Immunohistochemistry and Quantification

Lung samples were fixed overnight with 10% Neutral buffered formalin (Leica Biosystems Inc., Buffalo Grove, IL, United States), dehydrated using 30% sucrose overnight then embedded in optimal cutting temperature (OCT) compound, snap-frozen by liquid nitrogen, and sectioned at 7 μM. Heat mediated antigen retrieval was performed for 20 min in a steamer using Citrate Buffer (Abcam, Cambridge, MA, United States). Slides were incubated with mouse monoclonal anti-Arl13b antibody (75–287, clone N295B/66, UC Davis/NIH NeuroMab Facility, Davis, CA, United States) at 4°C overnight and then incubated with Cy3-rabbit anti-mouse antibody (Jackson ImmunoResearch Labs, West Grove, PA, United States) for 1 h at room temperature (RT), the following day. Nuclear staining and mounting were performed using Fluoroshield with DAPI (Sigma-Aldrich, St. Louis, MO, United States). The percent ciliation was expressed as percentage of Arl13b⁺ cells per section were quantified using ImageJ software from at least six sections that were 100 μm apart for each mouse offspring. This measurement was expressed as percent ciliation.

Total RNA was extracted from lung and liver of mice offspring using RNeasy Mini Kit (Qiagen, Valencia, CA, United States) and reverse-transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, United States). Quantitative PCR was performed with SYBR Green/ROX qPCR master mix (Fermentas, Glen Burnie, MD, United States) using primers for Man1a2 exon-2 (forward: 5′-CAAAGTAGCCCAAGCAATGAA 3′, reverse:5′-TCTTGAT CTCCATGTCTTCTGG-3′), Arl13b (forward: 5′-ACCAGTGGT CTGGCTGAGATTG-3′, reverse:5′-CATCACTGTCCTTCTCC ACGGT-3′), Ift74 (forward: 5′-GGCACAGATAGAAGCCAGC ATC-3′), reverse:5′-CAGCTCTTGGTTGGTGATGGAG-3′), and Dnah11 (forward: 5′-GCCATCACTTCGGTTCCAGAGA-3′, reverse:5′-GGTCTCTTTAGGACTGTCAGGAG-3′), and beta-actin (forward: 5′-ctaaggccaaccgtgaaaag-3′, reverse: 5′-accagaggcatacagggaca-3′) (IDT DNA technologies, Coralville, IA, United States). Target gene expression levels were normalized to beta-actin.