acidification rate ecar detection  (Elabscience Biotechnology)

Journal: Journal of Translational Medicine

Article Title: Vitamin D attenuates the progression of pulmonary fibrosis via inhibiting thymidine kinase 1/PFKFB3-driven glycolysis

doi: 10.1186/s12967-025-07298-1

Figure Lengend Snippet: Enhanced glycolysis contributes to pulmonary fibroblast activation. ( A ) Protein level detection of Fibronectin, Collagen I, and α-SMA levels in MRC-5 cells treated with 0, 1, 2, and 5ng/mL of TGF-β1 for 48 h. GAPDH was used as a loading control. The experiments were repeated three times and the results were similar. ( B ) Representative immunofluorescence staining of α-SMA (red) and DAPI (blue) was performed on MRC-5 cells treated with 5 ng/mL of TGF-β1 for 48 h. Scale bar = 125 μm. ( C ) DNA synthesis was assessed using the EdU assay in MRC-5 cells for the control and TGF-β1 (5 ng/mL) treatment groups. Green, EdU; blue, nuclei. Scale bar = 125 μm. ( D ) MRC-5 cells were stimulated with TGF-β1 for 48 h, and cell viability was determined by MTT assay ( n = 3), * P < 0.05. ( E - G ) Glucose consumption, lactate levels, and ATP concentration were measured ( n = 3), with * P < 0.05 and ** P < 0.01 vs. control. ( H ) MRC-5 cells were pretreated with 2-DG at 10 mM for 1 h, then exposed to TGF-β1 for 48 h. Graphic presentation depicting the immunostaining for Collagen I (green) in MRC-5 cells. The nucleus was stained with DAPI. Scale bar = 125 μm. ( I - J ) MTT assays were employed to evaluate cell viability, while the ECAR was utilized to assess glycolytic activity ( n = 3), * P < 0.05, ** P < 0.01 vs. the control group, # P < 0.05 vs. TGF-β1 + DMSO group. For D , E , F , G , I , and J , one-way ANOVA was used with the Bonferroni multiple comparisons test. Data are presented as mean ± SD. Source data are provided as a Source data file.

Article Snippet: Extracellular acidification rate (ECAR) assays (Elabscience, E-BC-F069) were performed using a microplate reader (BioTek, Winooski, VT, USA).

Techniques: Activation Assay, Control, Immunofluorescence, Staining, DNA Synthesis, EdU Assay, MTT Assay, Concentration Assay, Immunostaining, Activity Assay

Journal: Journal of Translational Medicine

Article Title: Vitamin D attenuates the progression of pulmonary fibrosis via inhibiting thymidine kinase 1/PFKFB3-driven glycolysis

doi: 10.1186/s12967-025-07298-1

Figure Lengend Snippet: Vitamin D exerts anti-fibrotic effects by reducing glycolysis. ( A ) Western blot analysis was used to examine the expression of Fibronectin, Collagen I, and α-SMA in MRC-5 cells treated with Vitamin D at 100 nM for 24 h before TGF-β1 treatment for 48 h. GAPDH was used as a loading control. The experiments were repeated three times and the results were similar. ( B ) Immunofluorescence staining of Collagen I (green) and α-SMA (red) in MRC-5 cells for the indicated groups. Blue represents nuclear DNA staining by DAPI. Scale bar = 125 μm. ( C - D ) The MTT and EdU assays were conducted on MRC-5 cells treated with Vitamin D at 100 nM for 24 h before TGF-β1 treatment for 48 h to assess cell proliferative ability ( n = 3), * P < 0.05 vs. the control group, and # P < 0.05 vs. TGF-β1 + DMSO group. Scale bar = 125 μm. ( E ) Contraction assay of the collagen gel complex in the presence or absence of Vitamin D. ( F ) Evaluating cell migration in TGF-β1-treated MRC-5 cells with and without co-incubation of Vitamin D, using the Transwell migration assay. ( G - J ) Glucose consumption, lactate concentration, ECAR, and ATP concentration were detected in MRC-5 cells for the indicated groups ( n = 3), * P < 0.05, ** P < 0.01 vs. the control group, # P < 0.05, and ## P < 0.01 vs. TGF-β1 + DMSO group. For C , G , H , I , and J , a one-way ANOVA was used, followed by the Bonferroni multiple comparisons test. Data are presented as mean ± SD. Source data are provided as a Source data file.

Article Snippet: Extracellular acidification rate (ECAR) assays (Elabscience, E-BC-F069) were performed using a microplate reader (BioTek, Winooski, VT, USA).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Contraction Assay, Migration, Incubation, Transwell Migration Assay, Concentration Assay

Journal: Journal of Translational Medicine

Article Title: Vitamin D attenuates the progression of pulmonary fibrosis via inhibiting thymidine kinase 1/PFKFB3-driven glycolysis

doi: 10.1186/s12967-025-07298-1

Figure Lengend Snippet: TK1 is involved in fibroblast activation and glycolysis. ( A ) Western blot and qRT-PCR analysis of TK1 in MRC-5 cells transfected with TK1 siRNA for 24 h, then treated with 5ng/mL TGF-β1 for 48 h ( n = 3), with ** P < 0.01 vs. the control group, and ## P < 0.01 vs. TGF-β1 + control siRNA group. GAPDH was used as a loading control. The experiments were repeated three times and the results were similar. ( B ) The expression of α-SMA (red) and Collagen I (green) was assessed via immunofluorescence staining to show fibroblast activation. Scale bar = 125 μm. ( C - D ) MTT and EDU assays evaluated the proliferative capacity of MRC-5 cells transfected with TK1 siRNA for 24 h, followed by treatment with 5 ng/mL TGF-β1 for 48 h ( n = 3). The results showed * P < 0.05 vs. the control group and # P < 0.05 vs. the TGF-β1 + control siRNA group. Scale bar = 125 μm. ( E ) MRC-5 cell contraction was measured using the collagen gel assay. ( F ) Cell migration ability in TGF-β1-treated MRC-5 cells with or without TK1 siRNA co-incubation using the Transwell migration assay. ( G - J ) Lactate concentration, glucose consumption, ECAR, and ATP concentration were assessed in MRC-5 cells across the experimental groups ( n = 3), with * P < 0.05, ** P < 0.01 vs. the control group, and # P < 0.05, ## P < 0.01 vs. TGF-β1 + control siRNA group. For A , C , G , H , I , and J , one-way ANOVA was used with the Bonferroni multiple comparisons test. Data are presented as mean ± SD. Source data are provided as a Source data file.

Article Snippet: Extracellular acidification rate (ECAR) assays (Elabscience, E-BC-F069) were performed using a microplate reader (BioTek, Winooski, VT, USA).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Transfection, Control, Expressing, Immunofluorescence, Staining, Migration, Incubation, Transwell Migration Assay, Concentration Assay