Journal: The EMBO Journal

Article Title: TRPML1 suppresses pulmonary fibrosis by limiting collagen and elastin deposition

doi: 10.1038/s44318-026-00712-4

Figure Lengend Snippet: Lung function measurements were performed using the SCIREQs FlexiVent system (see Methods) ( A ). Different maneuvers were applied. Single frequency forced oscillation technique (FOT) allows to study the subject’s response to a sinusoidal waveform, obtaining parameters such as elastance (E) and compliance (C). Broadband FOT measures the subject’s response to a signal including a broad range of frequencies, below and above the subject’s breathing frequency. Outcomes are, e.g., tissue elasticity (H). Deep Inflation inflates the lungs to a total lung capacity state. Initial and end volumes are used to calculate inspiratory capacity (IC). Pressure-volume (PV) loops capture the quasi-static mechanical properties of the respiratory system, such as quasi-static compliance (Cst) and total lung capacity ( A ). ( B ) In 3–5 months old female Trpml1 −/− mice ( Mcoln1 tm1Sasl/J ), a significant increase of elastance (E) of the whole respiratory system was observed, whereas the compliance (C) was reduced (basal, untreated). Likewise, other lung function parameters were changed, in line with a fibrosis-like phenotype. Data were mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001; Student’s t -test, unpaired, two-tailed. One single dot corresponds to one mouse, each. Exact p values were: Compliance, p = 0.0003; Elastance, p = 0.0003; Tissue elasticity, p = 0.0314; Inspiratory capacity, p = 0.0001; Total lung capacity, p < 0.0001; Quasi-static compliance, p = 0.0002. ( C , D ) Representative images, with scale bars in µm as indicated ( C ), and quantification of collagen deposition ( D ) in Masson-Trichrome-stained lung tissue sections from untreated BL6 WT and Trpml1 −/− mouse lungs. Collagen deposition (µm³/µm²) was quantified across 30–40 randomly selected fields of view per lung (6–11 mice per group), with each point representing the mean per mouse. Data were mean ± SEM. * p < 0.05; One-way ANOVA followed by Tukey’s post- hoc test. The exact p value was p = 0.0177. ( E – H ) Representative images and quantification as mean ± SEM of Sirius Red ( E , F ) or Col1a1 ( G , H ) stained lung tissue sections from untreated BL6 WT and Trpml1 −/− mouse lungs. Scale bars in µm as indicated. For quantification, 5–20 selected fields of view per lung (7–14 mice per group) were analysed (random, 20; peribronchovascular region (PBVR), 5–10), with each point representing the mean per mouse. ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA followed by Tukey’s post hoc test. Data were mean ± SEM. Exact p values were: SR ratio (%, PBVR), WT vs Het, p = 0.5073; WT vs KO, p < 0.0001; Het vs KO, p < 0.0001; SR Ratio (%, random), WT vs Het, p = 0.6870; WT vs KO, p = 0.0037; Het vs KO, p = 0.0081. Col1a1 ratio (%, random), WT vs Het, p = 0.9847; WT vs KO, p = 0.0003; Het vs KO, p < 0.0001; Col1a1 ratio (%, PBVR), WT vs Het, p = 0.9295; WT vs KO, p = 0.0002; Het vs KO, p < 0.0001. ( I ) Quantification of Verhoeff-Van Gieson-stained lung tissue sections from untreated BL6 WT and Trpml1 −/− mouse lungs, as shown in Fig. . For quantification, five selected fields of view per lung (7–14 mice per group) were analysed (counts of E-fibers per field), with each point representing the mean per mouse. **** p < 0.0001; One-way ANOVA followed by Tukey’s post hoc test. Data were mean ± SEM. Exact p values were: Counts of E-fibers, WT vs Het, p = 0.9266; WT vs KO, p < 0.0001; Het vs KO, p < 0.0001. ( J ) Quantification of the levels of desmosine in BALF isolated from WT and Trpml1 −/ − mice, using ELISA (normalized mean values ± SEM): One single dot corresponds to one biologically independent sample, i.e., one mouse. Statistical analysis was performed with Student’s t -test, unpaired, two-tailed, *** p < 0.001. Trpml1 −/− values were normalized to gender-matched WT controls within each experiment. Exact p value was p = 0.008. The data represent the combined results of three independent experiments. .

