Journal: Scientific Reports

Article Title: Mitochondrial proteomics reveals reductive metabolism dependent on glutamine in fibroblasts of idiopathic pulmonary fibrosis under hypoxia

doi: 10.1038/s41598-025-26911-3

Figure Lengend Snippet: Mitochondrial fraction isolation. ( A ) Electron microscopy images of mitochondria isolated from lung fibroblasts show inner membrane folds suggestive of opened or widened cristae (arrow). Few cristae with vesicular appearance (head arrow). Possible mitochondria without limiting membrane (*). ( B ) Western blot for triose phosphate isomerase (TPI) as a marker of the cytosol, COX IV, and VDAC1 in the mitochondrial fraction; Histone 3 (H3) as a marker of total lysate only. GAPDH was used as a loading control. ( C ) Electrophoresis protein integrity gels. D Mitochondrial load control for each cell line under different oxygenation conditions.

Article Snippet: Two lines of healthy human lung fibroblasts (CCL-201TM, CCL-204TM) and two lines of fibroblasts from patients (CCL-134TM, CCL-191TM) were purchased from American Type Culture Collection (ATCC), and therefore, informed consent was not required for their use.

Techniques: Isolation, Electron Microscopy, Membrane, Western Blot, Marker, Control, Electrophoresis

Journal: Scientific Reports

Article Title: Mitochondrial proteomics reveals reductive metabolism dependent on glutamine in fibroblasts of idiopathic pulmonary fibrosis under hypoxia

doi: 10.1038/s41598-025-26911-3

Figure Lengend Snippet: Analysis of mitochondrial proteome in IPF and controls fibroblasts under hypoxia. ( A ) Principal Component Analysis (PCA) of Control Hypoxia vs. Control Normoxia. ( B ) PCA of IPF Hypoxia vs. IPF Normoxia. Volcano plots show over- and under-expressed proteins in hypoxia for ( C ) healthy controls and ( D ) IPF fibroblasts, upregulated proteins are shown in red dots, and downregulated proteins are shown in green dots. The ( E ) Venn diagram shows differentially expressed mitochondrial proteins in Control Hypoxia vs. Control Normoxia and IPF Hypoxia vs. IPF Normoxia.

Article Snippet: Two lines of healthy human lung fibroblasts (CCL-201TM, CCL-204TM) and two lines of fibroblasts from patients (CCL-134TM, CCL-191TM) were purchased from American Type Culture Collection (ATCC), and therefore, informed consent was not required for their use.

Techniques: Control

Journal: Scientific Reports

Article Title: Mitochondrial proteomics reveals reductive metabolism dependent on glutamine in fibroblasts of idiopathic pulmonary fibrosis under hypoxia

doi: 10.1038/s41598-025-26911-3

Figure Lengend Snippet: Differentially expressed mitochondrial proteins in control fibroblasts under hypoxia. ( A ) Increased and ( B ) decreased protein expression in mitochondria. Box plots illustrate the differences in protein expression, highlighting deregulated proteins in normoxia (Control) and hypoxia (Control Hx), represented by blue and orange boxes, respectively. Additionally, the expression of IPF proteins in normoxia (IPF) and hypoxia (IPF Hx) is depicted by purple and green boxes, respectively.

Article Snippet: Two lines of healthy human lung fibroblasts (CCL-201TM, CCL-204TM) and two lines of fibroblasts from patients (CCL-134TM, CCL-191TM) were purchased from American Type Culture Collection (ATCC), and therefore, informed consent was not required for their use.

Techniques: Control, Expressing

Journal: Scientific Reports

Article Title: Mitochondrial proteomics reveals reductive metabolism dependent on glutamine in fibroblasts of idiopathic pulmonary fibrosis under hypoxia

doi: 10.1038/s41598-025-26911-3

Figure Lengend Snippet: Differentially expressed mitochondrial proteins in IPF fibroblasts under hypoxia. ( A ) Increased and ( B ) decreased protein expression in mitochondria. Box plots illustrate differences in protein expression, highlighting proteins deregulated in normoxia (Control) and hypoxia (Control Hx), represented by blue and orange boxes, respectively. Additionally, IPF protein expression in normoxia (IPF) and hypoxia (IPF Hx) is represented by purple and green boxes, respectively.

