Journal: bioRxiv
Article Title: Fetal neural progenitors process TLR signals from bacterial components to enhance proliferation and rework brain development
doi: 10.1101/2021.10.19.464985
Figure Lengend Snippet: A) Using the in vivo assay of , dams deficient in Tlr1, 2 , or 6 were challenged with bacteria or PBS at E10. Pictures show E16 cortex stained with cortical excitatory neuronal markers. Graphs show total numbers of neurons in the cortical plate from Nissl-stained sections analyzed by stereology at E16. Right and left sides of the cortex were measured, and the average of 3 sections per embryo was calculated (each symbol). Measurements were taken for 3-4 dams with 4-8 embryos/dam for each experimental group as indicated by value inside each bar: Amp (○) vs Spn + Amp (•). Values were combined from at least 3 different experiments. The scale bar represents 100 µm. B) Foxg1 transcript levels were measured by qPCR with RNA from brains exposed to Amp (blue) or Spn + Amp (red) at E10 and harvested at the indicated day. Values represent 3-4 dams and 3-5 embryo lysates/dam. Data are shown as mean ± SEM. C) WT and indicated Tlr deficient dams were challenged at E10 with PBS (blue) or Spn (red), treated with Amp 24 h later as in , and embryos were harvested at E16. Brain lysates were assessed by Western blot for FOXG1 and FOXO levels. Values are expressed as % of WT control set at 100, using 2-3 dams per condition with 2-4 embryos/dam. D) FOXG1 protein levels were measured in NPCs cultured in vitr o from E12.5 embryos. TLR antagonists were applied as indicated: GIT27 10ug/ml for TLR2/6 or CU-CPT22 5uM for TLR 2/1. After 2 h, cells were exposed to purified BCW (MOI 1) for 2hrs and harvested. Cell lysates were processed into nuclear and cytoplasmic fractions and assayed by Western blot for FOXG1. Protein levels are expressed as % of those in cells not treated with BCW or antagonist. Each bar represents the mean ± SEM of 4 independent experiments. E) pAKT and PI3K protein levels were measured in NPCs cultured from E12.5 embryos. TLR antagonists were applied as in panel D. Protein levels are expressed as % of controls not treated with BCW or antagonist. Each bar represents the mean ± SD of 4 independent experiments. F) We treated WT and Tlr2-/- dams at the indicated day with PBS (blue) or Spn (red), treated them with Amp 24 h later, and collected brains another 24 h later, as indicated. Brain lysates were assessed by Western blot for p-AKT, AKT or PI3K levels. Values are expressed as % of WT control. We used 2-3 dams per condition with 2-4 embryos/dam. P -values for all panels were determined by unpaired, two-tailed t-tests.
Article Snippet: 24 hours after seeding, cells were washed and pretreated with TLR 2/6 antagonist GIT27 10μg/mL (Tocris 3270) or TLR 2/1 antagonist CU-CPT22 5 μM (Tocris 4884) for 2 hours.
Techniques: In Vivo, Bacteria, Staining, Western Blot, Control, Cell Culture, Purification, Two Tailed Test
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