Article Snippet: The following ELISAs were used for the experiments: SP-A ELISA (NBP2-76693, Novus biologicals), Cathepsin K ELISA (NBP3-00426, Novus biologicals), TIMP1 ELISA (ab196265, Abcam), TIMP2 ELISA (ab227893, Abcam), MMP1 ELISA (NBP3-06885, Novus biologicals and ABIN6963621, Antibodies online), MMP2 ELISA (ab254516, Abcam), MMP3 ELISA (ab100731), MMP8 ELISA (ab206982, Abcam), MMP9 ELISA (MMPT90, R&D systems), MMP12 ELISA (ab213878, Abcam), MMP13 ELISA (NBP3-06930, Novus biologicals), MMP14 ELISA (NBP3-06941, Novus biologicals and ABIN6957687, Antibodies online), MMP19 ELISA (NBP3-06941, Novus biologicals), IL-17A ELISA (ab199081, Abcam), DESMOSINE ELISA (CSB-E14196m, Cusabio).

Techniques: Two Tailed Test, Staining, Isolation, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: TRPML1 suppresses pulmonary fibrosis by limiting collagen and elastin deposition

doi: 10.1038/s44318-026-00712-4

Figure Lengend Snippet: ( A – C ) Quantification of the levels of different inflammatory mediators in BALF, macrophage, and pmLF supernatants (SN) isolated from WT and Trpml1 −/− mice, using Multiplex (FirePlex) ( A – C ) as well as cathepsin K (CathK), surfactant protein A (SP-A), and TIMPs ( D , E ) using ELISA. BALF / pmLF SN: One single dot corresponds to one biologically independent sample i.e., one mouse. AMΦ SN: One single dot corresponds to one well. 8 WT and 8 Trpml1 −/− mice were lavaged to obtain the appropriate number of cells for all wells. Statistical analysis of datasets in ( A ) was performed with multiple t -test, corrected for multiple comparisons using the Holm–Šídák method, *** p < 0.001. Data were mean ± SEM. Statistical analysis of datasets in ( B , C ) was performed using Student’s t -test, unpaired, two-tailed. ( D ) Transcriptomics data of single-cell suspensions from whole WT mouse lungs. Featured plot shows the average expression levels of TIMPs based on unique molecular identifier (UMI) counts (coded by color grading). TIMP expression was determined in 32 different cell types. Shown are TIMP1, 2, 3, 4. .

Article Snippet: The following ELISAs were used for the experiments: SP-A ELISA (NBP2-76693, Novus biologicals), Cathepsin K ELISA (NBP3-00426, Novus biologicals), TIMP1 ELISA (ab196265, Abcam), TIMP2 ELISA (ab227893, Abcam), MMP1 ELISA (NBP3-06885, Novus biologicals and ABIN6963621, Antibodies online), MMP2 ELISA (ab254516, Abcam), MMP3 ELISA (ab100731), MMP8 ELISA (ab206982, Abcam), MMP9 ELISA (MMPT90, R&D systems), MMP12 ELISA (ab213878, Abcam), MMP13 ELISA (NBP3-06930, Novus biologicals), MMP14 ELISA (NBP3-06941, Novus biologicals and ABIN6957687, Antibodies online), MMP19 ELISA (NBP3-06941, Novus biologicals), IL-17A ELISA (ab199081, Abcam), DESMOSINE ELISA (CSB-E14196m, Cusabio).

