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: Bioengineering

Article Title: An Injectable, Osteoconductive Gelatin-Enabled GelMA/HAp Hydrogel Scaffold for Minimally Invasive Bone Tissue Engineering

doi: 10.3390/bioengineering13020139

Figure Lengend Snippet: ( a ) Immunofluorescence images of Runx2 in BMSCs co-cultured with hydrogels during osteogenic differentiation. ( b ) Number of Runx2-positive cells in BMSCs co-cultured with hydrogels. *** p < 0.005; **** p < 0.0001.

Article Snippet: The fixed samples were permeabilized and blocked, followed by incubation with a primary antibody against Runx2 (Shanghai, China, Proteintech, 20700-1-AP) and then with the corresponding fluorophore-conjugated secondary antibody (USA, Thermo Fisher Scientific, A-11011).

Techniques: Immunofluorescence, Cell Culture

Journal: Bioengineering

Article Title: An Injectable, Osteoconductive Gelatin-Enabled GelMA/HAp Hydrogel Scaffold for Minimally Invasive Bone Tissue Engineering

doi: 10.3390/bioengineering13020139

Figure Lengend Snippet: ( a – d ) qPCR analysis of OCN , COL-1 , Runx2 and Osterix in BMSCs on Day 7 and 14. * p < 0.05; **** p < 0.0001.

Article Snippet: The fixed samples were permeabilized and blocked, followed by incubation with a primary antibody against Runx2 (Shanghai, China, Proteintech, 20700-1-AP) and then with the corresponding fluorophore-conjugated secondary antibody (USA, Thermo Fisher Scientific, A-11011).

Techniques:

Journal: bioRxiv

Article Title: Influence of age on functional memory T cell diversity

doi: 10.1101/2021.05.29.446296

Figure Lengend Snippet: (A) Transcription Factor-Regulatory Element-Target Gene (TF-RE-TG) networks in CD73 − (left) or CD73 + cells (right) were modeled as described in Suppl. Figure 3. Red and yellow nodes represent transcriptional factors (TF) or chromatin regulators (CR); the green nodes represent their target genes (TG) that are differentially expressed in CD73 + and CD73 − memory T cells. The size of TF nodes corresponds to the number of TF connections. ( B-D ): Freshly isolated human total T cells were activated and infected with GFP+ lentivirus containing RUNX2 shRNA (B) RUNX2 cDNA (C), RUNX3 shRNA (D), and RUNX3 cDNA (E) respectively. TR30021, pCDH and Lenti-Control served as respective controls. Transduced cells were cultured for 7 days, before CD73 expression in gated GFP + cells was assessed. Results are compared by two-tailed paired t-test. N.S: not significant.

Article Snippet: To knockdown RUNX3 or RUNX2 in human T cells, we used RUNX3 or RUNX2 human shRNA plasmid containing GFP reporter gene (Origene, Cat#: TL309682 for RUNX3; Cat#: TL309683 for RUNX2).

Techniques: Isolation, Infection, shRNA, Control, Cell Culture, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Influence of age on functional memory T cell diversity

doi: 10.1101/2021.05.29.446296

Figure Lengend Snippet: (A and B) Freshly isolated memory T cells were activated in vitro by anti-CD3/CD28 Dynabeads for 4 days followed by culture with TGFβ /IL-15 for 3 days. CD4 (A) and CD8 (B) T cells were analyzed by flow cytometry for the T RM -associated markers CD69, CXCR6 and CD103 in CD73 + and CD73 − cells. ( C-F ): Freshly isolated human total T cells were activated and infected by GFP + lentivirus containing RUNX2 shRNA (C, TR30021 as a control), RUNX2 cDNA (D, Lenti-Control as a control), RUNX3 shRNA (E, TR30021 as a control) or RUNX3 cDNA (F, pCDH as a control) and differentiated under T RM development conditions for 7 days. GFP + c ells were gated and analyzed for CD69 and CD103 expression. (G) Expression profile of 16 of 19 T RM core genes in the CXCR6 + CD69 + and the CXCR6 − CD69 − CD4 T cell subsets that have the highest and the lowest CD73 expression, respectively. The remaining three genes (CX3CR1, S1PR5 and CRTAM) were undetectable and are not shown. qPCR results are shown as 2 (-delta Ct) *10 −5 . (H-K) Freshly isolated memory CD4 (H/J) and CD8 (I/K) T cells from young (<35y, red symbol) and older (>65y, black symbol) individuals were differentiated under 4 days of Dynabeads stimulation and 3 days of TGFβ treatment. Expression of CD73, CD69, CXCR6 and CD103 were analyzed by flow cytometry; results are summarized as box plots (H,I). Frequencies of CD73 + cells correlated with those of CD69 + CXCR6 + cells for CD4 T cells (J) and CD103 + cells for CD8 T cells (K) as determined by Pearson’s correlation analysis. Data were compared by two-tailed paired or unpaired ttest. One-way ANOVA was used for multi-group comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: To knockdown RUNX3 or RUNX2 in human T cells, we used RUNX3 or RUNX2 human shRNA plasmid containing GFP reporter gene (Origene, Cat#: TL309682 for RUNX3; Cat#: TL309683 for RUNX2).

