LAPTM4B levels are decreased in IPF and BLM-induced fibrotic lungs. (A) Representative images of IHC staining of LAPTM4B in paraffin sections of lung tissues from IPF patients and non-IPF donors. The boxed regions in images captured under 10× objective were showed in detail under 40× objective, scale bar: 50 μm. (B) Representative confocal images of immunofluorescent staining showing Laptm4b (green), Vimentin (red), and AGER (gray) in frozen sections of mouse lung tissues at 14 days post single-dose BLM challenge or saline treatment. Cell nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (C) Western blot and (D) densitometry analysis of Laptm4b and Collagen1 in lung homogenates from bleomycin-induced fibrotic models. β-Tubulin served as a loading control. (mean ±SD, n = 6 mice per group). (E) Western blot (F) and densitometry analysis of Laptm4b in lung homogenates from BLM-induced ALI model. β-actin served as a loading control. (mean ±SD, Saline: n = 4, and BLM: n = 5 mice). (G) Western blot and (H) densitometry analysis of Laptm4b in lung homogenates from LPS-induced ALI model. β-actin served as a loading control. (mean ±SD, n = 6 mice per group). (I) Uniform Manifold Approximation and Projection (UMAP) plot from the Human Lung Cell Atlas, showing the distribution of different lung cell types. Each color represents a distinct cell type. (J) UMAP plot and (K) dot plot illustrating the expression levels of LAPTM4B across various lung cell types. (L) Representative confocal images of immunofluorescent staining showing Laptm4b (green), and Sfptc-tdTomato (red) in mice lungs, DAPI (blue) was used to label cell nuclei. *** p < 0.001, **** p < 0.0001.

AAV shRNA-mediated Laptm4b knockdown exacerbates BLM-induced lung fibrosis. (A) Schematic diagram illustrating the experimental design. (B) Representative axial micro-CT scan images of the mouse lungs. (C) Representative images of hematoxylin and eosin (H&E) staining, and Masson’s trichrome staining of lung sections. Scale bar: 200 μm. (D) Ashcroft score analysis of H&E-stained lung sections (mean ± S.D., n = 6). (E) Quantification of the total number of WBC in BALF (mean ± S.D, n = 6). (F) Mouse whole lung weight vs body weight (mean ± S.D., n = 8). (G) Measurement of hydroxyproline content in the right lungs (mean ±S.D., n = 6-8). (H) Western blot analysis and (I) quantification of Fibronectin, Collagen 1, α-Sma and Laptm4b levels in mouse lung homogenates (mean ± S.D., n = 3). β-Tubulin was used as an internal control. All statistical analysis was performed using ANOVA followed by Tukey post hoc corrections. *p < 0.05, **p < 0.01,***p < 0.001, and ****p < 0.0001; ns, not significant.

LAPTM4B plays a crucial role in maintaining the homeostasis of epithelial and fibroblast cells and attenuates TGF-β1-induced pro-fibrotic response. (A) Confocal images of immunofluorescent staining of E-cadherin (green) and Vimentin (red) in sh-NC and sh-MUT A549 cells. DAPI (blue) was used to label cell nuclei, scale bar: 50 μm. (B) Representative images of scratch assay conducted on sh-NC and sh-LAPTM4B A549 cells. The red dotted lines delineated the wound edges. (C) Quantification of wound closure areas from (B), data presented as % of the original wound areas (mean ± S.D., n = 3). (D) Western blot and (E) densitometry analysis (mean ± SD, n = 3) of E-cadherin, N-cadherin, Vimentin, and LAPTM4B in sh-NC and sh-LAPTM4B A549 cells. β-actin was used as an internal control. (F) Representative confocal images of immunofluorescent staining of LAPTM4B and α-SMA in MRC-5 cells transfected with si-NC or si-LAPTM4B for 48 h. DAPI (blue) was used to label cell nuclei, scale bar: 50 μm. (G) Western blot and (H) densitometry analysis of Fibronectin, Collagen1, α-SMA, and LAPTM4B in NC and si-LAPTM4B MRC-5 cells. β-actin was used as an internal control. (I) Western blot and (J) densitometry analysis (mean ± SD, n = 3) of Fibronectin, Collagen1, and α-SMA in MRC-5 cells transfected with LAPTM4B-Flag followed by TGF-β1 treatment for 48 h. β-actin was used as an internal control. (K) Collagen gel contraction assay and (L) quantification (mean ± SD, n = 3) performed on MRC-5 cells transfected with LAPTM4B-Flag or vector control for 24 h, followed by treatment with TGF-β1 for an additional 24 h. (M) Collagen gel contraction assay and (N) quantification (mean ± SD, n = 3) performed on MRC-5 cells transfected with si-NC or si-LAPTM4B for 24 h, followed by treatment with TGF-β1 for an additional 24 h. Statistical analysis was performed using ANOVA followed by Dunnett (C, and E) or Tukey (J) post hoc corrections, and unpaired two-tailed t test (H). *p < 0.05, **p < 0.01,***p < 0.001, and ****p < 0.0001; ns, not significant.

