Journal: Frontiers in Oncology

Article Title: AMPK/mTORC2/AKT-473/RUNX2 signaling axis modulates epithelial-mesenchymal transition and bone tropism in breast cancer

doi: 10.3389/fonc.2026.1785903

Figure Lengend Snippet: RUNX2 is a substrate of AMPK in breast cancer cells. MDA-MB-231 cells were treated with either metformin (20mM) or compound C (5μM) for 6 hours or none and subjected to (A) Western blot analysis and (B) RT-PCR analysis. MDA-MB-231 cells were treated with either metformin (20mM) or compound C (5μM) for 6 hours or none and subjected to IP analysis by (C) RUNX2 pull down and levels of RUNX2, p-AMPK substrate and p-AMPK were analyzed or by (D) p-AMPK pull down and levels of RUNX2, p-AMPK were analyzed. (E) MCF-7 cells were transfected with either RUNX2 WT or RUNX2 S118A or RUNX2 S118D or none, along with or without treatment of metformin (20mM) and Compound C (5μM) for 6 hours post to 48 hours of transfection and subjected to IP analysis by p-AMPK pull down and levels of p-AMPK and RUNX2-RFP were analyzed. (F) MDA-MB-231cells were treated with metformin (20mM) or compound C for 6 hours and subjected to immunofluorescence by anti-RUNX2 (Alexa 594) and anti-AMPK (Alexa 488) antibodies, counterstained with DAPI. (G) Immunofluorescence data were quantified to assess the degree of colocalization between AMPK and RUNX2 using ImageJ software. Mean ± S.E.M.; N = 3. *p<0.1 versus control, NS p>0.1 versus control. The immunofluorescence and quantification experiments were carried out on three independent fields. The uncropped blots are provided in the . Cont, control; Met, metformin; Comp C, compound C; IP, immunoprecipitation; IB, immunoblotting; EV, empty vector; WT, wild type; NS, non-significant.

Article Snippet: RUNX2 cDNA ( NM_001024630.3 ) was procured from Genecopoeia, USA.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunofluorescence, Software, Control, Immunoprecipitation, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: AMPK/mTORC2/AKT-473/RUNX2 signaling axis modulates epithelial-mesenchymal transition and bone tropism in breast cancer

doi: 10.3389/fonc.2026.1785903

Figure Lengend Snippet: AMPK mediated phosphorylation of RUNX2 results in increased nuclear localization and transcriptional activity of RUNX2. MDA-MB-231 cells were treated with either metformin (20mM) or compound C (5μM) for 6 hours or none and subjected to nuclear- cytoplasmic extraction followed by (A) Western blot analysis and (B) IP analysis by RUNX2 pull down and levels of RUNX2, p-AMPK substrate and p-AMPK were analyzed. (C) MDA-MB-231 cells were treated with either metformin (20mM) or compound C (5μM) for 6 hours or none and nuclear extracts were subjected to EMSA. MDA-MB-231 cells were treated with either metformin (20mM) or compound C (5μM) for 6 hours and subjected to (D) Western blot analysis and (E) RT-PCR analysis. MDA-MB-231 cells were transfected with RUNX2 siRNA with or without metformin (20mM) treatment for 6 hours and subjected to (F) Western blot analysis and (G) RT-PCR analysis. MCF-7 cells were transfected with either RUNX2 WT or RUNX2 S118A or RUNX2 S118D or none, along with or without metformin (20mM) treatment for 6 hours post to 48 hours of transfection and subjected to (H) Western blot analysis and (I) RT-PCR analysis. Mean ± S.E.M.; N = 3. *p<0.1 versus control, **p<0.01 versus control, ***p<0.001 versus control, NS p>0.1 versus control. The immunofluorescence and quantification experiments were carried out on three independent fields. The uncropped blots are provided in the . Cont, control; met, metformin; Comp C, compound C; CE, cytoplasmic extract; NE, nuclear extract; RUNX2-KD, RUNX2 knock down by siRNA; Scr, scrambled; EV, empty vector; WT, wild type; RUNX2A, RUNX2 S118A; RUNX2D, RUNX2 S118D; NS, non-significant.

Article Snippet: RUNX2 cDNA ( NM_001024630.3 ) was procured from Genecopoeia, USA.