Techniques: Isolation, In Vitro, Flow Cytometry, Infection, shRNA, Control, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: The unique function of Runx1 in skeletal muscle differentiation and regeneration is mediated by an ETS interaction domain

doi: 10.1101/2023.11.21.568117

Figure Lengend Snippet: (A) Western blot analysis of Runx1and Runx2 in WT and Runx1KO C2C12 relative to β-actin. (B) Representative phase contrast microscopy images of WT and Runx1KO C2C12 cells in growth medium (GM) or after 6 d in differentiation medium (DM). (C) Quantification of fusion index of WT and Runx1KO C2C12 cells after 6 days in differentiation medium. ***p<0.001. Data present as means ± SEM. n=3 for each group. (D) Differential gene expression analysis reveal differences in GM (D0) and DM (D1,2,6). The affected genes in each group/day are displayed here and in Supplemental Table S1. (E) Heat maps of the changes in 368 transcription factors: above the clusters, relative Log2 fold change in each column is shown. Below the clusters, the relative abundance (LogCPM) is shown. Members of the Notch pathway, Ets, MRF gene families are indicated above their relative position. (F) GO terms of the up- and downregulated genes in Runx1KO. Details in Supplemental Table S1.

Article Snippet: The mouse Runx2 plasmid pCMV-Flag-mRunx2 was purchased from Origene.

Techniques: Western Blot, Microscopy, Gene Expression

Journal: bioRxiv

Article Title: The unique function of Runx1 in skeletal muscle differentiation and regeneration is mediated by an ETS interaction domain

doi: 10.1101/2023.11.21.568117

Figure Lengend Snippet: (A) Runx1KO C2C12 cells were transfected with 200ng Runx1 or Runx2 plasmids. After 24 hr, cells were induced to differentiate for 6 days followed by immunofluorescence staining for MHC (red) and DNA (Hoechst, blue). (B) Quantification of fusion index and of (C) MHC+ cell lengths in experiments shown in A. (D) Schematics of Runx1 and Runx2 chimera proteins. (E) Quantification of fusion index (F) and MHC+ cell lengths in experiments shown in G; (G) Runx1KO C2C12 cells were transfected with 200ng of five different chimera plasmids numbered in D. 24 h for after transfection, cells were induced to differentiate for 6 days followed by immunofluorescence staining for MHC (red) and DNA (Hoechst, Blue). Data presented as means ± SEM. n=3 for each group. ***p<0.001. n.s., not significant (Scale bars: 150μm.).

Article Snippet: The mouse Runx2 plasmid pCMV-Flag-mRunx2 was purchased from Origene.

Techniques: Transfection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The unique function of Runx1 in skeletal muscle differentiation and regeneration is mediated by an ETS interaction domain

doi: 10.1101/2023.11.21.568117

Figure Lengend Snippet: (A) Western blots from co-immunoprecipitation experiments in C2C12 cells transfected with Myc tagged Runx proteins and Flag-tagged Etv4. (B) Venn diagram of ATACseq peaks from C2C12 or Runx1KO cells. Most peaks are shared, 9,318 peaks that are only found to C2C12 and 2,607 peaks are only found in Runx1KO C2C12 cells. (C) Heatmaps showing ATACseq reads from WT or Runx1KO C2C12 cells mapped onto these 3 classes of ATACseq peaks (WT specific, Shared, and Runx1KO specific). (D) Graph displaying the -log pValue of transcription factor motif enrichment as determined by HOMER. Runx1 motifs (blue) are more highly enriched in WT C2C12 cells, whereas Mef2 motifs (red MADS-box) are more enriched in Runx1KO C2C12. The inset shows that AP1 motifs (purple) are similarly enriched in both WT and C2C12 cells as expected for a global enhancer binding factor. (E) Bar graph showing enrichment for the Ets:Runx composite motif in Runx1 ChIP data from myoblasts or T-ALL cells, less enrichment seen in Runx2 ChIP from preosteoblasts. The composite motif enrichment in each dataset is normalized to enrichment rate of the Runx-only motif in the respective ChIP from each cell types.

