Extended Data Fig. 5
From: Secreted fungal virulence effector triggers allergic inflammation via TLR4
(a) Arginase-1 FACS in Myd88+/+ or Myd88–/– BMDMs stimulated for 24 h with the indicated concentrations of rCPL1 alone (left) or in combination with IL-4 (10 ng/ml). n = three biologically independent samples. (b) Arginase-1 FACS gated on CD45.2+ BMDMs from the indicated genotypes co-cultured with a 50:50 mix of CD45.1 BoyJ BMDMs and stimulated for 24 h with IL-4 (10 ng/ml) or IL-4 + rCPL1 (111 nM). n = three biologically independent samples. (c) Arginase-1 FACS on BMDMs stimulated for 24 h with the indicated concentrations of LPS. (d) Measurement of pyroptosis by LDH release assay on BMDMs stimulated with the indicated concentrations of LPS alone or with 10 ug/ml cholera toxin B (CTB). n = three biologically independent samples. (e) Measurement of pyroptosis by LDH release assay on BMDMs stimulated with the indicated concentrations of rCPL1 alone or with 10 ug/ml CTB. n = three biologically independent samples. (f) Arginase-1 FACS on BMDMs stimulated with rCPL1 that was either kept on ice or boiled at 100 °C for 15 min. Cells were stimulated with either rCPL1 alone (left) or in combination with IL-4 (right). n = three biologically independent samples. (g)(h) Arginase-1 FACS in in BMDMs stimulated with the indicated concentrations of rCPL1 alone (g) or in combination with IL-4 (10 ng/ml) (h) that were either treated with control or polymyxinB. n = three biologically independent samples. (i) Arginase-1 FACS on BMDMs transduced with MSCV-empty or MSCV-CPL1 retrovirus and stimulated for 24 h with the indicated concentrations of IL-4. n = three biologically independent samples. (j) Silver stain on SDS-PAGE gel of rCPL1-6xHis or rCPL1(Y160A)-6xHis purified from P. pastoris. Image is representative of three independent experiments. Data are presented as mean values +/– SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.