LAPTM4B dampens the TGFβ/SMADs signaling pathway. (A) Western blot analysis of p-SMAD2, total SMAD2, and LAPTM4B in sh-NC and sh-LAPTM4B A549 cells treated with TGF-β1 (5 ng/mL) for indicated durations. GAPDH served as loading control. (B) Relative phosphorylation levels of SMAD2, represented as p-SMAD2/total SMAD2 (mean ± S.D., n = 3). (C) Western blot analysis of p-SMAD2, total SMAD2, and LAPTM4B in MRC-5 cells transfected with si-NC or si-LAPTM4B for 48 h, followed by TGF-β1 (5 ng/mL) treatment for indicated durations. β-Actin served as loading control. (D) Relative phosphorylation levels of SMAD2, represented as p-SMAD2/total SMAD2, were quantified (mean ± S.D., n = 3). (E) Western blot analysis of p-SMAD2, total SMAD2, and LAPTM4B in MRC-5 cells transfected with LAPTM4B-Flag for 48 h, followed by TGF-β1 (5 ng/mL) treatment for indicated durations, β-actin served as loading control. (F) Relative phosphorylation levels of SMAD2, represented as p-SMAD2/total SMAD2 (mean ± S.D., n = 3). (G) Representative confocal images of immunofluorescence staining showing LAPTM4B (green) and HA-tag (red) in sh-NC or sh-LAPTM4B A549 cells transfected with HA-SMAD3 overexpression plasmid for 48 hours. DAPI was used to label cell nuclei, scale bar: 10 μm. (H) Representative confocal images of immunofluorescence staining showing Flag-tag (green) and SMAD3 (red) in A549 cells transfected with LAPTM4B-Flag for 48 hours followed by treatment with or without 5 ng/mL TGF-β1 for an additional 30 min. DAPI was used to label cell nuclei, scale bar: 10 μm. (I) Relative pSBE4 luciferase activity in MRC-5 cells transfected with either si-NC or si-LAPTM4B, followed by followed by treatment with or without 5 ng/mL TGF-β1 (mean ± S.D., n = 3). (J) Relative pSBE4 luciferase activity in MRC-5 cells transfected with either vector control or LAPTM4B-Flag, followed by treatment with or without 5 ng/mL TGF-β1 (mean ±S.D., n = 3). All statistical analysis was performed using ANOVA followed by Tukey post hoc corrections. ***p < 0.001, and ****p < 0.0001; ns, not significant.

LAPTM4B interacts with and destabilizes TGFBR2 and SMAD3. (A) A549 cells were transfected with TGFBR2-HA for 48 hours, followed by treatment with or without 5 ng/mL TGF-β1 for an additional 30 min. Representative confocal microscopy images of LAPTM4B (green) and HA-tag (red), with cell nuclei stained with DAPI (blue). Detailed views of the boxed regions are shown. Scale bar: 5 μm. (B) A549 cells were co-transfected with LAPTM4B-Flag and TGFBR2-HA expression plasmids for 48 h. Representative N-SIM images of Flag (green) and HA-tag (red), with cell nuclei stained with DAPI (blue). Detailed views of the boxed regions are shown. Arrow heads indicate overlapped areas. (C) 293T cells were transfected with LAPTM4B-Flag and TGFBR2-HA, followed by treatment with or without 5 ng/mL TGF-β1 for an additional 30 min. Whole-cell lysates were immunoprecipitated with anti-Flag beads. (D) A549 cells were transfected with LAPTM4B-Flag for 48 hours, followed by treatment with 5 ng/mL TGF-β1 for an additional 30 min. Representative N-SIM images of Flag (red) and SMAD3 (green), with cell nuclei stained with DAPI (blue). Detailed views of the boxed regions are shown. (E) 293T cells were transfected with LAPTM4B-Flag, HA-SMAD3, and Myc-tagged ubiquitin, followed by treatment with 5 ng/mL TGF-β1 for an additional 30 min. Whole-cell lysates were immunoprecipitated with anti-HA beads. (F) Western blot and (G) densitometry analysis of p-SMAD2, total SMAD2, and LAPTM4B in A549 cells were transfected with LAPTM4B-Flag or vector for 48 hours, followed by treatment with MG132 (2 μM) for 2 hours before stimulation with 5 ng/mL TGF-β1 for an additional 30 min. β-Tubulin served as a loading control (mean ± S.D., n = 3). (H) 293T cells were transfected with LAPTM4B-Flag and TGFBR2-HA, or (I) HA-SMAD3, along with Myc-tagged UbK48O or Myc-tagged UbK63O, and incubated for 48 h. Subsequently, the cells were treated with 5 ng/mL TGF-β1 for an additional 30 min, followed by whole-cell lysis and immunoprecipitation with anti-HA beads. Statistical analysis was performed using ANOVA followed by Tukey post hoc corrections (G). **p < 0.01, ****p < 0.0001; ns, not significant.

