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. 2019 Sep 16;129(10):4433–4450. doi: 10.1172/JCI125669

Figure 8. SPDEF regulates ERN2 and mucin production in vivo and in vitro.

Figure 8

(A) WT (Spdef+/+) and Spdef-deficient (Spdef–/–) 6-week-old mice were exposed to sterile saline or murine recombinant IL-1β via intratracheal instillation. Ern2 mRNA in conducting airways was detected by RNAscope assay. Micrographs are representative of n = 3 mice/treatment/genotype. Arrows point to regions shown in inserts. (B) Ern2 and Ern1 mRNAs in whole lung were quantitatively measured by TaqMan assays. The scatter plots present data as mean ± SD; data were analyzed with 2-tailed unpaired t test. For Spdef+/+ mice, n = 5 and n = 6 mice were administered saline and IL-1β, respectively. For Spdef–/– mice, n = 5 and n = 7 mice were administered saline and IL-1β, respectively. (C) Non-CF HBE cells were infected with lentiviruses expressing GFP (control) or FLAG-Spdef fusion protein and cultured under ALI conditions for 1 week. Goblet cell differentiation and MUC5B/MUC5AC protein expression were revealed by AB-PAS and dual-immunofluorescent staining, respectively. (D) MUC5B and MUC5AC mRNA levels were quantitatively measured in GFP vs FLAG-Spdef–transduced cultures by TaqMan assays. ERN1/ERN2 mRNA expression was identified by RNAscope duplex assay and quantitatively determined by TaqMan assay (C and E). Data were analyzed with 2-way ANOVA followed by Šidák’s correction. Non-CF HBE cells from n = 5 donors (n = 3 independent cultures/donor) were used for lentiviruses infection, and the same donor cells infected with GFP or FLAG-Spdef viruses were labeled with color-matching dots. (F) Immortalized HBE cells (UNCN3T) were transfected with negative control or SPDEF-specific siRNA, and SPDEF, ERN1, ERN2, MUC5B, and MUC5AC mRNAs were quantitatively measured after 48 hours. Scatter plots present data as mean ± SD with n = 4 independent cell cultures, and data were analyzed by 2-tailed, unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001, compared with saline (B), GFP (D and E), and control siRNA (F) groups. Scale bars: 50 μm (A); 20 μm (C). Original magnification, ×60 (insets).