Techniques: Isolation, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transcriptomics, Single Cell, Expressing

Journal: The EMBO Journal

Article Title: TRPML1 suppresses pulmonary fibrosis by limiting collagen and elastin deposition

doi: 10.1038/s44318-026-00712-4

Figure Lengend Snippet: ( A , B ) Transcriptomics data of single-cell suspensions from whole WT mouse lungs. The featured plot shows the average expression level of MMPs based on unique molecular identifier (UMI) counts (coded by color grading). MMP expression was determined in 32 different cell types. Shown are MMP2, 3, 7, 8, 9, 12, 13, 14, 19. ( C – N ) Quantification of the levels of different MMPs in macrophage (AMΦ and IMΦ) and pmLF supernatants (SN) isolated from WT and Trpml1 −/− mice using ELISA. IMΦ/pmLF SN: One single dot corresponds to one biologically independent sample, i.e., one mouse. AMΦ SN: One single dot corresponds to one well. Six WT and six Trpml1 −/− mice were lavaged to obtain the appropriate number of cells for all wells. Statistical analysis of all datasets was performed using Student’s t -test, unpaired, two-tailed. * p < 0.05, *** p < 0.001. All data were mean ± SEM. Exact p values were: pmLF SN, MMP2, p = 0.0141; AM SN, MMP8, p = 0.0430; pmLF SN, MMP9, p = 0.0241; IM SN, MMP9, p = 0.0327; AM SN, MMP12, p = 0.0002; IM SN MMP12, p = 0.097; pmLF SN, MMP19, p = 0.0124. .

Article Snippet: The following ELISAs were used for the experiments: SP-A ELISA (NBP2-76693, Novus biologicals), Cathepsin K ELISA (NBP3-00426, Novus biologicals), TIMP1 ELISA (ab196265, Abcam), TIMP2 ELISA (ab227893, Abcam), MMP1 ELISA (NBP3-06885, Novus biologicals and ABIN6963621, Antibodies online), MMP2 ELISA (ab254516, Abcam), MMP3 ELISA (ab100731), MMP8 ELISA (ab206982, Abcam), MMP9 ELISA (MMPT90, R&D systems), MMP12 ELISA (ab213878, Abcam), MMP13 ELISA (NBP3-06930, Novus biologicals), MMP14 ELISA (NBP3-06941, Novus biologicals and ABIN6957687, Antibodies online), MMP19 ELISA (NBP3-06941, Novus biologicals), IL-17A ELISA (ab199081, Abcam), DESMOSINE ELISA (CSB-E14196m, Cusabio).

Techniques: Transcriptomics, Single Cell, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: TRPML1 suppresses pulmonary fibrosis by limiting collagen and elastin deposition

doi: 10.1038/s44318-026-00712-4

Figure Lengend Snippet: ( A ) Quantification of the levels of MMP2, 9, 19 in pmLF SN and MMP12 in AMΦ SN isolated from 3.5-month-old WT and Trpml1 −/− mice using ELISA after pretreatment with the TRPML1 agonist WR1-002 (C8). One single dot corresponds to one well. 7 WT and Trpml1 −/− mice were lavaged to obtain the appropriate number of cells for all wells. Statistical analysis of all datasets was performed using Student’s t -test, unpaired, two-tailed. ** p < 0.01. All data were mean ± SEM. Values were normalized to DMSO-treated WT controls. Exact p values were as follows: comparison of WT SN to WT SN treated with WR1-002 (C8) 5 µM - pmLF SN, MMP2, p = 0.0459; pmLF SN, MMP9, p = 0.0371; pmLF SN, MMP19, p < 0.0001; AM SN, MMP12, p = 0.0043. ( B – D ) Cartoons illustrating fibroblasts, macrophages and other cell types in the lung ( B , C ) as well as the trafficking and secretion dynamics of MMPs and TIMPs in WT, Trpml1 −/− , and Trpml3 −/− lung macrophages ( D ). In WT macrophages, MMPs and TIMPs are trafficking through the trans-Golgi network (TGN) and are being secreted either directly from the TGN or via lysosomes (LY). In Trpml1 −/− macrophages, lysosomal exocytosis of such latter MMPs is impaired, leading to reduced MMP levels in the extracellular space. Endocytosis and intracellular trafficking remain intact, and secretion of MMPs directly from the TGN is unaffected. This disruption results in a restrictive lung phenotype. Conversely, Trpml3 −/− macrophages show impaired endocytosis and intracellular trafficking, but lysosomal exocytosis is preserved. This leads to the accumulation of ELS (endolysosomal system) dependent MMPs in the extracellular space, contributing to an obstructive lung phenotype. Trpml1 −/− and Trpml3 −/− show contrasting roles in regulating ELS-dependent MMP secretion and their opposing effects on lung pathology. .

