Journal: Oncology Reports

Article Title: PINK1-mediated mitophagy enhances breast cancer proliferation through metabolic reprogramming

doi: 10.3892/or.2026.9117

Figure Lengend Snippet: Analysis of glycolysis-associated metabolite level changes in MCF-7 and MDA-MB-231 breast cancer cells. Levels of (A) PGK2, (B) pyruvate and (C) acetyl-CoA in MCF-7 cells, and (D) PGK2, (E) pyruvate and (F) acetyl-CoA in MDA-MB-231 cells transfected with vector control, PINK1-OE or PINK1-OE + siPGK2. Quantitative analysis was performed using ELISA in cells under the three indicated conditions. Statistical analysis was performed using a one-way ANOVA followed by Tukey's post-hoc test. Data are represented as the mean ± SEM from at least three independent experiments. ***P<0.001. PGK2, phosphoglycerate kinase 2; PINK1, PTEN-induced kinase 1; OE, overexpression; si, small interfering.

Article Snippet: Acetyl-CoA content was assessed using the Acetyl-CoA ELISA Kit (Wuhan Elabscience Biotechnology Co., Ltd.; cat. no. E-BC-F046-48T-ELS).

Techniques: Transfection, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Siah2 regulates lipid uptake in adipose tissue macrophages

doi: 10.1016/j.jbc.2026.111380

Figure Lengend Snippet: Lysosomal program is upregulated in ATMs independent of Siah2 . A , relative mRNA expression of lysosomal genes in fl/fl and Siah2 MacKO BMDMs and BM-ATMs. B , representative Western blot of lysosomal enzymes relative to β-actin in fl/fl and Siah2 MacKO BMDMs and BM-ATMs. The β-actin panel is the same as used in F . The blot shown in F was reprobed for the CTSK image in B . C , enzyme activity of CTSK and LAL in fl/fl and Siah2 MacKO BMDMs and BM-ATMs. D and E , relative mRNA expression of Atgl ( D ) and Hilpda ( E ) in fl/fl and Siah2 MacKO BMDMs and BM-ATMs. F , representative Western blot of ATGL and HILPDA relative to β-actin in fl/fl and Siah2 MacKO BMDMs and BM-ATMs. G , basal glycerol release in fl/fl and Siah2 MacKO BMDMs and BM-ATMs treated with vehicle or 50 μM Atglistatin or 0.1 μM Lalistat-1 for 24 h. H , glycerol release in fl/fl and Siah2 MacKO BM-ATMs treated with vehicle or 50 μM Atglistatin or 0.1 μM Lalistat-1 for 24 h with or without 0.5 mM IBMX (activated lipolysis). I and J , representative confocal images ( I ) and quantification ( J ) of fl/fl and Siah2 MacKO BM-ATMs stained for BODIPY 493/503 (LD, green ), LysoTracker (lysosomes, magenta ), and DAPI (nuclei, blue ) showing colocalization of LD and lysosomes. The scale bar represents 10 μm. Red signals of LysoTracker were converted to magenta using the LUT function in ImageJ. Statistics are reported as mean ± SD using an unpaired t test with Welch’s correction, and each dot denotes technical replicates representative of three to five independent experiments. A , D , E , G , H , and J , statistics are reported as mean ± SD using two-way ANOVA with Tukey’s multiple comparisons test ( C ). p Values are indicated on the graphs. ATGL, adipose triglyceride lipase; ATM, adipose tissue macrophage; BM-ATM, bone marrow–derived ATM; BMDM, bone marrow–derived macrophage; CTSK, cathepsin K; DAPI, 4′,6-diamidino-2-phenylindole; fl/fl, Siah2 flox/flox ; HILPDA, hypoxia-inducible LD-associated protein; IBMX, 3-isobutyl-1-methylxanthine; LAL, lysosomal acid lipase; LD, lipid droplet; MacKO, Siah2 MacKO ; Siah2, seven in absentia homolog 2.

Article Snippet: CTSK activity in BMDMs and BM-ATMs was measured using the Cathepsin K assay kit (Novus Biologicals, catalog no.: NBP2-54842) according to the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Activity Assay, Staining, Derivative 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: 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