Fig 2: Protein expression analysis of E15.5 wild-type (+/+), heterozygote (Jf/+) and homozygote (Jf/Jf) lungs. Equal amounts of protein lysates were subjected to PAGE, transferred and probed with antibodies against FBXO11 (A), p21 (B), PAI-1 (C), MMP-2 (D), BCL-6 (E), CDT2 (F) and SET8 (G). RAW 264.7 whole-cell lysate was used as a positive control for all of the antibodies except for MMP2, where middle ear effusion lysate was used, and for CDT2, where adult mouse spleen lysate was used. Graphs show comparisons of the western blots for each antibody after normalizing with an anti-actin antibody. The results presented in the graphs are from three independent experiments for all the antibodies except for p21, where four independent experiments were used to present the data. Bars: standard error of mean (s.e.m.). *P<0.05.

Fig 3: Phenotype of the Fbxo11tm2b⁣/ + mice. (A) Hematoxylin-eosin stained transverse sections through the middle ear of 3-weeks and 2-months-old mice showing air-filled middle ear cavity for both heterozygote Fbxo11tm2b⁣/ + and wild-type Fbxo11+/+ mice. Scale bars: 1 mM. MEC: middle ear cavity. (B) Comparison of the weight of the mice at the age of 2-months. The weights were taken from six mice from each sex and genotype except for the female heterozygote mice for which we had seven mice. (C) Comparison of the thickness of the epithelial lining of the middle ear of 2-months-old mice. The measurements were taken from three mice (six ears) from each genotype. (D) Broadband click stimuli ABR thresholds in the right ears of 2-months-old wild-type Fbxo11+/+ and heterozygote Fbxo11tm2b⁣/ + mice. Two wild-type males, three wild-type females, two heterozygote males and three heterozygote females were used for the test. As the data was exactly the same (40dB) for both heterozygote males used for this study, the error bars are not well visible in the graph. Bars: standard error of mean. P-values were determined using two-tailed t-test, ∗p ≤ 0.05.

Fig 4: OM phenotype of the Fbxo11tm2b⁣/ + mice after inoculation with NTHi. (A) Hematoxylin-eosin stained transverse sections through the middle ear of 2-months-old wild-type Fbxo11+/+ and heterozygote Fbxo11tm2b⁣/ + mice after the challenge. Scale bars: 100 μM. (B) Comparison of the thickness of the epithelial lining of the middle ear for each genotype after the challenge. The measurements were taken from two mice (one male and one female, three ears) from each genotype. Bars: standard error of mean. P-values were determined using two-tailed t-test. ∗∗∗p ≤ 0.001. (C) Immunohistochemistry to detect different cell types in the middle ear fluid: cleaved caspase 3 antibody (cl. casp. 3) detecting apoptotic cells; F4/80 detecting foamy macrophages; myeloperoxidase antibody (MPO) detecting neutrophils and antibody against NTHi162sr (NTHi) detecting NTHi bacteria in the middle ear fluid. Scale bars: 100 μM. (D) Negative controls for the staining for antibodies raised in rat (for F4/80) or rabbit (all the other). Scale bars: 100 μM. (E) The AB-PAS staining detected goblet cells in the epithelial lining of the Fbxo11tm2b⁣/ + mice middle ear cavity. Scale bars: 50 μM.

Fig 5: Generation of the Fbxo11 knock-out mice. (A) Schematic representation of the knock-out strategy used by EUCOMM to produce the mice (not to scale), illustrating the wild type structure of the Fbxo11 gene from exons 2–5 and the structure of the mutant tm2a allele generated (see results). The tm2b allele is derived by cre excision of the tm2a allele, deleting exon 4. (B) Protein levels of FBXO11 in E18.5 embryonic lungs. Neither the 177A antibody which detects the large isoform of mouse FBXO11 (FBXO11-001, 930 aa, 104 kDa) nor the 178A, which detects both the large and small isoforms (FBXO11-001, 930 aa, 104 kDa and FBXO11-004, 855 aa, 94 kDa) detected the protein in the homozygote embryonic lung tissue. Actin was used as loading control. Truncated protein, 18 kDa, was detected in the mutant tissues. Cross- reactive bands are indicated by an asterisk. (C) Alignment of the two FBXO11 isoforms (FBXO11-001 and FBXO11-004), the fragments of FBXO11 protein used to produce the A301-177A and A301-178A antibodies for FBXO11 and the targeted region in the knock-out (not to scale).