Article Snippet: The following ELISAs were used for the experiments: SP-A ELISA (NBP2-76693, Novus biologicals), Cathepsin K ELISA (NBP3-00426, Novus biologicals), TIMP1 ELISA (ab196265, Abcam), TIMP2 ELISA (ab227893, Abcam), MMP1 ELISA (NBP3-06885, Novus biologicals and ABIN6963621, Antibodies online), MMP2 ELISA (ab254516, Abcam), MMP3 ELISA (ab100731), MMP8 ELISA (ab206982, Abcam), MMP9 ELISA (MMPT90, R&D systems), MMP12 ELISA (ab213878, Abcam), MMP13 ELISA (NBP3-06930, Novus biologicals), MMP14 ELISA (NBP3-06941, Novus biologicals and ABIN6957687, Antibodies online), MMP19 ELISA (NBP3-06941, Novus biologicals), IL-17A ELISA (ab199081, Abcam), DESMOSINE ELISA (CSB-E14196m, Cusabio).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison, Disruption

Journal: The EMBO Journal

Article Title: TRPML1 suppresses pulmonary fibrosis by limiting collagen and elastin deposition

doi: 10.1038/s44318-026-00712-4

Figure Lengend Snippet: Lung function measurements were performed using the SCIREQs FlexiVent system (see Methods) ( A ). Different maneuvers were applied. Single frequency forced oscillation technique (FOT) allows to study the subject’s response to a sinusoidal waveform, obtaining parameters such as elastance (E) and compliance (C). Broadband FOT measures the subject’s response to a signal including a broad range of frequencies, below and above the subject’s breathing frequency. Outcomes are, e.g., tissue elasticity (H). Deep Inflation inflates the lungs to a total lung capacity state. Initial and end volumes are used to calculate inspiratory capacity (IC). Pressure-volume (PV) loops capture the quasi-static mechanical properties of the respiratory system, such as quasi-static compliance (Cst) and total lung capacity ( A ). ( B ) In 3–5 months old female Trpml1 −/− mice ( Mcoln1 tm1Sasl/J ), a significant increase of elastance (E) of the whole respiratory system was observed, whereas the compliance (C) was reduced (basal, untreated). Likewise, other lung function parameters were changed, in line with a fibrosis-like phenotype. Data were mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001; Student’s t -test, unpaired, two-tailed. One single dot corresponds to one mouse, each. Exact p values were: Compliance, p = 0.0003; Elastance, p = 0.0003; Tissue elasticity, p = 0.0314; Inspiratory capacity, p = 0.0001; Total lung capacity, p < 0.0001; Quasi-static compliance, p = 0.0002. ( C , D ) Representative images, with scale bars in µm as indicated ( C ), and quantification of collagen deposition ( D ) in Masson-Trichrome-stained lung tissue sections from untreated BL6 WT and Trpml1 −/− mouse lungs. Collagen deposition (µm³/µm²) was quantified across 30–40 randomly selected fields of view per lung (6–11 mice per group), with each point representing the mean per mouse. Data were mean ± SEM. * p < 0.05; One-way ANOVA followed by Tukey’s post- hoc test. The exact p value was p = 0.0177. ( E – H ) Representative images and quantification as mean ± SEM of Sirius Red ( E , F ) or Col1a1 ( G , H ) stained lung tissue sections from untreated BL6 WT and Trpml1 −/− mouse lungs. Scale bars in µm as indicated. For quantification, 5–20 selected fields of view per lung (7–14 mice per group) were analysed (random, 20; peribronchovascular region (PBVR), 5–10), with each point representing the mean per mouse. ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA followed by Tukey’s post hoc test. Data were mean ± SEM. Exact p values were: SR ratio (%, PBVR), WT vs Het, p = 0.5073; WT vs KO, p < 0.0001; Het vs KO, p < 0.0001; SR Ratio (%, random), WT vs Het, p = 0.6870; WT vs KO, p = 0.0037; Het vs KO, p = 0.0081. Col1a1 ratio (%, random), WT vs Het, p = 0.9847; WT vs KO, p = 0.0003; Het vs KO, p < 0.0001; Col1a1 ratio (%, PBVR), WT vs Het, p = 0.9295; WT vs KO, p = 0.0002; Het vs KO, p < 0.0001. ( I ) Quantification of Verhoeff-Van Gieson-stained lung tissue sections from untreated BL6 WT and Trpml1 −/− mouse lungs, as shown in Fig. . For quantification, five selected fields of view per lung (7–14 mice per group) were analysed (counts of E-fibers per field), with each point representing the mean per mouse. **** p < 0.0001; One-way ANOVA followed by Tukey’s post hoc test. Data were mean ± SEM. Exact p values were: Counts of E-fibers, WT vs Het, p = 0.9266; WT vs KO, p < 0.0001; Het vs KO, p < 0.0001. ( J ) Quantification of the levels of desmosine in BALF isolated from WT and Trpml1 −/ − mice, using ELISA (normalized mean values ± SEM): One single dot corresponds to one biologically independent sample, i.e., one mouse. Statistical analysis was performed with Student’s t -test, unpaired, two-tailed, *** p < 0.001. Trpml1 −/− values were normalized to gender-matched WT controls within each experiment. Exact p value was p = 0.008. The data represent the combined results of three independent experiments. .

Article Snippet: SP-A ELISA kit , Novus biologicals , NBP2-76693.

Techniques: Two Tailed Test, Staining, Isolation, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: TRPML1 suppresses pulmonary fibrosis by limiting collagen and elastin deposition

doi: 10.1038/s44318-026-00712-4

Figure Lengend Snippet: ( A – C ) Quantification of the levels of different inflammatory mediators in BALF, macrophage, and pmLF supernatants (SN) isolated from WT and Trpml1 −/− mice, using Multiplex (FirePlex) ( A – C ) as well as cathepsin K (CathK), surfactant protein A (SP-A), and TIMPs ( D , E ) using ELISA. BALF / pmLF SN: One single dot corresponds to one biologically independent sample i.e., one mouse. AMΦ SN: One single dot corresponds to one well. 8 WT and 8 Trpml1 −/− mice were lavaged to obtain the appropriate number of cells for all wells. Statistical analysis of datasets in ( A ) was performed with multiple t -test, corrected for multiple comparisons using the Holm–Šídák method, *** p < 0.001. Data were mean ± SEM. Statistical analysis of datasets in ( B , C ) was performed using Student’s t -test, unpaired, two-tailed. ( D ) Transcriptomics data of single-cell suspensions from whole WT mouse lungs. Featured plot shows the average expression levels of TIMPs based on unique molecular identifier (UMI) counts (coded by color grading). TIMP expression was determined in 32 different cell types. Shown are TIMP1, 2, 3, 4. .

Article Snippet: SP-A ELISA kit , Novus biologicals , NBP2-76693.