Article Snippet: Two lines of healthy human lung fibroblasts (CCL-201TM, CCL-204TM) and two lines of fibroblasts from patients (CCL-134TM, CCL-191TM) were purchased from American Type Culture Collection (ATCC), and therefore, informed consent was not required for their use.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Mitochondrial proteomics reveals reductive metabolism dependent on glutamine in fibroblasts of idiopathic pulmonary fibrosis under hypoxia

doi: 10.1038/s41598-025-26911-3

Figure Lengend Snippet: Validation of the glutaminase response under hypoxic conditions. ( A ) Western blot for GLS in total lysate (Control Normoxia n = 3 and IPF Normoxia n = 5. ® actin was used as a loading control. ( B ) Graph of the densitometric analysis. ( C ) Representative immunoblot of GLS and ® actin was used as a loading Control (normoxia and hypoxia) and IPF 1, 2 and 3 (normoxia and hypoxia). ( D ) Representative images showing the colocalization of α-SMA (red) and Ki67 (green) in control and IPF fibroblasts under normoxic and hypoxic conditions.

Article Snippet: Two lines of healthy human lung fibroblasts (CCL-201TM, CCL-204TM) and two lines of fibroblasts from patients (CCL-134TM, CCL-191TM) were purchased from American Type Culture Collection (ATCC), and therefore, informed consent was not required for their use.

Techniques: Biomarker Discovery, Western Blot, Control

Journal: Scientific Reports

Article Title: Mitochondrial proteomics reveals reductive metabolism dependent on glutamine in fibroblasts of idiopathic pulmonary fibrosis under hypoxia

doi: 10.1038/s41598-025-26911-3

Figure Lengend Snippet: Summary scheme. This scheme illustrates the deregulated proteins in the mitochondria of control fibroblasts and fibroblasts from IPF patients under hypoxic conditions, emphasizing the metabolic pathways and cellular mechanisms these proteins may influence. The fatty acid metabolic pathway is indicated to be active in hypoxic control fibroblasts (represented by blue mitochondria). At the same time, glutaminolysis is suggested to play a critical role in the mitochondria of IPF fibroblasts (represented by pink mitochondria). Created in BioRender.

Article Snippet: Two lines of healthy human lung fibroblasts (CCL-201TM, CCL-204TM) and two lines of fibroblasts from patients (CCL-134TM, CCL-191TM) were purchased from American Type Culture Collection (ATCC), and therefore, informed consent was not required for their use.

Techniques: Control

Journal: Scientific Reports

Article Title: ACPA prevents lung fibroblast-to-CAF transformation by reprogramming the tumor microenvironment through NSCLC-derived exosomes

doi: 10.1038/s41598-025-29726-4

Figure Lengend Snippet: Control and IC50 ACPA-applied NSCLC cell exosomes were successfully characterized. a , b Micrographs representing phase-contrast (PC, scale bar, 50 μm) view of ( a ) A549 NSCLC and ( b ) LL24 healthy human lung fibroblasts, 400x. ( c ) Flow cytometric analysis of tetraspanin CD9, CD63 and CD81 markers in control and ACPA-applied A549 cell exosomes. Fluorescence (CD9-APC, CD63-FITC and CD81-PE) on the x-axis vs. number of events (Count (%)) on the y-axis ( n = 3). d–f TEM analysis. ( d ) TEM images and ( e , f ) size distribution diameters from TEM images of control and ACPA-applied A549 cell exosomes ( n = 20). g–j Representative NTA of ( g , h ) control and ( i , j ) ACPA-applied A549 cell exosomes under 100x dilutions. Graphs g and i depict the finite track length adjustment (FTLA) size per concentration as triplicates and graphs h and j depict the average FTLA size per concentration.

Article Snippet: A549 non-small cell lung adenocarcinoma cell line A549 (CCL- 185) and LL24 healthy lung fibroblasts (CCL-151) (all from ATCC, USA) were cultured with DMEM High Glucose and Ham’s F12K, respectively, supplemented with 10–15% fetal bovine serum (FBS, Capricorn, USA), or 2% L-glutamine (Capricorn, USA) and 1% penicillin-streptomycin (Capricorn, USA).

Techniques: Control, Fluorescence, Concentration Assay

Journal: Scientific Reports

Article Title: ACPA prevents lung fibroblast-to-CAF transformation by reprogramming the tumor microenvironment through NSCLC-derived exosomes

doi: 10.1038/s41598-025-29726-4

Figure Lengend Snippet: ACPA-applied NSCLC cell exosomes at a lower dose diminish LL24 HLFs compared to control within 12 h. ( a ) qRT-PCR analysis for relative miRNA fold change values of miR-21, miR-23a and miR-23b relative to U6 gene ( n = 4). b–d Proliferation indices of LL24 HLFs following the application of control or ACPA-applied A549 NSCLC cell exosomes at doses of 10, 50 and 100 µg/ml for ( b ) 1, ( c ) 2 or ( d ) 3 days by MTT assay in mean ± SEM ( n = 5, * p < 0.05 by one-way analysis of variance (ANOVA) and post-hoc Duncan’s test). All proliferation data are presented in absorbance (A570 nm). e–h Real-time normalized proliferation indices of ( e ) control or ACPA-applied A549 NSCLC cell exosome-administered HLFs presented with control and exosomes at doses of 10, 50 and 100 µg/ml along with the IC50 graphs of ( f ) control and ( g ) ACPA-applied A549 NSCLC cell exosomes for 4 days ( n = 3 for exosome-applied groups and n = 6 for controls) and ( h ) LL24 fibroblasts following co-culture with A549 cells at ratio of 1:1, 1:5 or 5:1 ( n = 6). ( i ) Treatment response time points of LL24 fibroblasts following co-culture with A549 cells when cell index (CI) is at 50 or 80%.

