous protein atau monitoring efisiensi transfeksi. Penelitian ini bertujuan untuk membandingkan tingkat akurasi, taraf kepercayaan dan reprodusibilitas GFP untuk memonitor pertumbuhan sel.Kurva baku dibuat dengan serial dilusi sel CHO-K1-EGFP dalam media 10% FCS di 96 well plate. Jumlah sel dalam tiap sumuran dihubungkan dengan signal fluorescence. Untuk memonitor pertumbuhan sel, signal fluoresensi dibandingkan dengan metode Trypan Blue Exclusion yang jumlah sel dalam tiap sumuran dihitung selama periode waktu tertentu (n=3). Untuk monitoring pertumbuhan sel, signal dari GFP memperlihatkan korelasi yang baik dengan jumlah sel dengan tingkat linieritas 0,9866 dalam kisaran jumlah sel 1250 – 1 x 105 sell/sumuran (standar error maksimum 11%). Metode ini terbukti memungkinkan pengukuran langsung signal fluoresensi sehingga mampu menurunkan kemungkinan kesalahan yang terjadi pada saat preparasi sel yang dapat mempengaruhi akurasi data yang diperoleh. Sekali klones permanen (stable clones) diperoleh klons ini dapat digunakan untuk banyak aplikasi.Kata Kunci: Green Fluorescent Protein (GFP), Fluorescence, proliferasi sel, Trypan Blue Exclusio

Apoptosis is induced in Chlamydia trachomatis-infected HEp-2 cells by the addition of a combination innate immune activation compounds and the inhibitor wedelolactone

Problem: Innate immune activation of human cells, for some intracellular pathogens, is advantageous for vacuole morphology and pathogenic viability. It is unknown whether innate immune activation is advantageous to Chlamydia trachomatis viability. ----- ----- Method of study: Innate immune activation of HEp-2 cells during Chlamydia infection was conducted using lipopolysaccharide (LPS), polyI:C, and wedelolactone (innate immune inhibitor) to investigate the impact of these conditions on viability of Chlamydia. ----- ----- Results: The addition of LPS and polyI:C to stimulate activation of the two distinct innate immune pathways (nuclear factor kappa beta and interferon regulatory factor) had no impact on the viability of Chlamydia. However, when compounds targeting either pathway were added in combination with the specific innate immune inhibitor (wedelolactone) a major impact on Chlamydia viability was observed. This impact was found to be due to the induction of apoptosis of the HEp-2 cells under these conditions. ----- ----- Conclusion: This is the first time that induction of apoptosis has been reported in C. trachomatis-infected cells when treated with a combination of innate immune activators and wedelolactone

Cyclin A/cdk2 Regulates Adenomatous Polyposis Coli-dependent Mitotic Spindle Anchoring*

Mutations in adenomatous polyposis coli (APC) protein is a major contributor to tumor initiation and progression in several tumor types. These mutations affect APC function in the Wnt-β-catenin signaling and influence mitotic spindle anchoring to the cell cortex and orientation. Here we report that the mitotic anchoring and orientation function of APC is regulated by cyclin A/cdk2. Knockdown of cyclin A and inhibition of cdk2 resulted in cells arrested in mitosis with activation of the spindle assembly checkpoint. The mitotic spindle was unable to form stable attachments to the cell cortex, and this resulted in the spindles failing to locate to the central position in the cells and undergo dramatic rotation. We have demonstrated that cyclin A/cdk2 specifically associates with APC in late G2 phase and phosphorylates it at Ser-1360, located in the mutation cluster region of APC. Mutation of APC Ser-1360 to Ala results in identical off-centered mitotic spindles. Thus, this cyclin A/cdk2-dependent phosphorylation of APC affects astral microtubule attachment to the cortical surface in mitosis