Techniques: Phospho-proteomics, Activity Assay, Extraction, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Immunofluorescence, Knockdown, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: AMPK/mTORC2/AKT-473/RUNX2 signaling axis modulates epithelial-mesenchymal transition and bone tropism in breast cancer

doi: 10.3389/fonc.2026.1785903

Figure Lengend Snippet: mTORC2 is crucial for AMPK/RUNX2 axis. MDA-MB-231 cells were transfected with siRNA’s against RICTOR and RAPTOR or none and 48 hours post transfection subjected to (A) Western blot analysis and (B) IP analysis by RUNX2 pull down and levels of RUNX2, GSK3β and p-AMPK were analyzed. MDA-MB-231 cells were transfected with siRNA’s against RICTOR and RAPTOR or none and 48 hours post transfection subjected to (C) Immunofluorescence data were quantified to assess the degree of colocalization between RUNX2 and GSK3β using ImageJ software. (D) immunofluorescence by anti-RUNX2 (Alexa 594) and anti-GSK3β (Alexa 488) antibodies counterstained with DAPI. (E) MDA-MB-231 cells were transfected with RICTOR siRNA with or without metformin (20mM) treatment for 6 hours and subjected to IP by RUNX2 pull down and levels of RUNX2, GSK3β and p-AMPK were analyzed. (F) MDA-MB-231 cells were transfected with RICTOR siRNA with or without metformin (20mM) or LiCl (0.5M) or MG-132 (3mM) treatment for 6 hours and subjected to Western blot analysis. Mean ± S.E.M.; N = 3. *p<0.1 versus scrambled, NS p>0.1 versus scrambled. The immunofluorescence and quantification experiments were carried out on three independent fields. The uncropped blots are provided in the . Cont, control; Met, metformin; Comp C, compound C; IP, immunoprecipitation; IB, immunoblotting; Scr, scrambled; RAP, RAPTOR; RIC, RICTOR; NS, non-significant.

Article Snippet: RUNX2 cDNA ( NM_001024630.3 ) was procured from Genecopoeia, USA.

Techniques: Transfection, Western Blot, Immunofluorescence, Software, Control, Immunoprecipitation

Journal: Frontiers in Oncology

Article Title: AMPK/mTORC2/AKT-473/RUNX2 signaling axis modulates epithelial-mesenchymal transition and bone tropism in breast cancer

doi: 10.3389/fonc.2026.1785903

Figure Lengend Snippet: Metformin promotes EMT and induces osteoblast like phenotype to breast cancer cells through p-AMPK/RUNX2/mTORC2 axis. MDA-MB-231 cells were treated with either metformin (20mM) or compound C (5μM) for 6 hours or none and subjected to (A) RT-PCR analysis and (B) Western blot analysis. MDA-MB-231 cells were transfected with siRNA against RUNX2 or none, and 48 hours post-transfection with or without metformin (20mM) treatment for 6 hours, and subjected to (C) RT-PCR analysis and (D) Western blot analysis. MCF-7 cells were transfected with either RUNX2 WT or RUNX2 S118A or RUNX2 S118D or none, along with or without metformin (20mM) treatment for 6 hours post to 48 hours of transfection and subjected to (E) RT-PCR analysis and (F) Western blot analysis. (G) MDA-MB-231 cells were treated with either metformin (20mM) or compound C (5μM) for 6 hours or none and subjected to RT-PCR analysis. (H) MDA-MB-231 cells were transfected with siRNA against RUNX2 or none and 48 hours post-transfection with or without metformin (20mM) treatment for 6 hours and subjected to RT-PCR analysis. (I) MCF-7 cells were transfected with either RUNX2 WT or RUNX2 S118A or RUNX2 S118D or none, along with or without metformin (20mM) treatment for 6 hours post to 48 hours of transfection and subjected to RT-PCR analysis. Mean ± S.E.M.; N = 3. *p<0.1 versus scrambled or control, **p<0.01 versus scrambled or control, ***p<0.001 versus scrambled or control, NS p>0.1 versus scrambled or control. The immunofluorescence and quantification experiments were carried out on three independent fields. The uncropped blots are provided in the . Cont, control; met, metformin; Comp C, compound C; IP, immunoprecipitation; IB, immunoblotting; Scr, scrambled; RUNX2KD, knock down of RUNX2 using siRNA; WT, wild type; RUNX2A, RUNX2 S118A; RUNX2D, RUNX2 S118D; NS, non-significant; POSTN, periostin; CTSK, cathepsin K; COL1A1, type I collagen.

