runx2 promoter  (Genecopoeia)

Journal: The Journal of Clinical Investigation

Article Title: TET2 suppresses vascular calcification by forming an inhibitory complex with HDAC1/2 and SNIP1 independent of demethylation

doi: 10.1172/JCI186673

Figure Lengend Snippet: ( A and B ) Heatmap showing TET2 , SMC marker ( Myh11, Tagln ), and osteogenic marker ( Spp1, IL6, IL1a, Bmp1 ) mRNA expression in control and apoE –/– calcified mouse aorta ( A ) and β-GP treatment–induced calcified mouse aorta ( B ). ( C ) Leukocyte TET2 mRNA expression in patients with CKD with calcified ( n = 21) or noncalcified arteries ( n = 12). ( D ) Correlation between leukocyte TET2 mRNA expression and calcific score ( D ), or RUNX2 mRNA expression ( E ) in CKD patients with calcification (VC, n = 21). ( F ) Von Kossa staining and immunohistochemical images of TET2 expression in control and calcified arteries from patients with CKD. Scale bars: 50 μm. n = 6. ( G ) Von Kossa staining and immunohistochemical images of Tet2 expression in control and calcified mouse arteries. Scale bars: 100 μm. n = 3. ( H and I ) Western blot analysis and quantification of TET2 and RUNX2 expression in calcified mouse and control arteries ( H ) ( n = 3) or in hVSMCs induced by Pi for the indicated time ( I ) ( n = 3). All values are presented as mean ± SEM. * P < 0.05, ** P < 0.01. Statistical significance was assessed using 2-tailed t tests ( C ), 1-way ANOVA followed by Dunnett’s test ( H and I ), and Pearson’s correlation coefficient analysis ( D and E ).

Article Snippet: In order to examine the function of TET2 binding to the RUNX2 promoter in the context of VC, we carried out CUT&Tag coupled with qPCR (CUT&Tag-qPCR, NovoNGS; CUT&Tag High-Sensitivity Kit, Cell Signaling Technology).

Techniques: Marker, Expressing, Control, Staining, Immunohistochemical staining, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: TET2 suppresses vascular calcification by forming an inhibitory complex with HDAC1/2 and SNIP1 independent of demethylation

doi: 10.1172/JCI186673

Figure Lengend Snippet: ( A and B ) Alizarin red staining of hVSMCs transfected with Lenti-sh-Scr or with Lenti-sh-TET2 ( A ) and transfected with Ad-Vector or Ad-TET2 ( B ) ( n = 3). ( C and D ) ALP activity assay ( C ) and quantification of calcium content ( D ) in hVSMCs transfected with Lenti-sh-Scr or Lenti-sh-TET2 ( n = 5). ( E and F ) ALP activity assay ( E ) and quantification of calcium content ( F ) in hVSMCs transfected with Ad-Vector or Ad-TET2 ( n = 5). ( G and H ) Western blot analysis and quantification of TET2, RUNX2, OPN, smoothelin, and SM22α expression in hVSMCs transfected with Lenti-sh-Scr or with Lenti-sh-TET2 ( G ) and transfected with Ad-Vector or Ad-TET2 ( H ) ( n = 3). All values are presented as mean ± SEM. * P < 0.05, ** P < 0.01. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s test ( C – H ).

Article Snippet: In order to examine the function of TET2 binding to the RUNX2 promoter in the context of VC, we carried out CUT&Tag coupled with qPCR (CUT&Tag-qPCR, NovoNGS; CUT&Tag High-Sensitivity Kit, Cell Signaling Technology).

Techniques: Staining, Transfection, Plasmid Preparation, ALP Activity Assay, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: TET2 suppresses vascular calcification by forming an inhibitory complex with HDAC1/2 and SNIP1 independent of demethylation

doi: 10.1172/JCI186673

Figure Lengend Snippet: ( A ) Representative alizarin red S staining images of whole aortas from control mice, mice injected with vitamin D3, and mice injected with vitamin D3 together with AAV-sh-Scr or AAV-sh-Tet2 . n = 3. ( B ) Representative von Kossa staining of aortic sections from control mice, mice injected with vitamin D3, and mice injected with vitamin D3 together with AAV-sh-Scr or AAV-sh-Tet2 . Scale bars: 100 μm. n = 3. ( C ) Representative immunohistochemical images of Runx2 expression in aortic sections from control mice, mice injected with vitamin D3, and mice injected with vitamin D3 together with AAV-sh-Scr or AAV-sh-Tet2 . Scale bars: 100 μm. n = 3. ( D ) Western blot analysis and quantification of Tet2 and osteogenic phenotypic marker (Runx2 and Opn) and contractile phenotype marker (smoothelin and SM22α) expression in aortas from control mice, mice injected with vitamin D3, and mice injected with vitamin D3 together with AAV-sh-Scr or AAV-sh-Tet2 . n = 3. ( E ) Representative alizarin red S staining images of whole aortas from control mice, CKD model mice, and CKD model mice injected with AAV-sh-Scr or AAV-sh-Tet2 . n = 3. ( F ) Representative von Kossa staining of aortic sections from control mice, CKD model mice, and CKD model mice injected with AAV-sh-Scr or AAV-sh-Tet2. Scale bars: 100μm. n = 3. ( G ) Western blot analysis and quantification of Tet2 and osteogenic phenotypic marker (Runx2 and Opn) and contractile phenotype marker (smoothelin and SM22α) expression in aortas from control mice, CKD model mice, and CKD model mice injected with AAV-sh-Scr or AAV-sh-Tet2. n = 3. All values are presented as mean ± SEM. * P < 0.05, ** P < 0.01. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s test ( D and G ).

Article Snippet: In order to examine the function of TET2 binding to the RUNX2 promoter in the context of VC, we carried out CUT&Tag coupled with qPCR (CUT&Tag-qPCR, NovoNGS; CUT&Tag High-Sensitivity Kit, Cell Signaling Technology).

Techniques: Staining, Control, Injection, Immunohistochemical staining, Expressing, Western Blot, Marker

Journal: The Journal of Clinical Investigation

Article Title: TET2 suppresses vascular calcification by forming an inhibitory complex with HDAC1/2 and SNIP1 independent of demethylation

doi: 10.1172/JCI186673

Figure Lengend Snippet: ( A and B ) Quantitative real-time PCR analysis of RUNX2 expression in hVSMCs transfected with Lenti-sh-Scr or Lenti-sh-TET2 ( A ), or Ad-Vector or Ad-TET2 ( B ) ( n = 4). ( C ) ChiP-Seq analysis for TET2 enrichment on the RUNX2 gene. ( D and E ) DNA methylation quantified by MethylCap-qPCR in the RUNX2 P2 promoter from hVSMCs with TET2 overexpression ( D ) or TET2 knockdown ( E ) ( n = 5). ( F and G ) Luciferase activity analyzed after cotransfection with control Renilla luciferase plasmid and constructs of the RUNX2 P1 promoter ( F ) or P2 promoter–driven luciferase reporters ( G ), and cotransfection with control, TET2 -WT, or enzyme activity locus – mutated TET2 ( TET2 -MUT) ( n = 6). ( H ) ATAC-Seq analysis for RUNX2 gene transposase-accessible chromatin in the TET2 -WT and TET2 -KO groups. ( I ) ATAC-Seq analysis for RUNX2 gene transposase-accessible chromatin in the TET2 -WT and TET2 -MUT groups. ( J and K ) TET2 CUT&Tag-qPCR ( J ) and H3K27ac CUT&Tag-qPCR ( K ) at the RUNX2 (CUT1) P1 and (CUT2) P2 promoter in either control or Pi-exposed hVSMCs ( n = 3). ( L and M ) H3K27ac CUT&Tag-qPCR at the RUNX2 (CUT1) P1 and (CUT2) P2 promoter in hVSMCs with either TET2 knockdown ( L ) or TET2 overexpression ( M ) ( n = 3). All values are presented as mean ± SD. * P < 0.05, ** P < 0.01. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s test ( A , B , D – G and J – M ).

Article Snippet: In order to examine the function of TET2 binding to the RUNX2 promoter in the context of VC, we carried out CUT&Tag coupled with qPCR (CUT&Tag-qPCR, NovoNGS; CUT&Tag High-Sensitivity Kit, Cell Signaling Technology).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, ChIP-sequencing, DNA Methylation Assay, Over Expression, Knockdown, Luciferase, Activity Assay, Cotransfection, Control, Construct

Journal: The Journal of Clinical Investigation

Article Title: TET2 suppresses vascular calcification by forming an inhibitory complex with HDAC1/2 and SNIP1 independent of demethylation

doi: 10.1172/JCI186673

Figure Lengend Snippet: ( A ) Co-IP analysis to detect the interaction between TET2 and HDAC1/2 in hVSMCs. ( B ) In VSMCs pretreated with control or HDAC1/2 knockdown, luciferase activities were analyzed after cotransfection with control Renilla luciferase plasmid and constructs of RUNX2 P2 promoter–driven luciferase reporters, and cotransfection with control, TET2-WT, or enzyme activity locus–mutated TET2 ( n = 6 per group). ( C ) H3K27ac CUT&Tag-qPCR at the RUNX2 P2 promoter in hVSMCs transfected with si-Scr , si-HDAC1 , HDAC2 , or HDAC1/2 , together with subjection to TET2 overexpression ( n = 3 per group). ( D – I ) HDAC1 CUT&Tag-qPCR ( D – F ) or HDAC2 CUT&Tag-qPCR ( G – I ) at the RUNX2 P2 promoter in hVSMCs with either Pi exposure ( D and G ), TET2 knockdown ( E and I ), or TET2 overexpression ( F and H ) ( n = 3 per group). ( J ) HDAC2 CUT&Tag-qPCR at the RUNX2 P2 promoter in TET2-overexpressing hVSMCs with either control or HDAC1 knockdown ( n = 3 per group). All values are presented as mean ± SEM. * P < 0.05, ** P < 0.01. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s test ( B – J ).

Article Snippet: In order to examine the function of TET2 binding to the RUNX2 promoter in the context of VC, we carried out CUT&Tag coupled with qPCR (CUT&Tag-qPCR, NovoNGS; CUT&Tag High-Sensitivity Kit, Cell Signaling Technology).

Techniques: Co-Immunoprecipitation Assay, Control, Knockdown, Luciferase, Cotransfection, Plasmid Preparation, Construct, Activity Assay, Transfection, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: TET2 suppresses vascular calcification by forming an inhibitory complex with HDAC1/2 and SNIP1 independent of demethylation

doi: 10.1172/JCI186673

Figure Lengend Snippet: ( A ) Alizarin red staining of hVSMCs transfected with si-Scr or si-HDAC1/2 together with Ad-Vector or Ad-TET2 ( n = 3 per group). ( B and C ) ALP activity assay ( B ) and quantification of calcium content ( C ) in hVSMCs transfected with si-Scr or si-HDAC1/2 together with Ad-Vector or Ad-TET2 ( n = 5 per group). ( D and E ) Western blot analysis and quantification of TET2, RUNX2, OPN, smoothelin, SM22, and HDAC1/2 expression in hVSMCs transfected with si-Scr or si-HDAC1/2 together with Ad-Vector or Ad-TET2 ( n = 3 per group). All values are presented as mean ± SEM. * P < 0.05, ** P < 0.01. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s test ( B , C , and E ).

Article Snippet: In order to examine the function of TET2 binding to the RUNX2 promoter in the context of VC, we carried out CUT&Tag coupled with qPCR (CUT&Tag-qPCR, NovoNGS; CUT&Tag High-Sensitivity Kit, Cell Signaling Technology).

Techniques: Staining, Transfection, Plasmid Preparation, ALP Activity Assay, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: TET2 suppresses vascular calcification by forming an inhibitory complex with HDAC1/2 and SNIP1 independent of demethylation

doi: 10.1172/JCI186673

Figure Lengend Snippet: ( A ) ChIP-Seq analysis for SNIP1 enrichments on the RUNX2 gene. ( B ) SMAD2 binding motif. ( C ) Co-IP to detect the interaction between SNIP1, TET2, and HDAC1/2 in hVSMCs. ( D ) Co-IP analysis of hVSMCs pretransfected with Lenti-sh-Scr or Lenti-sh-SNIP1 to detect the interaction between TET2 and HDAC1/2. ( E ) Luciferase activity analysis of cells preinfected with Lenti-sh-Scr or Lenti-sh-SNIP1, after cotransfection with control Renilla luciferase plasmid and constructs of the P2 promoter–driven luciferase reporters; and cotransfection with control, TET2-WT, or enzyme activity locus–mutated TET2 ( n = 6 per group). ( F – I ) TET2 ( F ), HDAC1 ( G ), HDAC2 ( H ), and H3K27ac ( I ) CUT&Tag-qPCR at the RUNX2 P2 promoter in hVSMCs transfected with Lenti-sh-Scr or Lenti-sh-SNIP1 together with TET2 overexpression ( n = 3 per group). All values are presented as mean ± SEM. * P < 0.05, ** P < 0.01. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s test ( E – I ).

Article Snippet: In order to examine the function of TET2 binding to the RUNX2 promoter in the context of VC, we carried out CUT&Tag coupled with qPCR (CUT&Tag-qPCR, NovoNGS; CUT&Tag High-Sensitivity Kit, Cell Signaling Technology).

Techniques: ChIP-sequencing, Binding Assay, Co-Immunoprecipitation Assay, Luciferase, Activity Assay, Cotransfection, Control, Plasmid Preparation, Construct, Transfection, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: TET2 suppresses vascular calcification by forming an inhibitory complex with HDAC1/2 and SNIP1 independent of demethylation

doi: 10.1172/JCI186673

Figure Lengend Snippet: ( A ) Western blot analysis and quantification of Snip1 expression in aortas from mice injected with AAV-sh-Scr and AAV-sh-Snip1 ( n = 3 per group). ( B ) Representative alizarin red S staining images of whole aortas from control mice, mice injected with vitamin D3, and mice injected with AAV-sh-Scr or AAV-sh-Snip1 ( n = 3 per group). ( C ) Representative von Kossa staining of aortic sections from control mice, mice injected with vitamin D3, and mice injected with AAV-sh-Scr or AAV-sh-Snip1 . Scale bars: 100 μm. n = 3 per group. ( D ) Western blot analysis and quantification of Snip1, osteogenic phenotypic marker Runx2, and contractile phenotype marker (Smoothelin and SM22α) expression in the aortas from control mice, mice injected with vitamin D3, and mice injected with AAV-sh-Scr or AAV-sh-Snip1 ( n = 3 per group). All values are presented as mean ± SEM. * P < 0.05, ** P < 0.01. Statistical significance was assessed using 2-tailed t tests ( A ) and 1-way ANOVA followed by Dunnett’s test ( D ).

Article Snippet: In order to examine the function of TET2 binding to the RUNX2 promoter in the context of VC, we carried out CUT&Tag coupled with qPCR (CUT&Tag-qPCR, NovoNGS; CUT&Tag High-Sensitivity Kit, Cell Signaling Technology).

