Journal: Respiratory Research
Article Title: Downregulation of SLC40A1 leads to iron accumulation in fibrotic lung fibroblasts
doi: 10.1186/s12931-025-03479-0
Figure Lengend Snippet: Transcriptional regulation of SLC40A1 by TGF-β1. a HPFs were treated for 48 h with different cytokines [TGF-β1 (5 ng/ml), WNT-1(50 ng/ml), WNT-3a (50 ng/ml), WNT-5a (50 ng/ml), TNFα (10 ng/ml), LPS (1,000 ng/ml), IL-1β (10 ng/ml), IL-4 (20 ng/ml), IL-5 (20 ng/ml), IL-13 (25 ng/ml), IL-21 (25 ng/ml), IL-25 (25 ng/ml), IL-33 (30 ng/ml), IFNβ (1 ng/mL), or IFNγ (20 ng/ml)]. SLC40A1 mRNA levels were determined by real-time PCR and expressed a ratio of the control (CON). b HPFs were treated with 50 ng/ml IL-6 for 48 h or serum-starved for 4 h or 24 h and then treated with 10 nM thrombin or 100 nM factor Xa for 48 h and SLC40A1 mRNA levels were determined by real-time PCR. c HPFs were co-transfected with the pGL3-Basic vector containing the SLC40A1 promoter fragment and firefly luciferase gene (180 ng) along with pRL-TK plasmid containing Renilla luciferase gene (20 ng) and then treated with 5 ng/ml TGF-β1 for 48 h. Firefly and Renilla luciferase activities in cell lysates were measured. The relative firefly luciferase activities were normalized to the respective Renilla luciferase activities. The results are presented as a ratio CON. d HPFs were treated with shSMAD2, shSMAD3, or shCON lentivirus at a MOI of 100 for 48 h. SMAD2 and SMAD3 protein levels were determined by western blot and quantitated. e HPFs were treated with shSMAD2, shSMAD3, or shCON lentivirus at a MOI of 100 for 48 h and then treated with 5 ng/ml TGF-β1 for 48 h. SLC40A1 mRNA levels were determined by real-time PCR. f HPFs were treated with SIS3 (10 µM, Milipore Sigma, Cat# 566405) for 2 h and then with TGF-β1 (5 ng/ml) for 48 h. SLC40A1 mRNA levels were determined by real-time PCR. g HPFs were treated with TGF-β1 (5 ng/ml) for 48 h. The cell lysates were subjected to chromatin immunoprecipitation (ChIP) using anti-SMAD3 antibodies. The SMAD3 binding site on the SLC40A1 promoter was detected by real-time PCR. n =3, * p < 0.05, ** p < 0.01, *** p < 0.001 v.s CON or shCON. ## p < 0.01 vs ShCon/TGF-β1. Student t-test ( c ), one-way ANOVA ( a and d ) or two-way ANOVA ( b , e , f and g ). followed by uncorrected Fisher’s least significant difference (LSD) test ( e , f and g ) or Turkey’s multiple comparison test ( b )
Article Snippet: HPFs (1x10 5 /well) were seeded into 12-well plates and treated with different cytokines and other compounds for 48 h: TNF-α (10 ng/ml, #300-01A, PeproTech, Cranbury, NJ, USA), lipopolysaccharides (LPS) (1,000 ng/ml, #00–4976-93, Invitrogen), TGF-β1 (5 ng/ml, #240-B, R&D Systems, Minneapolis, MN, USA) , IL-4 (20 ng/ml, #200-04, PeproTech), IFN-γ (20 ng/ml, #300-02, PeproTech) , IL-21 (25 ng/ml, #200-21, PeproTech), IL-13 (25 ng/ml, #200-13, PeproTech), IFN-β (1 ng/mL, #300-02BC, PeproTech), IL-25 (25 ng/ml, #1258-IL, R&D Systems), IL-33 (30 ng/ml, #200-33, PeproTech), WNT-1 (50 ng/ml, #120-17, PeproTech), IL-1β (10 ng/ml, #200-01B, PeproTech), IL-5 (20 ng/ml, #200-05, PeproTech), WNT-3a (50 ng/mL, #5036-WN, R&D Systems), WNT-5a (50 g/mL, #645-WN, R&D Systems), IL-6 (50 ng/ml, #200-06, Gibco), thrombin (10 nM, #RP-43100, Invitrogen), factor Xa (100 nM, #RP-43114, Invitrogen), ferric ammonium citrate (FAC) (200 μM, # RES20400 -A7, Sigma), deferoxamine (DFO) (10 μM #D9533, Sigma).
Techniques: Real-time Polymerase Chain Reaction, Control, Transfection, Plasmid Preparation, Luciferase, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Comparison
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![Transcriptional regulation of SLC40A1 by TGF-β1. a HPFs were treated for 48 h with different cytokines [TGF-β1 (5 ng/ml), WNT-1(50 ng/ml), WNT-3a (50 ng/ml), WNT-5a (50 ng/ml), TNFα (10 ng/ml), LPS (1,000 ng/ml), IL-1β (10 ng/ml), IL-4 (20 ng/ml), IL-5 (20 ng/ml), IL-13 (25 ng/ml), IL-21 (25 <t>ng/ml),</t> <t>IL-25</t> (25 ng/ml), IL-33 (30 ng/ml), IFNβ (1 ng/mL), or IFNγ (20 ng/ml)]. SLC40A1 mRNA levels were determined by real-time PCR and expressed a ratio of the control (CON). b HPFs were treated with 50 ng/ml IL-6 for 48 h or serum-starved for 4 h or 24 h and then treated with 10 nM thrombin or 100 nM factor Xa for 48 h and SLC40A1 mRNA levels were determined by real-time PCR. c HPFs were co-transfected with the pGL3-Basic vector containing the SLC40A1 promoter fragment and firefly luciferase gene (180 ng) along with pRL-TK plasmid containing Renilla luciferase gene (20 ng) and then treated with 5 ng/ml TGF-β1 for 48 h. Firefly and Renilla luciferase activities in cell lysates were measured. The relative firefly luciferase activities were normalized to the respective Renilla luciferase activities. The results are presented as a ratio CON. d HPFs were treated with shSMAD2, shSMAD3, or shCON lentivirus at a MOI of 100 for 48 h. SMAD2 and SMAD3 protein levels were determined by western blot and quantitated. e HPFs were treated with shSMAD2, shSMAD3, or shCON lentivirus at a MOI of 100 for 48 h and then treated with 5 ng/ml TGF-β1 for 48 h. SLC40A1 mRNA levels were determined by real-time PCR. f HPFs were treated with SIS3 (10 µM, Milipore Sigma, Cat# 566405) for 2 h and then with TGF-β1 (5 ng/ml) for 48 h. SLC40A1 mRNA levels were determined by real-time PCR. g HPFs were treated with TGF-β1 (5 ng/ml) for 48 h. The cell lysates were subjected to chromatin immunoprecipitation (ChIP) using anti-SMAD3 antibodies. The SMAD3 binding site on the SLC40A1 promoter was detected by real-time PCR. n =3, * p < 0.05, ** p < 0.01, *** p < 0.001 v.s CON or shCON. ## p < 0.01 vs ShCon/TGF-β1. Student t-test ( c ), one-way ANOVA ( a and d ) or two-way ANOVA ( b , e , f and g ). followed by uncorrected Fisher’s least significant difference (LSD) test ( e , f and g ) or Turkey’s multiple comparison test ( b )](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2364/pmc12922364/pmc12922364__12931_2025_3479_Fig3_HTML.jpg)