Article Snippet: RUNX2 cDNA ( NM_001024630.3 ) was procured from Genecopoeia, USA.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Control, Immunofluorescence, Immunoprecipitation, Knockdown

Journal: Frontiers in Oncology

Article Title: AMPK/mTORC2/AKT-473/RUNX2 signaling axis modulates epithelial-mesenchymal transition and bone tropism in breast cancer

doi: 10.3389/fonc.2026.1785903

Figure Lengend Snippet: Metformin promotes chemotaxis/metastasis of transformed breast cancer cells. (A) MDA-MB-231 cells were treated with either metformin (20mM) or compound C (5μM) for 6 hours or none and subjected to Western blot analysis. (B) MDA-MB-231 cells were transfected with siRNA against RICTOR or none and 48 hours post transfection with or without metformin (20mM) treatment for 6 hours and subjected to Western blot analysis. (C) MCF-7 cells were transfected with either RUNX2 WT or RUNX2 S118A or RUNX2 S118D or none, along with or without metformin (20mM) treatment for 6 hours post to 48 hours of transfection and subjected to Western blot analysis. (D) MDA-MB-231 cells were treated with either metformin (20mM) or compound C (5μM) for 6 hours or none and subjected to immunofluorescence stained using Rhodamine-phalloidin (540), counter stained by DAPI. (E) Quantification of fluorescence signal using ImageJ. (F) Quantification of number of migrated cells by electron microscopy. (G) MCF-7 cells were transfected with either RUNX2 WT or RUNX2 S118A or RUNX2 S118D or none, along with or without metformin (20mM) treatment for 6 hours post to 48 hours of transfection and subjected to migration through collagen coated membrane, with lower chambers coated with either HEK-293T cells or U2OS cells. (H) Breast tumor tissue along with adjacent normal tissue were subjected to protein isolation followed by Western blot analysis and (I) IP by RUNX2 pull down and levels of p-AMPK, RUNX2 and p-AMPK substrate-specific motif were analyzed. Mean ± S.E.M.; N = 3. *p<0.1 versus control or WT, NS p>0.1 versus control. The immunofluorescence and quantification experiments were carried out on three independent fields. The uncropped blots are provided in the . Cont, control; Met, metformin; Comp C, compound C; IP, immunoprecipitation; IB, immunoblotting; Scr, scrambled; EV, empty vector; WT, wild type. **p<0.01 versus control or WT, ***p<0.001 versus control or WT.

Article Snippet: RUNX2 cDNA ( NM_001024630.3 ) was procured from Genecopoeia, USA.

Techniques: Chemotaxis Assay, Transformation Assay, Western Blot, Transfection, Immunofluorescence, Staining, Fluorescence, Electron Microscopy, Migration, Membrane, Isolation, Control, Immunoprecipitation, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: AMPK/mTORC2/AKT-473/RUNX2 signaling axis modulates epithelial-mesenchymal transition and bone tropism in breast cancer

doi: 10.3389/fonc.2026.1785903

Figure Lengend Snippet: RUNX2-Dependent Bone Metastatic Outgrowth of MCF-7 Cells in NOD-SCID Mice. This figure illustrates the experimental setup and outcomes of intravenous injections of MCF-7 cell lines with distinct RUNX2 expressions, emphasizing the impact on bone metastatic outgrowth in NOD-SCID mice (n=4 per group). (A) Intravenous Injection and Tumor Growth Observation. MCF-7 cell lines overexpressing RUNX2 WT, RUNX2 S118A, and RUNX2 S118D mutants were intravenously injected into the tail vein of NOD-SCID mice. Metastatic outgrowth was visually monitored for tumor growth. (B) Quantification of tumornodules observed in the lungs of WT and mutant phenotypes. (C) Bone Marrow Cell Collection and GFP Intensity Assessment. Bone marrow cells were obtained through bone flush, and the GFP intensity was assessed through immunoblot analysis. (D) Lung tissue protein isolation and GFP intensity Check. GFP intensity was evaluated by isolating proteins from lung tissues and subjecting them to immunoblot analysis. (E) Tumor and normal tissue collection for GFP intensity analysis. Tumors, along with adjacent normal tissues, were collected. Proteins were isolated, and GFP intensity was checked through immunoblot analysis. (F) Histopathological examination by hematoxylin and eosin staining of lung tissues (G) Visual observation table. The table provides a visual summary of tumor observations and lung metastatic nodules in different groups of NOD-SCID mice. The uncropped blots are provided in the . EV, Empty vector; WT, Wild type.

Article Snippet: RUNX2 cDNA ( NM_001024630.3 ) was procured from Genecopoeia, USA.

Techniques: Injection, Mutagenesis, Western Blot, Isolation, Staining, Plasmid Preparation

Journal: Molecules

Article Title: Lipid Droplets as Cellular Sensors of Lipid Metabolic Reprogramming in Colon Cancer: Insights from Essential Amino Acid Supplementation Using Raman Spectroscopy and Imaging

doi: 10.3390/molecules31050762

Figure Lengend Snippet: Raman images of CCD-18Co cells for control cells and cells incubated with leucine, threonine, and arginine, with corresponding Raman spectra. The colors of spectra correspond to the colors of classes in the Raman maps and represent specific organelles: nucleus (red), lipid droplets (orange), endoplasmic reticulum (blue), mitochondria (magenta), cytoplasm (green), and membrane (light gray).

