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. Author manuscript; available in PMC: 2009 Oct 3.
Published in final edited form as: Cell. 2009 Apr 3;137(1):47–59. doi: 10.1016/j.cell.2009.01.038

Fig. 2. Microglia initiate LPS-mediated inflammation and astrocytes propagate the production of neurotoxic factors.

Fig. 2

A. Expression of TNFα mRNA upon LPS stimulation in primary microglia, astrocytes and Neuro2A cells. B. Effect of LPS or TNFα + cycloheximide (CHX) on viability of the indicated neuronal cell lines determined using a TUNEL ELISA assay. *, p<0.01 compared to untreated sample (no Tx) (white). C. Effect of Nurr1 knockdown in Neuro2A cells on sensitivity to TNFα + CHX assessed by TUNEL ELISA assay. D–E. Effect of Nurr1 knockdown inBV2 cells on LPS-induced expression of TNFα (D), iNOS (E) and IL1β (F) mRNA.*, p<0.01 compared to no stimulation (Ctrl); **, p<0.01 compared to LPS stimulation of shCtrl-BV2 cells. G. Scheme of conditioned media (CM) and cell death assay. CMs were harvested from shCtrl- and shNurr1-BV2 cells that were stimulated with LPS for 24h. Neurons or glial cells were assayed for specific markers by immunostaining and for cell death by TUNEL assay. H. Effect of CM from LPS-treated shCtrl-BV2 cells and a mixture of the CMs from shNurr1-1- and shNurr1-2-infected BV2 cells (shNurr1) on neurons and glial cells derived from in vitro differentiated neural stem cells (NSC). TUNEL assay was performed on TH-, GABA- or GFAP-positive cells derived from mouse NSC. Numbers of TUNEL-negative live cells are shown. TH-positive cells are indicated in red. I. The percentages of TUNEL-positive population are shown. *, p<0.01 and **, p<0.001 compared to no treatment (no TX). J. Effect of Nurr1 knockdown in microglia and astrocytes on the production of neurotoxic factors. Primary mouse microglia and astrocytes were infected with shCtrl- or shNurr1-lentivirus. Cells were treated with LPS for 2h and washed extensively with PBS. Cells were cultured for another 24h to generate CM. For sequential CM assay, CMs harvested from microglia were cultured with lentivirus-infected astrocytes for 24 hours. Then, CMs were harvested and tested for effect on viability of Neuro2A cells.