Figure 8: TDP-43 is essential for IL-6 RNA processing.

(a–e) In vivo TDP-43 depletion impairs IL-6 production. (a) Strategy for depletion of TDP-43 in vivo and development of inflammation by LPS. C57BL/6N mice were divided into two different groups, namely control mice injected with the control-Invivofectamine complex (n=4) and an experimental group injected with the TDP-43 siRNA-Invivofectamine complex (n=4). Each siRNA-Invivofectamine complex was intraperitoneally administered and then the mice were treated intraperitoneally with LPS (5 mg kg⁻¹) to induce inflammation. Serum was collected at 0, 6 and 9 h for ELISA. After 9 h, mice were euthanized and the splenic macrophages were collected for RT–PCR, qRT–PCR or immunoblot analysis. (b) The efficiency of TDP-43 depletion in splenic macrophages was confirmed by immunoblot analysis using anti-TDP-43 or anti-β-actin antibody. (c) qRT–PCR with Taqman showed a significant reduction in IL-6 mRNA production. All qRT–PCR data were normalized to the expression of GAPDH mRNA. (n=4), *P<0.005 (Student’s t-test). (d) RT–PCR using primer pairs (P1) for the exon 1–exon 2 junction of IL-6 RNA was performed from control or TDP-43-depleted mice. Confirmation of TDP-43 knockdown and measurement of the quantity of RNA were achieved by RT–PCR with primer pairs P4, P6 and P7. Arrowheads and arrows indicate mRNA and pre-mRNA, respectively. (e) ELISA assay was carried out to measure IL-6 levels in serum from control or TDP-43-depleted mice. (n=4), *P<0.01 (Student’s t-test). (f) Model of TDP-43-mediated subnuclear foci function in IL-6 and IL-10 pre-mRNA processing. Data are representative of at least three independent experiments (mean±s.e.m. in c; mean±s.d. in e).