LAPTM4B-mediated recruitment of NEDD4L to endosomes facilitates ubiquitination of active TGFBR2 and SMAD3. (A) Confocal images of immunofluorescence staining for LAPTM4B-Flag (green) and HA-NEDD4L (red) in A549 cells, with cell nuclei stained with DAPI (blue). Scale bar: 10 μm. Detailed views of the boxed regions are shown. (B) N-SIM images of immunofluorescence staining for LAPTM4B-Flag (green) and endogenous NEDD4L (red) in A549 cells. Representative N-SIM images show Flag (green) and HA-tag (red), with cell nuclei stained with DAPI (gray). Detailed views of the boxed regions are shown. (C) 293T cells were transfected with LAPTM4B-Flag and HA-NEDD4L, and whole-cell lysates were immunoprecipitated with anti-Flag or anti-HA beads. (D) 293T cells were transfected with Flag-NEDD4L, HA-SMAD3, and Myc-tagged ubiquitin, followed by treatment with 5 ng/mL TGF-β1 for an additional 30 min. Whole-cell lysates were immunoprecipitated with anti-HA beads. (E) Western blot analysis of p-SMAD2, total SMAD2, and HA in A549 cells transfected with vector or HA-NEDD4L for 48 hours, followed by TGF-β1 (5 ng/mL) treatment for the indicated durations. β-Tubulin served as a loading control. (F) Western blot analysis of p-SMAD2, total SMAD2, and NEDD4L in A549 cells transfected with si-NC or si-NEDD4L for 48 hours, followed by TGF-β1 (5 ng/mL) treatment for the indicated durations. β-actin served as a loading control. (G) Western blot and (H) densitometry analysis of p-SMAD2, total SMAD2, NEDD4L, and Flag-tag in A549 cells transfected with the indicated siRNAs and plasmids for 48 hours, followed by TGF-β1 (5 ng/mL) treatment for 30 min. GAPDH served as a loading control (mean ± S.D., n = 3). (I) Relative pSBE4 luciferase activity in A549 cells transfected with the indicated siRNAs and plasmids, followed by TGF-β1 treatment (mean ± S.D., n = 3). (J) Western blot and (K) densitometry analysis of p-SMAD2, total SMAD2, LAPTM4B, and HA-tag in A549 cells transfected with the indicated siRNAs and plasmids for 48 hours, followed by TGF-β1 (5 ng/mL) treatment for 30 min. β-actin served as a loading control. (I) Relative pSBE4 luciferase activity in A549 cells transfected with the indicated siRNAs and plasmids, followed by TGF-β1 treatment (mean ± S.D., n = 3). All statistical analysis was performed using ANOVA followed by Tukey post hoc corrections. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

AAV-mediated LAPTM4B overexpression attenuates BLM-induced lung fibrosis. (A) Schematic diagram illustrating the experimental design. (B) Body weight changes of AAV Laptm4b or AAV-vector-infected mice treated with saline control or BLM (mean ±SD, n = 8 mice in Saline group, and 10 mice per group in other groups). (C) Representative Micro-CT images of mouse lungs (D) Hematoxylin and eosin (H&E) staining and Masson’s trichrome staining of lung sections (scale bar = 200 μm). (E) Ashcroft score analysis of H&E-stained lung sections (mean ±S.D, n = 6). (F) Quantification of the total number of WBC in BALF (mean ± SD, n = 6). (G) Mouse lung wet weight vs body weight (mean ± S.D., n = 8). (H) Measurement of hydroxyproline content in the right lungs (mean ±S.D., n = 8). (I) Western blot and (J) quantification of Fibronectin, Collagen1, α-Sma and Laptm4b levels in mouse lung homogenates (mean ±S.D., n = 3), Gapdh was used as loading control. All statistical analysis was performed using ANOVA followed by Tukey post hoc corrections. *p < 0.05, **p < 0.01,***p < 0.001, and ****p < 0.0001; ns, not significant.