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. 2020 May 15;201(10):1249–1262. doi: 10.1164/rccm.201908-1513OC

Figure 7.

Figure 7.

Wnt5a expression in the setting of hyperoxia injury is mediated by NF-κB (nuclear factor-κB) in the developing lung. (A) Quantification of Wnt5a expression normalized to cell number for each condition, analyzed by two-way ANOVA and secondary analysis by Tukey’s test for multiple comparisons. N = 4, *P < 0.05 and **P < 0.01. (B) Cytokine analysis on tissue lysate samples from precision-cut lung slices (PCLS) from P4 lungs cultured in normoxia, hyperoxia, normoxia + BAY11–7082, and hyperoxia + BAY11–7082 (n = 4 mice per group). **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way ANOVA with secondary analysis by Tukey’s test for multiple comparisons. (C) Mean linear intercept of PCLS cultured in normoxia or hyperoxia, with or without BAY11–7082, analyzed by two-way ANOVA and secondary analysis by Tukey’s test for multiple comparisons. ****P < 0.0001. (D) Hematoxylin and eosin staining of PCLS cultured in normoxia or hyperoxia, with or without BAY11–7082. Scale bars, 100 μm. (E) Airspace volume density of PCLS cultured in normoxia and hyperoxia, with or without BAY11–7082. ****P < 0.0001. (F) Multiplex RNA in situ hybridization comparing expression of Wnt5A (red) in hyperoxia-exposed pups treated with BAY11–7082 on P12–13 and untreated hyperoxia pups; Sftpc expression is in green. Scale bars, 10 μm. (G) Quantification of RNA in situ hybridization normalized to nuclear surface area shows significantly decreased expression of Wnt5a in hyperoxia-exposed pups treated with BAY11–7082 when compared with untreated hyperoxia controls. **P < 0.01. (H) Expression of inducible dominant-negative inhibitor (with doxycycline treatment) of NF-κB signaling in mouse lung fibroblasts showed decrease in Wnt5a expression by real-time quantitative PCR (qPCR) when NF-κB signaling was inhibited. ****P < 0.0001. qPCR confirmed expression of IκBα gene Nfkbia with treatment of doxycycline. ****P < 0.0001. Data are representative of ≥4 biological replicates for each condition, with 3 technical replicates for histology and 4 replicates used for qPCR. Dox = treated with doxycycline; NS = nonsignificant (P > 0.05).