Fig 1: Endocytosis in WT and Trpml3-/- AMF.a Shown are representative confocal images obtained from endocytosis experiments using dextran coupled to Alexa Fluor 568. Images show AMF (WT vs. Trpml3-/-) that have been pulsed with fluorescently labelled dextran for different time periods. Scale bar 5 µm. b Quantification of dextran uptake showing significantly decreased rates of endocytosis in Trpml3-/- AMF compared to WT AMF at various time points. A sum of at least 130 cells were analysed per timepoint and genotype deriving from five biologically independent experiments for both Trpml3-/- lines (Mcoln3tm1.2Hels and Mcoln3tm1.1Jga), respectively. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Two-way ANOVA followed by Bonferroni’s post hoc test. c Effect of different endocytosis inhibitors on MMP-12 levels in WT and Trpml3-/- AMF supernatants (SN). *p < 0.05, **p < 0.01, ****p < 0.0001; One-way ANOVA followed by Dunnett’s post hoc test. One single dot corresponds to the AMF SN from one well, each. 11 WT and 11 Trpml3-/- mice were lavaged to obtain the number of cells for all wells. Data are mean ± SEM. d Cartoon showing endocytosis of MMP-12 via three different endocytosis pathways (CME, CIE, MP) and the effect of endocytosis inhibitors on MMP-12 uptake: According to the results shown in (c) the MMP-12 uptake in AMF corresponds to CIE and MP, resulting in higher concentrations of MMP-12 in the extracellular fluid after inhibition of these pathways. CME seems to be not involved. e Effect of the selective TRPML3 agonist ML3-SA1 (incubation o.n., 30 µM) on MMP-12 levels in WT and Trpml3-/- AMF supernatants (SN). *p < 0.05, Student’s t test, unpaired, two-tailed. One single dot corresponds to the AMF SN from one well, each. 5 WT and 5 Trpml3-/- mice were lavaged to obtain the appropriate number of cells for all wells. Data are mean ± SEM. Source data are provided as a Source Data file.
Fig 2: Increased MMP-12 levels in WT and Trpml3-/-.a Quantification of the levels of different chemokines/cytokines and MMPs in BALF isolated from 4-month old WT and Trpml3-/- mice using Multiplex analysis. b Repeated Multiplex analysis of MMP-12 levels in BALF. c, d MMP-12 quantification in BALF isolated from 4-month old WT and Trpml3-/- mice using ELISA. One single dot corresponds to BALF from one mouse, each in c, d. e, f qRT-PCR data showing mRNA expression levels of Mmp-12 in AMF (WT and Trpml3-/-). g Quantification of total cell numbers in BALF using the CASY1 cell counter. h Quantification of cell numbers in BALF using morphological criteria on May-Grünwald-Giemsa-stained cytospins. i MMP-12 quantification in the supernatant of cultured AMF isolated from 4-month old WT and Trpml3-/- mice using ELISA. One single dot corresponds to the AMF SN from one well, each. Statistical analysis of datasets a-i was performed by using Student’s t test, unpaired, two-tailed (**p < 0.01, ****p < 0.0001). j, k MMP quantification in the supernatant of cultured AMF isolated from 4-month old WT and Trpml3-/- mice using Multiplex and ELISA. One single dot corresponds to the AMF SN from one well, each. Two-way ANOVA followed by Tukey’s post hoc test; ***p < 0.001 (j) or Student’s t test, unpaired, two-tailed; **p < 0.01 (k). l Desmosine ELISA of BALF isolated from WT and Trpml3-/- mice. One single dot corresponds to BALF from one mouse. Student’s t test, unpaired, two-tailed; **p < 0.01. m Verhoeff-Van Gieson (VVG) staining of formalin-fixed, paraffin-embedded lung sections of female, 4-month old WT or Trpml3-/- mice (Mcoln3tm.1.1Jga) treated either with PBS or porcine pancreatic elastase. Elastic fibers are stained blue-black, collagen appears red, and other tissue elements yellow. Scale bar 100 µm. n Quantification of elastin fibers as counts per field in VVG stained lung tissue sections from 6–8 mice per group. One dot corresponds to the mean count of elastin fibers in 8-10 fields of view per mouse lung. *p < 0.05, ****p < 0.0001; One-way ANOVA followed by Tukey’s post hoc test. In all figures, each single dot corresponds to one biologically independent sample. Data are mean ± SEM. Source data are provided as a Source Data file.
Fig 3: p16−/− lungs maintain function and structure when challenged with CS. a Lung compliance (**p < 0.0177), pressure/volume, and area (**p < 0.0001) between the inflation and deflation limb of the PV loop measured using Flexi-Vent. b Representative Masson’s trichrome staining of lungs exposed to RA or CS (scale bar = 40 µm), mean linear intercept was determined c from these images and quantified (*p < 0.0001 and **p < 0.0001 vs. p16−/− RA). d Body weight increases over 4 months, values are the percentage increase from day 0 (*p = 0.0031, **p < 0.0001). Quantitative RT-PCR analysis e of MMP-12 (*p = 0.029 vs. p16+/+ RA, **p = 0.0077 vs. p16+/+ CS), IL-33 (*p = 0.012 vs. p16+/+ RA, **p = 0.045 vs. p16+/+ CS), and TGFβ1 (*p = 0.0021 vs. p16+/+ RA, **p = 0.0036). After 4 months of CS, all samples were normalized to mouse GAPDH. f MMP-12 (*p = 0.0004 vs. p16+/+ RA, **p = 0.0088 vs. p16+/+ CS), IL-33 (*p = 0.004 vs. p16+/+ RA, **p = 0.023 vs. p16+/+ CS, ***p = 0.0452 vs. p16−/− RA), and TGFβ1 (*p = 0.0005 vs. p16+/+ RA, **p = 0.0097, ***p = 0.0036 vs. p16+/+ RA), protein levels determined by ELISA. All protein levels are normalized to total protein in the lysate. All data is expressed as mean ± SEM, n = 4–14 mice per group
Fig 4: TIMPs in WT and Trpml3-/- BALF and schematic of emphysema development in Trpml3-/- lungs.a, b TIMP-1 and TIMP-2 levels in BALF obtained from WT and Trpml3-/- mice measured by ELISA. Data are mean ± SEM collected from up to 8 mice per genotype per mouse line. Statistical analysis was performed using Student’s t test, unpaired, two-tailed. One single dot corresponds to one mouse, each in a, b. c Scheme showing the mechanism of emphysema development in Trpml3-/- mouse lungs. In WT lungs the amount of MMP-12 outside the AMF is regulated by TIMP-1/2, as well as endocytosis of MMP-12 and lysosomal degradation. We observed increased MMP-12 levels in BALF and lower endocytosis rates in Trpml3-/- AMF. Vice versa selective activation of TRPML3 resulted in reduced MMP-12 levels in BALF. Therefore, it is postulated that loss of TRPML3 results in extracellular matrix (ECM) remodeling and emphysema characterized by destruction of the alveolar walls as depicted. Source data are provided as a Source Data file.
Fig 5: Analysis of selected transcripts and protein levels in miR151 or miR5100 transfected mouse BMSCs. (A) Gene expression levels of selected genes in control or miR151 or miR5100 transfected mouse BMSCs. (B) ELISA analysis of MMP12 amount in medium from in vitro cultures of control or transfected mouse BMSCs. (C) The level of IGFBP2 in control or transfected BMSCs. The differences were considered statistically significant when p < 0.05 (marked with asterisks, * - p < 0.05; ** - p < 0.005; *** - p < 0.001)
Supplier Page from Abcam for Mouse MMP-12 ELISA Kit