|
| Status |
Public on Nov 25, 2019 |
| Title |
Activated gametocyte BIX-treated (G9a) 1 h |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Activated gametocyte RNA
|
| Organism |
Plasmodium falciparum |
| Characteristics |
strain: NF54 Wildtype
|
| Treatment protocol |
P. falciparum Percoll enriched immature (stages II-IV) gametocytes (imGC), mature (stage V) gametocytes (mGC), and gametocytes at 1 h post-activation with 100 µM Xanthurenic acid at RT (aGC) were obtained. Each enriched gametocyte sample was treated with BIX-01294 at IC90 concentrations (0.12 µM) or with 0.5% vol. DMSO (untreated control) for 1 h and 6 h.
|
| Growth protocol |
P. falciparum NF54 strain were cultivated in vitro in RPMI 1640 medium supplemented with 10% heat-inactivated human serum [50] and cultures were maintained at 37°C in nitrogen containing 5% O2 and 5% CO2. Cultures were synchronized by repeated sorbitol treatment as described [51]. To generate gametocytes, the cultures were kept at high parasitaemia and gametocytogenesis was induced following addition of lysed RBCs. As soon as stage I gametocytes emerged in the culture, the culture medium was supplemented with 50 mM N-acetyl glucosamine (GlcNac) for approximately 5 days to kill the asexual blood stages [52]. The gametocyte culture was then maintained in normal culture medium without GlcNac until immature (stage II -IV) or mature stage V gametocytes were harvested and enriched by Percoll gradient centrifugation [53]. In order to obtain activated gametocytes, Percoll enriched mature gametocytes were incubated with 100 µM XA (Xanthurenic acid) 1 h or 6 h at room temperature (RT).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gametocytes samples were purified from different cultures, treated as indicated and stored in Trizol at –80°C. Samples treated in the same way were then pooled together and total RNA extracted according to the manufacturer´s instructions (Invitrogen).
|
| Label |
Cy5
|
| Label protocol |
Painter, H. J., Altenhofen, L. M., Kafsack, B. F. C. & Llinás, M. Whole-genome analysis of Plasmodium spp. Utilizing a new agilent technologies DNA microarray platform. Methods Mol. Biol. 923, 213–219 (2013).
|
| |
|
| Channel 2 |
| Source name |
Mixed gametocyte and asexual blood stage reference pool
|
| Organism |
Plasmodium falciparum |
| Characteristics |
strain: NF54 and 3D7
|
| Treatment protocol |
P. falciparum Percoll enriched immature (stages II-IV) gametocytes (imGC), mature (stage V) gametocytes (mGC), and gametocytes at 1 h post-activation with 100 µM Xanthurenic acid at RT (aGC) were obtained. Each enriched gametocyte sample was treated with BIX-01294 at IC90 concentrations (0.12 µM) or with 0.5% vol. DMSO (untreated control) for 1 h and 6 h.
|
| Growth protocol |
P. falciparum NF54 strain were cultivated in vitro in RPMI 1640 medium supplemented with 10% heat-inactivated human serum [50] and cultures were maintained at 37°C in nitrogen containing 5% O2 and 5% CO2. Cultures were synchronized by repeated sorbitol treatment as described [51]. To generate gametocytes, the cultures were kept at high parasitaemia and gametocytogenesis was induced following addition of lysed RBCs. As soon as stage I gametocytes emerged in the culture, the culture medium was supplemented with 50 mM N-acetyl glucosamine (GlcNac) for approximately 5 days to kill the asexual blood stages [52]. The gametocyte culture was then maintained in normal culture medium without GlcNac until immature (stage II -IV) or mature stage V gametocytes were harvested and enriched by Percoll gradient centrifugation [53]. In order to obtain activated gametocytes, Percoll enriched mature gametocytes were incubated with 100 µM XA (Xanthurenic acid) 1 h or 6 h at room temperature (RT).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gametocytes samples were purified from different cultures, treated as indicated and stored in Trizol at –80°C. Samples treated in the same way were then pooled together and total RNA extracted according to the manufacturer´s instructions (Invitrogen).
|
| Label |
Cy3
|
| Label protocol |
Painter, H. J., Altenhofen, L. M., Kafsack, B. F. C. & Llinás, M. Whole-genome analysis of Plasmodium spp. Utilizing a new agilent technologies DNA microarray platform. Methods Mol. Biol. 923, 213–219 (2013).
|
| |
|
| |
| Hybridization protocol |
Painter, H. J., Altenhofen, L. M., Kafsack, B. F. C. & Llinás, M. Whole-genome analysis of Plasmodium spp. Utilizing a new agilent technologies DNA microarray platform. Methods Mol. Biol. 923, 213–219 (2013).
|
| Scan protocol |
Agilent G2600D Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version 11.5.1.1).
|
| Data processing |
Agilent Feature Extraction Software (v 11.5.1.1) was used for background subtraction.
|
| |
|
| Submission date |
Aug 19, 2019 |
| Last update date |
Nov 25, 2019 |
| Contact name |
Manuel Llinas |
| E-mail(s) |
manuel@psu.edu
|
| Phone |
8148673444
|
| Organization name |
Penn State University
|
| Department |
Biochemistry and Molecular Biology
|
| Lab |
Manuel Llinas Lab
|
| Street address |
Millennium Science Complex, Pollock Rd
|
| City |
University Park |
| State/province |
Pennsylvania |
| ZIP/Postal code |
16802 |
| Country |
USA |
| |
|
| Platform ID |
GPL15130 |
| Series (1) |
| GSE136008 |
The G9a histone methyltransferase inhibitor BIX-01294 modulates gene expression during gametocyte development and transmission |
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