Fig. 5: NonO is involved in Neat1 mediated antigen-specific Th17 cell responses.

A Cytoplasmic and nuclear levels of Neat1 in CD4⁺ T cells from EAU mice (n = 3 per group). B Representative images of paraspeckles in naïve and EAU CD4⁺ T cells under confocal microscopy (n = 30 cells in each group). Red, Cy3-labeled Neat1 probes; green, Alexa-488-labeled secondary antibody for NonO protein. Scale bars, 2 μm. C Representative confocal images of paraspeckles in EAU CD4⁺ T cells upon Neat1 knockdown (n = 30 cells in each group). D Prediction of NonO and SFPQ binding sites in the Il17 and Il23r promoter regions by MEME analysis. E–H, N–O CD4⁺ T cells isolated from immunized mice were transfected with indicated oligonucleotides and stimulated with IRBP_1-20 in the presence of irradiated APCs under Th17-polarizing conditions. E Real-time qRT-PCR analysis of NonO expression (n = 4 per group). F Flow cytometric analysis of the percentages of Th17 cells (n = 4 per group). G, O Real-time qRT-PCR analysis of Il17 and Il23r expression (n = 4 per group). H, N ELISA analysis of IL-17 secretion in the culture supernatants (n = 4 per group). I Luciferase activity analysis of reporter containing Il17 or Il23r promoter transfected into HEK293T with the indicated plasmids (n = 4 per group). J–M ChIP analysis of NonO occupancy at the promoter of the Il17 or Il23r gene was performed in CD4⁺ T cells (n = 3 per group). Blue lines 1 to 4 indicate regions detected by ChIP-qPCR. Regions without NonO binding sites (no binding site) were used as negative control. Data present at least three independent experiments. *p < 0.05, **p < 0.01.