Article Snippet: A549 non-small cell lung adenocarcinoma cell line A549 (CCL- 185) and LL24 healthy lung fibroblasts (CCL-151) (all from ATCC, USA) were cultured with DMEM High Glucose and Ham’s F12K, respectively, supplemented with 10–15% fetal bovine serum (FBS, Capricorn, USA), or 2% L-glutamine (Capricorn, USA) and 1% penicillin-streptomycin (Capricorn, USA).

Techniques: Control, Quantitative RT-PCR, MTT Assay, Co-Culture Assay

Journal: Scientific Reports

Article Title: ACPA prevents lung fibroblast-to-CAF transformation by reprogramming the tumor microenvironment through NSCLC-derived exosomes

doi: 10.1038/s41598-025-29726-4

Figure Lengend Snippet: ACPA-applied NSCLC cell exosomes at a lower dose inhibited PDPN, FAP and α-SMA expressions in HLFs compared to control. Representative univariate histograms ( a–c ) and bar graphs ( d–f ) of FCM depicting the expressions of ( a , d ) PDPN, ( b , e ) FAP and ( c , f ) α-SMA in LL24 fibroblasts following control or ACPA-applied A549 NSCLC cell exosomes at doses of 100–50 µg/ml, respectively ( n = 3). g-h Secretome profile of LL24 fibroblasts including ( g ) IL-6 and ( h ) IL-8 on days 1, 2 and 3 following the application of control or ACPA-applied A549 NSCLC cell exosomes at doses of 10, 50 and 100 µg/ml by ELISA. ( n = 3, * p < 0.05 by one-way analysis of variance (ANOVA) and post-hoc Duncan’s test).

Article Snippet: A549 non-small cell lung adenocarcinoma cell line A549 (CCL- 185) and LL24 healthy lung fibroblasts (CCL-151) (all from ATCC, USA) were cultured with DMEM High Glucose and Ham’s F12K, respectively, supplemented with 10–15% fetal bovine serum (FBS, Capricorn, USA), or 2% L-glutamine (Capricorn, USA) and 1% penicillin-streptomycin (Capricorn, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: ACPA prevents lung fibroblast-to-CAF transformation by reprogramming the tumor microenvironment through NSCLC-derived exosomes

doi: 10.1038/s41598-025-29726-4

Figure Lengend Snippet: ACPA-applied NSCLC cell exosomes reduce fibroblast growth via carbohydrate, lipid and amino acid metabolic pathways. ( a–b ) PCA plots, ( c ) VIP scores, ( d ) heat maps and ( e–i ) mean-standard deviation graphs of metabolites of LL24 fibroblasts following treatment of ACPA-applied and control NSCLC cell exosomes and control medium containing exosome-depleted FBS ( n = 3, * p < 0.05 by one-way analysis of variance (ANOVA) and post-hoc Duncan’s test).

Article Snippet: A549 non-small cell lung adenocarcinoma cell line A549 (CCL- 185) and LL24 healthy lung fibroblasts (CCL-151) (all from ATCC, USA) were cultured with DMEM High Glucose and Ham’s F12K, respectively, supplemented with 10–15% fetal bovine serum (FBS, Capricorn, USA), or 2% L-glutamine (Capricorn, USA) and 1% penicillin-streptomycin (Capricorn, USA).

Techniques: Standard Deviation, Control

Journal: Medicina

Article Title: Evaluation of TAM Receptor Targeting in Pathophysiology of Idiopathic Pulmonary Fibrosis

doi: 10.3390/medicina61101837

Figure Lengend Snippet: Relative mRNA expression levels of Axl, Tyro3, and Gas6 genes in IPF FBs and HPFs quantified by RT-qPCR. No Mer expression was detected in both fibroblast types. TBP gene was used as housekeeping gene. N = 4.

Article Snippet: IPF fibroblasts (IPF FBs CCL-134, ATCC) were cultured in Ham’s F12K medium supplemented with 10% FBS and 1% penicillin/streptomycin solution, and cells were used between passage 11 and 24.

Techniques: Expressing, Quantitative RT-PCR