Fig 2: The DROSHA and AIM2 expression levels were elevated in alveolar macrophages of patients with IPF. (A) Representative immunofluorescence image of CD68 (Green), DROSHA (Red), and DAPI (Blue) staining in lung tissues from patients from IPF (IPF) or healthy subjects (Control). Positive area and cells are indicated by white arrows. Scale bars, 200 µm. Quantification of co-localization positive cells between DROSHA and CD68 (The percent of co-localization positive cells in total 100 cells in 10 individual images per group) (right) in lung tissues from patients with IPF (IPF, n = 5) or non-IPF patients (Control, n = 5). (B) Representative immunofluorescence image of CD68 (Green), AIM2 (Red) and DAPI (Blue) staining in lung tissues from patients with IPF or non-IPF patients (Control). Positive area and cells are indicated by white arrows. Scale bars, 200 µm. Quantification of co-localization positive cells between AIM2 and CD68 (the percent of co-localization positive cells in total 100 cells in 10 individual images per group) (right) in lung tissues from patients with IPF (IPF, n = 5) or non-IPF patients (Control, n = 5). (C) Representative immunofluorescence image of AIM2 (Green), DROSHA (Red), and DAPI (Blue) staining in lung tissues from patients with IPF or non-IPF patients (Control). Positive area and cells are indicated by white arrows. Scale bars, 200 µm. Quantification of co-localization positive cells between DROSHA and AIM2 (the percent of co-localization positive cells in total 100 cells in 10 individual images per group) (right) in lung tissues from patients with IPF (IPF, n = 3) or non-IPF patients (Control, n = 3). Data are mean ± SEM. *** p <0.001, ** p <0.01; by Student’s two-tailed t-test.

Fig 3: Deficiency of DROSHA suppresses AIM2 inflammasome activation in alveolar macrophages. (A) Quantification of interleukin (IL)-1ß (left), IL-18 (middle), and TNF-a (right) secretion from wild-type (WT) alveolar macrophages transduced with DROSHA-targeting gRNA (Drosha gRNA), or with a control plasmid (Control), and stimulated with lipopolysaccharide (LPS) and poly(dA:dT). (n = 9 mice per group). (B) Representative immunoblot analysis for caspase-1 and IL-1ß (left) and densitometry quantification of caspase-1 p10 and IL-1ß p17 levels (normalized to levels of ß-actin) (right) from WT alveolar macrophages transduced with DROSHA-targeting gRNA (Drosha gRNA), or with a control plasmid (Control), and stimulated with LPS and poly(dA:dT). For immunoblots, ß-actin was used as loading control. (n = 3 mice per group). (C) Quantification of IL-1ß and IL-18 secretion from WT alveolar macrophages transduced with DROSHA-targeting gRNA (Drosha gRNA), or with a control plasmid (Control), and stimulated with LPS and either ATP, flagellin, or MDP (n = 9 mice per group). (D) Representative immunofluorescence images (total 100 cells in 10 individual images per group) (left) and quantification (right) of ASC speck formation (white arrows) (the number of ASC speck positive cells in 10 individual images per group) in WT alveolar macrophages transduced with DROSHA-targeting gRNA (Drosha gRNA), or with a control plasmid (Control), and stimulated with LPS and poly(dA:dT). (n = 6 mice per group). Scale bars, 20 µm. Data are mean ± SEM. ** p <0.01; by Student’s two-tailed t-test or ANOVA.

Fig 4: Deficiency of DROSHA suppresses AIM2 inflammasome activation in macrophages. (A) Quantification of IL-1ß (left), IL-18 (middle) and TNF-a (right) secretion from WT bone marrow-derived macrophages (BMDMs) transduced with DROSHA -targeting small interfering RNA (Drosha siRNA), or with a control plasmid (Control), and stimulated with LPS and poly(dA:dT) (n = 9 mice per group). (B) Representative immunoblot analysis for caspase-1 and IL-1ß (left) and densitometry quantification of caspase-1 p10 and IL-1ß p17 levels (normalized to levels of ß-actin) (right) from WT BMDMs transduced with DROSHA -targeting siRNA (Drosha siRNA), or with a control plasmid (Control), and stimulated with LPS and poly(dA:dT). For immunoblots, ß-actin was used as loading control (n = 3 mice per group). (C) Quantification of IL-1ß and IL-18 secretion from WT BMDMs transduced with DROSHA -targeting siRNA (Drosha siRNA), or with a control plasmid (Control), and stimulated with LPS and either ATP, flagellin, or MDP (n = 9 mice per group). (D) Representative immunofluorescence images (total 100 cells in 10 individual images per group) (left) and quantification (right) of ASC speck formation (white arrows) (the number of ASC speck positive cells in 10 individual images per group) in WT BMDMs transduced with DROSHA -targeting siRNA (Drosha siRNA), or with a control plasmid (Control), and stimulated with LPS and poly(dA:dT). (n = 6 mice per group). Scale bars, 20 µm. Data are mean ± SEM. ** p <0.01; by Student’s two-tailed t-test or ANOVA.

Fig 5: The DROSHA protein levels were elevated in lung tissues of patients with idiopathic pulmonary fibrosis (IPF). (A) Representative immunohistochemistry image of fibrosis by Masson’s trichrome (M/T) staining in lung tissues from patients with IPF (IPF) or non-IPF patients (Control). Positive area and cells are indicated by black arrow. In the low magnification image (top), scale bars are 200 µm. In the high magnification image (bottom) scale, bars are 50 µm. Results are representative of three independent experiments. (B) Representative immunohistochemistry image (left) of DROSHA and quantification of DROSHA-positive cells (the percent of DROSHA-positive cells in total 100 cells in 10 individual images per group) (right) in lung tissues from patients with IPF (IPF, n = 12) or non-IPF patients (Control, n = 9). Positive area and cells are indicated by a black arrow. In low magnification image (top), scale bars are 200 µm. In high magnification image (bottom) scale, bars are 50 µm. (C) Representative immunoblot analysis for DROSHA and DGCR8 (left) and densitometry quantification of DROSHA and DGCR8 levels (normalized to levels of a-tubulin) (right) from lung tissues from patients with IPF (IPF, n = 11) or non-IPF patients (Control, n = 11). (D) Representative immunoblot analysis for absent in melanoma 2 (AIM2) (left) and densitometry quantification of AIM2 levels (normalized to levels of ß-actin) (right) from lung tissues from patients with IPF (IPF, n = 12) or non-IPF patients (Control, n = 12). For immunoblots, a-tubulin or ß-actin was used as loading control. Data are representative of three independent experiments. Data are mean ± SEM. *** p <0.001, * p <0.05; by Student’s two-tailed t-test.