Article Snippet: The mouse Runx2 plasmid pCMV-Flag-mRunx2 was purchased from Origene.

Techniques: Western Blot, Immunoprecipitation, Transfection, Binding Assay

Journal: Cell death & disease

Article Title: Glucocorticoid-mediated BIM induction and apoptosis are regulated by Runx2 and c-Jun in leukemia cells.

doi: 10.1038/cddis.2012.89

Figure Lengend Snippet: Figure 2 BIM, c-Jun, and Runx2 are induced by Dex in CCRF-CEM (T-ALL) and RS4;11 (B-ALL) cells. (a) CEM cells were treated with 0.3 mM Dex for 0.5, 2, 3, 6, 16, and 24 h and total RNAs were subjected to qPCR to determine the levels of Bim, c-Jun and Runx2 mRNA. Values represent the mean±S.D. of three independent experiments. (b) CEM cells were treated with 0.3 mM Dex for 24 h and equal amounts of total cell extracts were subjected to western blotting with the indicated antibodies. (c and d) RS4;11 cells were treated with 0.3 mM Dex for 24 h. Relative Bim, c-Jun, and Runx2 mRNAs were determined by qPCR (c) and equal amounts of total cell extracts were subjected to western blotting with the indicated antibodies (d)

Article Snippet: Flag-tagged c-Jun and Runx2 expression plasmids were purchased from OriGene (Rockville, MD, USA).

Techniques: Western Blot

Journal: Cell death & disease

Article Title: Glucocorticoid-mediated BIM induction and apoptosis are regulated by Runx2 and c-Jun in leukemia cells.

doi: 10.1038/cddis.2012.89

Figure Lengend Snippet: Figure 4 Runx2 regulates BIM expression and cell death induced by Dex. (a) CEM cells were infected with lentiviruses expressing shRNAs for non-targeting control or Runx2. Puromycin-resistant cells were pooled after each infection. Cells were treated with 0.3 mM Dex for 24 h and total RNAs were subjected to qPCR to determine the levels of Bim, c-Jun, and Runx2 mRNA. (b) Cells in (a) were treated with 0.3 mM Dex for 24 h and total cell extracts were subjected to western blotting with the indicated antibodies. (c) Cells in (a) were treated with 0.3 mM Dex for 72 h and percentage of cell death was determined by AnnexinV-PI staining followed by FACS analysis. Values represent the mean±S.D. of three independent experiments. (d) CEM/shRunx2 clone was transfected with Flag-c-Jun cDNA or a vector. The established clones (CEM/shRunx2/Jun3 or CEM/shRunx2/control) were treated with 0.3 mM Dex for 24 h and total cell extracts were subjected to western blotting with the indicated antibodies. (e) Cells in (d) were treated with 0.3 mM Dex for 24 h and total RNAs were subjected to qPCR to determine the levels of Bim mRNA. (f) Cells in (d) were treated with 0.3 mM Dex for 72 h and percentage of cell death was determined by AnnexinV-PI staining followed by FACS analysis. Values represent the mean±S.D. of three independent experiments. We analyzed another c-Jun overexpressing clone and the results were reproducible

Article Snippet: Flag-tagged c-Jun and Runx2 expression plasmids were purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Infection, Control, Western Blot, Staining, Transfection, Plasmid Preparation, Clone Assay

Journal: Cell death & disease

Article Title: Glucocorticoid-mediated BIM induction and apoptosis are regulated by Runx2 and c-Jun in leukemia cells.

