RIP3 is required for PFT induced necroptosis.

<p><b>A)</b> LDH release assay of THP-1 cells transfected with siRNAs targeting RIP3, caspase-1 (Casp-1), caspase-8 (Casp-8) and a scramble control, infected with (A) <i>Sma</i> or challenged with <b>B)</b> recombinant pneumolysin (rPly). <b>C</b>) LDH release assay of MH-S macrophages infected with <i>Sma</i> at an MOI of 0.1 mock or following pretreatment with RIP3 inhibitor GSK’872. <b>D)</b> LDH release assay of BMDM from WT-C57BL/6, Caspase 1/11 KO, Caspase 3 KO and RIP3 KO mice, infected with <i>Sma</i> at an MOI of 0.5 or 0.1. <b>E)</b> LDH release of BMDM from wildtype mice, Caspase 1/11 (Casp1/11) KO, RIP3 KO, and BMDM from wildtype mice pretreated with necrostatin-5 (100μM) following their infection with <i>S</i>. <i>aureus</i> (MOI 10), <i>L</i>. <i>monocytogenes</i> (MOI 10) and <i>S</i>. <i>pneumoniae</i> (MOI 100). <b>F)</b> LDH release assay of BMDM from wild type C57BL/6 or RIP3 KO mice following their challenge with recombinant pneumolysin (rPly). <b>G)</b> LDH release assay of MH-S macrophages challenged with rPly with and without pretreatment with GSK’872 (10 μM). <b>H)</b> Alveolar macrophage numbers in BALF of RIP3 KO mice 18h after intratracheal infection with <i>S</i>. <i>marcescens</i>. Mann-Whitney tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, <i>P</i> ≤ 0.05. Data are representative of ≥3 separate experiments, each with 8 biological replicates.