Extended Data Fig. 10: Analysis of thymic cell death in vivo and effects of supernatants during arthritis.
From: Metabolites released from apoptotic cells act as tissue messengers
a, Analysis of thymic populations used for experimental data in Fig. 4a. After thymus isolation, the CD11b−CD11c− population that contained thymocytes was used for mRNA isolation to test the efficiency of deletion of the Panx1 allele. qPCR analysis of Panx1 mRNA in control mice (Panx1fl/flCd4-cre−/−) (n = 6) or mice in which PANX1 has been knocked out in thymocytes (Panx1fl/flCd4-cre+/−) (n = 7). CD11b+CD11c+ myeloid cells obtained from the thymus of Panx1fl/flCd4-cre+/− mice were analysed for Panx1 expression to demonstrate that PANX1 was not deleted. PANX1 deletion was deleted only from thymocytes and not the myeloid cells that do not express CD4. Data are mean ± s.d. **P = 0.0015, unpaired two-tailed Student’s t-test. b, Representative flow cytometric plots showing the extent of apoptosis induced by dexamethasone in control and Panx1fl/fl CD4-Cre+ mice. After thymus isolation, cells were stained with 7AAD and annexin V to determine the percentage of live, apoptotic or necrotic cells, as in Extended Data Fig. 1a. c, Quantitative analysis of apoptosis (left) and secondary necrosis (right) of CD11b−CD11c− thymic populations from Panx1fl/fl CD4-Cre− (PBS n = 4, Dex n = 10) or Panx1fl/fl CD4-Cre+ (PBS n = 3, Dex n = 9) mice treated with PBS or dexamethasone. Data are mean ± s.e.m. ****P < 0.0001, ordinary one-way ANOVA with Turkey’s multiple comparison test. d, Representative flow cytometry plots demonstrating the purity of CD11b+CD11c+ population after magnetic separation from the different mice and treatment conditions. e, Comparison of the CD11b+CD11c+ cells isolated under different conditions (cre−/−: PBS n = 4, Dex n = 7; cre+/−: PBS n = 3, Dex n = 6). Data are mean ± s.e.m. P > 0.05 (n.s.), ordinary one-way ANOVA with Turkey’s multiple comparison test. f, Apoptotic supernatants alleviate arthritic disease induced by serum from KBx/N mice. C57BL/6J mice were injected with serum from K/BxN mice to induce arthritis. Live (n = 4) or apoptotic (n = 5) supernatant was given for five days after arthritis induction. Paw swelling was measured using a calliper and reported as the percentage change compared with day 0. Data are mean ± s.e.m. *P = 0.0131, two-way ANOVA.