doi: 10.1038/cddis.2012.89

Figure Lengend Snippet: Figure 5 c-Jun binds to and activates the Bim promoter. (a) Human Bim promoter upstream of the start site of transcription contains a Runx-, AP-1-, and FOXO-binding sites. (b) The luciferase constructs indicated were cotransfected with a c-Jun and/or a Runx2 expression vectors into 293T cells for 48 h. Luciferase activity was normalized to Renilla activity for each well to control for transfection efficiency. Values represent the mean±S.D. of three independent experiments. (c) A luciferase reporter construct containing an intact Bim AP-1 and Runx site (Bim intact), a mutated AP-1 site (Bim AP-1 mut), or a mutated Runx site (Bim Runx mut) was cotransfected with a c-Jun and/or a Runx2 expression vectors into 293T cells for 48 h. Luciferase activities were determined as (b). Values represent the mean±S.D. of three independent experiments. minP: minimal promoter. (d) The same plasmids in (c) were cotransfected into CEM cells. Twenty-four hours after transfection, cells were split into half and continued in culture for another 24 h in the presence or absence of 0.3 mM Dex. Luciferase activities were determined as (b). Values represent the mean±S.D. of three independent experiments. (e) Direct binding of c-Jun on the AP-1 site of human Bim promoter. Left panel: a probe containing the intact AP-1 site (AP-1 wt) or a probe with mutated AP-1 site (AP-1 mut) was incubated with the nuclear extracts from CEM cells treated in the presence or absence of 0.3 mM Dex for 24 h. An arrow indicates a specific DNA–protein complex. Right panel: anti-c-Jun antibodies were incubated in the reaction mixture. An arrow indicates a probe DNA–c-Jun complex

Article Snippet: Flag-tagged c-Jun and Runx2 expression plasmids were purchased from OriGene (Rockville, MD, USA).

Techniques: Binding Assay, Luciferase, Construct, Expressing, Activity Assay, Control, Transfection, Incubation

Journal: Cell death & disease

Article Title: Glucocorticoid-mediated BIM induction and apoptosis are regulated by Runx2 and c-Jun in leukemia cells.

doi: 10.1038/cddis.2012.89

Figure Lengend Snippet: Figure 6 BIM, c-Jun, and Runx2 expression induced by Dex is mediated through the GC receptor-p38-MAPK pathway. (a) CEM cells were pretreated with 1 mM RU486 for 30 min and were treated with 0.3 mM Dex for 24 h. Total RNAs were subjected to qPCR to determine the levels of c-Jun, Runx2, and Bim mRNA. Values represent the mean±S.D. of three independent experiments. (b) CEM cells were treated as (a), and equal amounts of total cell extracts were subjected to western blotting with the indicated antibodies. (c) CEM cells were treated as (a) except Dex treatment was performed for 48 h, and percentage of cell death was determined by AnnexinV-PI staining followed by FACS analysis. Values represent the mean±S.D. of three independent experiments. (d) CEM cells were pretreated with 2 mM SB203580 for 1 h and were treated with 0.3 mM Dex for 24 h. Total RNAs were subjected to qPCR to determine the levels of c-Jun, Runx2, and Bim mRNA. Values represent the mean±S.D. of three independent experiments. (e) CEM cells were treated as (d), and equal amounts of total cell extracts were subjected to western blotting with the indicated antibodies. (f) CEM cells were treated as (d) except Dex treatment was performed for 48 h, and percentage of cell death was determined by AnnexinV-PI staining followed by FACS analysis. Values represent the mean±S.D. of three independent experiments. (g) A luciferase reporter construct containing AP-1-binding sites (pAP-1-luc) was transfected into CEM cells. Twenty-four hours after transfection, cells were split into half, pretreated with 2 mM SB203580 for 1 h, and were treated with 0.3 mM Dex for another 24 h. Luciferase activity was normalized to Renilla activity for each well to control for transfection efficiency. Values represent the mean±S.D. of three independent experiments

Article Snippet: Flag-tagged c-Jun and Runx2 expression plasmids were purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Western Blot, Staining, Luciferase, Construct, Binding Assay, Transfection, Activity Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: METTL14 promotes migration and invasion of choroidal melanoma by targeting RUNX2 mRNA via m6A modification

doi: 10.1111/jcmm.17577

Figure Lengend Snippet: RUNX2 is the key target of METTL14 in CM. (A) Correlation of METTL14 and RUNX2 expression in the TCGA database. (B) qRT‐PCR analysis of RUNX2 mRNA after METTL14 inhibition or overexpression. (C) WB results indicate that protein expression of RUNX2 is significantly downregulated or upregulated after METTL14 knockdown or overexpression, respectively.

Article Snippet: The slides were incubated overnight at 4 °C with the primary antibodies anti‐METTL14 (1:1000, HPA038002, Sigma‐Aldrich) and anti‐RUNX2 (1:1000, MAB2006, RD), followed by addition of secondary antibodies to an avidin‐biotin‐peroxidase complex (e.g. biotinylated anti‐rabbit and anti‐mouse antibodies in goats).