Article Snippet: CCD-18Co (ATCC ® CRL-1459TM) and Caco-2 cell lines were acquired from ATCC (Manassas, VA, USA) and cultured according to their standard procedures.

Techniques: Control, Incubation, Membrane

Journal: Molecules

Article Title: Lipid Droplets as Cellular Sensors of Lipid Metabolic Reprogramming in Colon Cancer: Insights from Essential Amino Acid Supplementation Using Raman Spectroscopy and Imaging

doi: 10.3390/molecules31050762

Figure Lengend Snippet: Raman images of CCD-18Co cells for control cells and cells incubated with leucine, threonine, and arginine obtained by the integration of the Raman bands over the characteristic spectral regions—organic matter (2820–3030 cm −1 ), nucleus (2935–3005 cm −1 ), saturated lipids (2820–2880 cm −1 ), and unsaturated lipids (3000–3030 cm −1 )—SAT/UNSAT lipid images, and microscope images.

Article Snippet: CCD-18Co (ATCC ® CRL-1459TM) and Caco-2 cell lines were acquired from ATCC (Manassas, VA, USA) and cultured according to their standard procedures.

Techniques: Control, Incubation, Microscopy

Journal: Molecules

Article Title: Lipid Droplets as Cellular Sensors of Lipid Metabolic Reprogramming in Colon Cancer: Insights from Essential Amino Acid Supplementation Using Raman Spectroscopy and Imaging

doi: 10.3390/molecules31050762

Figure Lengend Snippet: The average Raman spectra +/− SD (SD—standard deviation) in fingerprint region and in the high-frequency region for lipid droplets obtained for control CCD-18Co (blue line), CCD-18Co supplemented with leucine (olive line), CCD-18Co supplemented with threonine (magenta line), CCD-18Co supplemented with arginine (orange line), control Caco-2 (red line), Caco-2 supplemented with leucine (turquoise line), Caco-2 supplemented with threonine (green line), and Caco-2 supplemented with arginine (gray line). 20 mW laser power, 0.5 s integration time in the fingerprint region, and 0.3 s integration time in the high-frequency region.

Article Snippet: CCD-18Co (ATCC ® CRL-1459TM) and Caco-2 cell lines were acquired from ATCC (Manassas, VA, USA) and cultured according to their standard procedures.

Techniques: Standard Deviation, Control

Journal: Molecules

Article Title: Lipid Droplets as Cellular Sensors of Lipid Metabolic Reprogramming in Colon Cancer: Insights from Essential Amino Acid Supplementation Using Raman Spectroscopy and Imaging

doi: 10.3390/molecules31050762

Figure Lengend Snippet: PLS-DA score plots (model: mean center) for Raman spectra for lipid droplets for control CCD-18Co cell line (navy diamond), CCD-18Co cell line supplemented with leucine (blue circle), CCD-18Co cell line supplemented with threonine (ocean square), CCD-18Co cell line supplemented with arginine (turquoise pentagram) and for Raman spectra for control Caco-2 cell line (purple diamond), Caco-2 cell line supplemented with leucine (red circle), Caco-2 cell line supplemented with threonine (magenta square), Caco-2 cell line supplemented with arginine (light pink pentagram), and the loadings plots of LV1–LV3. One point in the PLS-DA score plot represents the average Raman spectrum of lipid droplets in one cell.

Article Snippet: CCD-18Co (ATCC ® CRL-1459TM) and Caco-2 cell lines were acquired from ATCC (Manassas, VA, USA) and cultured according to their standard procedures.

Techniques: Control

Journal: Molecules

Article Title: Lipid Droplets as Cellular Sensors of Lipid Metabolic Reprogramming in Colon Cancer: Insights from Essential Amino Acid Supplementation Using Raman Spectroscopy and Imaging

doi: 10.3390/molecules31050762

Figure Lengend Snippet: Response for lipid droplets of normal CCD-18Co (blue bar) colon cells to leucine (olive bar), threonine (magenta bar), and arginine (orange bar) supplementation and of cancer Caco-2 (red bar) colon cells to leucine (turquoise bar), threonine (green bar), and arginine (gray bar) supplementation. Normalized Raman intensities of the ratios: 2845/3015, 2845/2930, 3015/2888, and 1444/1256. *** means statistically significant results, p -value ≤ 0.001; ** means statistically significant results, p -value ≤ 0.01; * means statistically significant results, p -value ≤ 0.05. The number of cells for each culture condition is n = 10.

Article Snippet: CCD-18Co (ATCC ® CRL-1459TM) and Caco-2 cell lines were acquired from ATCC (Manassas, VA, USA) and cultured according to their standard procedures.

Techniques: