| Autor: |
Kieran, Nicholas W., Suresh, Rahul, Dorion, Marie-France, MacDonald, Adam, Blain, Manon, Wen, Dingke, Fuh, Shih-Chieh, Ryan, Fari, Diaz, Roberto J., Stratton, Jo Anne, Ludwin, Samuel K., Sonnen, Joshua A., Antel, Jack, Healy, Luke M. |
| Rok vydání: |
2022 |
| DOI: |
10.6084/m9.figshare.17982578 |
| Popis: |
Additional file 3: Figure S2. Primary human fetal astrocytes respond to in vitro stresses, and mice upregulate miR-210 around ischemic lesions. (A) Primary human astrocytes, oligodendrocytes, and microglia were separately cultured and analyzed for expression of canonically expressed genes of glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) and ionized calcium binding adaptor molecule 1 (IBA1) by RT-qPCR. n = 3���4. (B) Primary human fetal astrocytes were immunostained with anti-GFAP, anti-O4, and anti-PU.1 antibodies. A CX7 automated microscope was used to quantify the percent positive astrocytes for each marker, n = 4. (C) Primary human astrocytes were treated in inflammatory (���I���) metabolic (���M���) or hypoxic (���H���) stress conditions induced by IL1b, glucose-free media, or a 1% oxygen chamber for 24 h compared to untreated control. RT-qPCR assessment of CXCL10, HMOX1, and MCT4 expression was measured to confirm the astrocytic response to inflammatory, metabolic, and hypoxic stress conditions, respectively, n = 4���6. (D) HeLa cells were left in normoxic (���N���) conditions for 48 h or were treated in 1% O2 (hypoxia, H) for the noted time. After 2���48 h, the cells were removed and were immediately lysed with RIPA buffer for subsequent Western blot. n = 3. (E) Graphical representation of the area investigated for miR-210 expression using miRscope. miR-210 was quantified in the highlighted region around the infarct site (left), and in the respectively highlighted region on the contralateral side of the brain (right). 300���500 cells were counted per brain section. (F) Representative images and quantification of microRNAscope (miRscope) of miR-210 around MCAO lesions (labeled as infarct) or on the contralateral side of the brain (labeled as control). Scale bar = 20 ��m. Quantification was performed by manual counting of the number of nuclei positive for one or more red puncta. Red dots show miR-210 expression, and DAPI (blue) was used as a counterstain. Each dot in image F represents a separate tissue section all derived from one mouse, resulting in five sections imaged per brain region. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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