Techniques: Expressing, Quantitative RT-PCR, Inhibition, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: METTL14 promotes migration and invasion of choroidal melanoma by targeting RUNX2 mRNA via m6A modification

doi: 10.1111/jcmm.17577

Figure Lengend Snippet: RUNX2 mRNA can be methylated by METTL14 and it promotes migration and invasion of CM cells in vitro. (A–E) RUNX2 mRNA can be methylated by METTL14. A, MeRIP‐qPCR analysis was used to determine m6A modification enrichment in RUNX2 mRNA after overexpressing METTL14 in MUM‐2B cells. (B) C, RUNX2 methylation site. (D) Mutations at the RUNX2 methylation site. (E) Luciferase activities in MUM‐2B cells transfected with RUNX2‐WT or RUNX2‐Mut + vector or METTL14. (F–J) RUNX2 promotes migration and invasion of CM cells in vitro. (F) Kaplan–Meier survival analysis of CM tumour samples suggest that high RUNX2 expression levels are related to reduced overall survival (OS). (G) Protein expression of RUNX2 in CM and normal choroidal tissues. (H) IHC staining of RUNX2 in CM and normal choroidal tissues. (I, J) Transwell assays were used to determine the effects of RUNX2 on migration and invasion capability in OCM1 and MUM‐2B cells.

Article Snippet: The slides were incubated overnight at 4 °C with the primary antibodies anti‐METTL14 (1:1000, HPA038002, Sigma‐Aldrich) and anti‐RUNX2 (1:1000, MAB2006, RD), followed by addition of secondary antibodies to an avidin‐biotin‐peroxidase complex (e.g. biotinylated anti‐rabbit and anti‐mouse antibodies in goats).

Techniques: Methylation, Migration, In Vitro, Modification, Luciferase, Transfection, Plasmid Preparation, Expressing, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: METTL14 promotes migration and invasion of choroidal melanoma by targeting RUNX2 mRNA via m6A modification

doi: 10.1111/jcmm.17577

Figure Lengend Snippet: Role of METTL14 and RUNX2 in metastasis. (A) Knockdown of RUNX2 inhibits lung metastasis in nude mice models. WB verified the successful construction of knockdown stabilized MUM‐2B cells. WT and RUNX2 knockdown MUM‐2B stable cells were injected via the tail vein. Representative images of metastatic lung tumours. (B, C) Transwell assay demonstrates the effects of METTL14 and RUNX2 on the migration and invasion capability of CM cells. Overexpression of RUNX2 partially reversed the reduction in cell migration and invasion caused by decreased METTL14 expression. All data are presented as mean ± SD from three independent replicates. Student's t ‐test was used to assess inter‐group differences.

Article Snippet: The slides were incubated overnight at 4 °C with the primary antibodies anti‐METTL14 (1:1000, HPA038002, Sigma‐Aldrich) and anti‐RUNX2 (1:1000, MAB2006, RD), followed by addition of secondary antibodies to an avidin‐biotin‐peroxidase complex (e.g. biotinylated anti‐rabbit and anti‐mouse antibodies in goats).

Techniques: Injection, Transwell Assay, Migration, Over Expression, Expressing

Journal: Biomaterials advances

Article Title: In vitro development and optimization of cell-laden injectable bioprinted gelatin methacryloyl (GelMA) microgels mineralized on the nanoscale.

doi: 10.1016/j.bioadv.2024.213805

Figure Lengend Snippet: Fig. 5. Osteocyte morphology and functionality in mineralized and non-mineralized microgels. A-B and E-F represent the confocal images of non-mineralized (A-B) and mineralized (E-F) samples. Microgels stained with actin (green), DAPI (blue), and SOST (red) and SOST intensity (normalized fluorescence per cell) in mineralized microgels (A-H, K-L). Also, the dendrite length in μm (I) and the number of dendrites per cell (I-J) were not significantly different between mineralized and non-mineralized samples. Statistical differences are represented by **** p < 0.0001 after the One-way ANOVA test post-Turkey’s corrections.

Article Snippet: Cells were permeabilized with 0.1 % Triton X100, blocked with 1.5 % bovine serum albumin in PBS, and incubated at room temperature for 1 h. Samples were then washed in PBS and incubated with the primary antibody (rabbit M.G. da Costa Sousa et al. Biomaterials Advances 159 (2024) 213805 polyclonal SOST antibody (Novus Biologicals, NBP1-77461)) (1:100 dilution), overnight at 4 ◦C.

Techniques: Staining, Fluorescence