Fig. 2. Characterization of INTS13 variants using patients’ cells.

a RT-qPCR shows that endogenous INTS13 transcript levels were significantly reduced relative to control cells in primary dermal fibroblasts of affected individual II.4 from Family 1. Data are mean + /- SD, n = 3 replicate cell samples. Statistical significance was calculated using a two-tailed unpaired t-test. p = 0.000223. b Primary dermal fibroblasts were treated with cycloheximide (CHX) (100 g/ml). Following treatment, qRT-PCR was done at 8 h and shows that INTS13 mRNA level was increased in treated cells indicating a non-sense mediated decay. Data are mean ± s.e.m., *P < 0.05; **P < 0.005; ***P < 0.001; one-tailed Student’s t-test. c Western blot analysis of primary dermal fibroblasts of affected individual II.4 from Family 1 using two polyclonal antibodies with antibody directed against the C-terminus of INTS13 (C) and the second antibody directed against the center of INTS13 (M). Actin was used as a loading control and molecular weight markers shown are in kilodaltons. d RT-qPCR shows that INTS13 transcript levels were significantly reduced in primary dermal fibroblast cells of affected individual II.4 from Family 2 relative to control cells. Data are mean + /- SD, n = 3 replicate cell samples. Statistical significance was calculated using a two-tailed unpaired t-test. p = 0.003833. e Western blot analysis of primary dermal fibroblasts of affected individual II.4 from Family 2 shows reduced levels of INTS13 protein compared to control cells. f Western blot analysis of primary dermal fibroblasts from individual II.4 from Family 2 treated with MG132. Treated cells show a level of INTS13 protein comparable to control cells. * indicates a non-specific signal. Multiple western blots were developed (> 3), and the results shown are representative of the data obtained. g Multiciliated airway cells were obtained from nasal biopsies of three members of Family 1: the unaffected carrier mother (Con.) and the two affected children (II.4 and II.5). Probing for acetylated α-tubulin shows significantly shorter, less dense, and disorganized cilia in the affected individuals compared to the mother’s cells. scale bar is 5 μm. h The average length of nasal cilia is significanty reduced in the two affected sisters compared to that of their mother. Data are mean ± s.e.m., ***P < 4 × 10⁻⁹, n ≥ 31 cells for each patient in one biological replicate. Source data are provided as a Source Data file.