Figure 3.

Necroptosis during Spn colonization is pneumolysin dependent. (A) FaDu pharyngeal cell death as measured by percent LDH release from cells pre-treated with normal media or media containing 10 μM of either the MLKL inhibitor Necrosulfonamide (NSA), the general caspase inhibitor ZVAD, or the ferroptosis inhibitor Liproxstatin-1 (Liprox), for 1 h then challenged overnight (15 h) with TIGR4 or TIGR4Δply at an MOI of 10. One-way analysis of variance (F = 11.86, p < 0.0001); P-values listed on graph; * indicates a value <0.05 (See also Figure S3B). (B) Representative immunofluorescent images of nasal turbinates from wildtype mice colonized with TIGR4 or TIGR4Δply, at day 7 post inoculation with 10⁵ CFU. Turbinates fixed and stained for collagen-la (red), Spn (yellow), pMLKL (green), and DAPI (Blue). Imaged at 40X magnification and Tile Scan assembled. White scale bars indicate 250 μm. (C) Western blot and densitometry for pMLKL and actin in nasal homogenates from mice colonized with TIGR4 or TIGR4Δply. Nasal lavage at 7-days post intra-nasal inoculation with TIGR4 or TIGR4Δply analyzed for (D) nEC sloughing, (E) lactate dehydrogenase (LDH), and (F) IL-1α. LDH Absorbance at 490 nm LOD normalized to absorbance of uninfected control NALF. Mann-Whitney U-test used for two-way comparisons and Kruskal-Wallis test with Dunn's post-test for multiple comparisons (Two infection experiments; total n = 10–14 animals per genotype). P-values listed on graph; * indicates a value < 0.05. (See also Figures S2, S3 and Table S2).