Techniques: Isolation, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transcriptomics, Single Cell, Expressing

Journal: The EMBO Journal

Article Title: TRPML1 suppresses pulmonary fibrosis by limiting collagen and elastin deposition

doi: 10.1038/s44318-026-00712-4

Figure Lengend Snippet: ( A , B ) Transcriptomics data of single-cell suspensions from whole WT mouse lungs. The featured plot shows the average expression level of MMPs based on unique molecular identifier (UMI) counts (coded by color grading). MMP expression was determined in 32 different cell types. Shown are MMP2, 3, 7, 8, 9, 12, 13, 14, 19. ( C – N ) Quantification of the levels of different MMPs in macrophage (AMΦ and IMΦ) and pmLF supernatants (SN) isolated from WT and Trpml1 −/− mice using ELISA. IMΦ/pmLF SN: One single dot corresponds to one biologically independent sample, i.e., one mouse. AMΦ SN: One single dot corresponds to one well. Six WT and six Trpml1 −/− mice were lavaged to obtain the appropriate number of cells for all wells. Statistical analysis of all datasets was performed using Student’s t -test, unpaired, two-tailed. * p < 0.05, *** p < 0.001. All data were mean ± SEM. Exact p values were: pmLF SN, MMP2, p = 0.0141; AM SN, MMP8, p = 0.0430; pmLF SN, MMP9, p = 0.0241; IM SN, MMP9, p = 0.0327; AM SN, MMP12, p = 0.0002; IM SN MMP12, p = 0.097; pmLF SN, MMP19, p = 0.0124. .

Article Snippet: SP-A ELISA kit , Novus biologicals , NBP2-76693.

Techniques: Transcriptomics, Single Cell, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: TRPML1 suppresses pulmonary fibrosis by limiting collagen and elastin deposition

doi: 10.1038/s44318-026-00712-4

Figure Lengend Snippet: ( A ) Quantification of the levels of MMP2, 9, 19 in pmLF SN and MMP12 in AMΦ SN isolated from 3.5-month-old WT and Trpml1 −/− mice using ELISA after pretreatment with the TRPML1 agonist WR1-002 (C8). One single dot corresponds to one well. 7 WT and Trpml1 −/− mice were lavaged to obtain the appropriate number of cells for all wells. Statistical analysis of all datasets was performed using Student’s t -test, unpaired, two-tailed. ** p < 0.01. All data were mean ± SEM. Values were normalized to DMSO-treated WT controls. Exact p values were as follows: comparison of WT SN to WT SN treated with WR1-002 (C8) 5 µM - pmLF SN, MMP2, p = 0.0459; pmLF SN, MMP9, p = 0.0371; pmLF SN, MMP19, p < 0.0001; AM SN, MMP12, p = 0.0043. ( B – D ) Cartoons illustrating fibroblasts, macrophages and other cell types in the lung ( B , C ) as well as the trafficking and secretion dynamics of MMPs and TIMPs in WT, Trpml1 −/− , and Trpml3 −/− lung macrophages ( D ). In WT macrophages, MMPs and TIMPs are trafficking through the trans-Golgi network (TGN) and are being secreted either directly from the TGN or via lysosomes (LY). In Trpml1 −/− macrophages, lysosomal exocytosis of such latter MMPs is impaired, leading to reduced MMP levels in the extracellular space. Endocytosis and intracellular trafficking remain intact, and secretion of MMPs directly from the TGN is unaffected. This disruption results in a restrictive lung phenotype. Conversely, Trpml3 −/− macrophages show impaired endocytosis and intracellular trafficking, but lysosomal exocytosis is preserved. This leads to the accumulation of ELS (endolysosomal system) dependent MMPs in the extracellular space, contributing to an obstructive lung phenotype. Trpml1 −/− and Trpml3 −/− show contrasting roles in regulating ELS-dependent MMP secretion and their opposing effects on lung pathology. .

Article Snippet: SP-A ELISA kit , Novus biologicals , NBP2-76693.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison, Disruption