Fig 2: DCLK1 knockout preserves lung function, mitigates morphological damage, and decreases inflammation in BLM-treated mice. A Lung compliance, normalized work of breathing, and pressure–volume loops measured using the FlexiVent FX system (n = 7–9 per group). B–D microCT scans, three-dimensional reconstructions, and quantification of lung volume on day 21 (n = 4 per group). E Mean HU density histograms derived from segmented lung maps on day 21 (n = 4 per group). F Percentage of peripheral blood neutrophils quantified after euthanasia (n = 5–9 per group). n represents the number of independent animals per group. Data are presented as means ± SEMs. A two-way ANOVA and Bonferroni’s multiple comparisons test were used to determine significance relative to BLM-treated control mice. *p < 0.05 was considered significant. ANOVA, analysis of variance. BLM, bleomycin. Crs, lung compliance. HU, Hounsfield unit. microCT, micro computed tomography. nWoB, normalized work of breathing. SEM, standard error of the mean

Fig 3: Akt/DCLK1/Smad3 signaling is associated with TGF-β-induced CTGF expression in NHLFs. A–B Immunohistochemistry results for phospho-Akt, Akt, phospho-Smad3, and Smad3 in lung tissues from PBS-treated and BLM-treated control mice with or without DCLK1 deletion (n = 7–9 per group; 3 nonoverlapping fields analyzed per animal; original magnification, 4 × and 10 ×). A two-way ANOVA and Bonferroni’s multiple comparisons test were used to determine significance. C Western blot analysis of phospho-Akt in NHLFs stimulated with TGF-β (10 ng/mL) for 30 min (n = 3). An unpaired t test was used to determine significance. D–F Western blot analysis of phospho-DCLK1, fibronectin, and CTGF in NHLFs transfected with pcDNA or DN-Akt, followed by TGF-β stimulation for 30 min (DCLK1), 2 h (CTGF), or 24 h (fibronectin; n = 3 per experiment). A one-way ANOVA and Dunnett’s test were used to determine significance. G Western blot confirmation of Akt inhibition (n = 3). An unpaired t test was used to determine significance. H Western blot analysis of phospho-Smad3 in NHLFs transfected with DCLK1 siRNA or scrambled siRNA and stimulated with TGF-β for 30 min (n = 4). A one-way ANOVA and Dunnett’s test were used to determine significance. I Western blot analysis of Smad3 phosphorylation in NHLFs overexpressing DCLK1 and stimulated with TGF-β for 30 min (n = 4). An unpaired t test was used to determine significance. J Coimmunoprecipitation of DCLK1 and Smad3 after TGF-β stimulation for 30 min (n = 6). An unpaired t test was used to determine significance. K Chromatin immunoprecipitation analysis using a DCLK1 antibody targeting the Smad3-binding region of the CTGF promoter after TGF-β stimulation for 30 min (n = 3). An unpaired t test was used to determine significance. L Immunofluorescence results for DCLK1 (green), Smad3 (red), and DAPI (blue) in NHLFs after TGF-β stimulation for 30 min (n = 4; original magnification, 40 ×). M Western blot analysis of CTGF expression in NHLFs pretreated with DCLK1-IN-1 (10 μM, 30 min) before TGF-β stimulation for 2 h (n = 3). A one-way ANOVA and Dunnett’s test were used to determine significance. n represents the number of independent animals per group (in vivo) or independent biological replicates derived from separate cell cultures (in vitro). Data are presented as means ± SEMs. *p < 0.05 was considered significant. ANOVA, analysis of variance. BLM, bleomycin. Co-IP, coimmunoprecipitation. CTGF, connective tissue growth factor. DAPI, 4′,6-diamidino-2-phenylindole. DCLK1, doublecortin-like kinase 1. DN-Akt, dominant negative Akt. DCLK1-IN-1, doublecortin-like kinase 1 inhibitor 1. NHLF, normal human lung fibroblast. PBS, phosphate-buffered saline. SEM, standard error of the mean. siRNA, small interfering RNA. TGF-β, transforming growth factor beta

Fig 4: DCLK1-IN-1 preserves lung function, reduces morphological damage, and decreases inflammation in BLM-treated mice. A Lung compliance, normalized work of breathing, and pressure–volume loops measured by using the FlexiVent FX system (n = 5–8 per group). B–D MicroCT scans, three-dimensional reconstructions, and quantification of lung volume on day 28 (n = 4–5 per group). E Mean HU density histograms derived from segmented lung maps at day 28 (n = 4–5 per group). F Percentage of peripheral blood neutrophils quantified after euthanasia (n = 5–9 per group). G Schematic of signaling pathways indicating TGF-β–induced DCLK1 expression through Smad3 and NF-κB and proposed model linking TGF-β-induced DCLK1 expression with Akt/DCLK1/Smad3-associated signaling and profibrotic marker expression. n represents the number of independent animals per group. Data are presented as means ± SEMs. A one-way ANOVA and Dunnett’s test were used to determine significance compared with BLM-treated control mice. *p < 0.05 was considered significant. α-SMA, alpha–smooth muscle actin. ANOVA, analysis of variance. BLM, bleomycin. Crs, lung compliance. CTGF, connective tissue growth factor. DCLK1-IN-1, doublecortin-like kinase 1 inhibitor 1. HU, Hounsfield unit. microCT, micro computed tomography. NF-κB, nuclear factor kappa B. nWoB, normalized work of breathing. SEM, standard error of the mean. TGF-β, transforming growth factor beta

Fig 5: DCLK1 knockout attenuates PF and reduces fibrotic protein expression in BLM-treated mice. A Experimental design revealing whole-body DCLK1 deletion and BLM intratracheal administration. Tamoxifen (80 mg/kg, i.p.) was administered daily for 7 days before BLM or PBS administration and twice weekly from day 1 to 21 after administration. B H&E and Masson trichrome staining of lung tissue (n = 7–9 per group; original magnification, 4 × and 10 ×). C Immunofluorescence results for DCLK1 (green), α-SMA (red), FSP (purple), and DAPI (blue; n = 7 per group; original magnification, 20 ×). D Immunohistochemistry results for fibronectin, α-SMA, CTGF, DCLK1, and phospho-DCLK1 (n = 7–9 per group; 3 nonoverlapping fields analyzed per animal; original magnification, 4 × and 10 ×). E Western blot analysis of lung lysates for fibronectin, α-SMA, CTGF, DCLK1, and phospho-DCLK1 (n = 7–9 per group). n represents the number of independent animals per group. Data are presented as means ± SEMs. A two-way ANOVA and Bonferroni’s multiple comparisons test were used to determine significance relative to BLM-treated control mice. *p < 0.05 was considered significant. α-SMA, alpha–smooth muscle actin. ANOVA, analysis of variance. BLM, bleomycin. CTGF, connective tissue growth factor. DAPI, 4′,6-diamidino-2-phenylindole. DCLK1, doublecortin-like kinase 1. FSP, fibroblast surface protein. H&E, hematoxylin and eosin. i.p., intraperitoneal. i.t., intratracheal. SEM, standard error of the mean