Techniques: Western Blot, Expressing, Injection, Staining, Control, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Gli1-mediated tumor cell-derived bFGF promotes tumor angiogenesis and pericyte coverage in non-small cell lung cancer

doi: 10.1186/s13046-024-03003-0

Figure Lengend Snippet: A schema showing that Gli1-mediated tumor cell-derived bFGF promotes tumor angiogenesis by regulating bFGF expression. The bFGF bind with FGFR1 in the endothelial cells and FGFR2 in the pericytes, and then promotes endothelial cell mediated- and pericytes-mediated motility for angiogenesis, leading to tumor angiogenesis and growth of NSCLC

Article Snippet: Antibodies against Ki67 (#12,202), α-SMA (#19,245), bFGF (#20,102), FGFR1 (#76,123), FGFR2 (#23,328), β-actin (#23,328) and HRP-conjugated anti-rabbit IgG (#7074) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Derivative Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Crosstalk between protein kinase C α and transforming growth factor β signaling mediated by Runx2 in intestinal epithelial cells

doi: 10.1016/j.jbc.2023.103017

Figure Lengend Snippet: Runx2 is required for PKCα-induced upregulation of TGFβR1 . A and B , IEC-18 cells were transfected with nontargeting or Runx2 (rat, mouse, human) targeting ONTARGETplus SMARTPool siRNA for 72 h prior to treatment with 100 nM PMA or 20 μg/ml DiC 8 . After 2 h, expression of the indicated proteins was determined by Western blotting ( Ai,ii ) and TGFβR1 mRNA levels (normalized to 18S rRNA) were determined by RT-qPCR ( B ). C , IEC-18 cells were transfected with nontargeting or rat-specific Runx2-targeting siRNA for 72 h prior to treatment with 100 nM PMA for 2 h, and expression of the indicated proteins was determined by Western blotting ( i ). IEC-18 cells stably expressing human Runx2 (Origene, RC212936) were transfected with nontargeting siRNA, rat/mouse/human Runx2-targeting ONTARGETplus SMARTPool siRNA, or rat-specific Runx2 siRNA for 72 h prior to treatment with 100 nM PMA for 2 h. Expression of the indicated proteins was determined by Western blotting. Endogenous rat Runx2 ( arrowhead ) and exogenous human Runx2 ( arrow ) are indicated ( ii ). Cells generated as in ( i ) and ( ii ) were treated with PMA for 2 h as indicated and TGFβR1 mRNA levels (normalized to 18 S rRNA) were determined by RT-qPCR ( iii ). Data in A and Ci , and Cii are representative of at least three independent experiments, and data in B and Ciii are the average ± SEM of at least three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01. DiC8, 1,2-dioctanoyl- sn -glycerol; IEC-18, intestinal crypt-like cells; PKCα, protein kinase C α; PMA, phorbol 12-myristate 13-acetate; Runx2, runt-related transcription factor 2; TGFβR1, transforming growth factor-β receptor 1.

Article Snippet: For rescue experiments, IEC-18 cells were transfected with a plasmid in which human Runx2 expression is driven by the CMV promoter (RC212936, Origene), and stable transfectants were selected with 1.5 mg/ml G418.

Techniques: Transfection, Expressing, Western Blot, Quantitative RT-PCR, Stable Transfection, Generated

Journal: Cell reports

Article Title: Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning

doi: 10.1016/j.celrep.2021.109293

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To assay human RUNX2 isoform overexpression we subcloned human RUNX2-I (MRIPV isoform, GeneCopoeia #EX-I2457-Lv105) into pCIB (Addgene #119863), and the human-equivalent ERK-target sites were made using Quikchange and subcloning: wild-type pCIB-hsRUNX2, pCIB-hsRUNX2-S280A-S298A (RUNX2-SA) and hsRUNX2-S280E-S298E (RUNX2-SE) which were used for additional in vitro validations (Figures S9B and S9C; note that human RUNX2-SA does not run faster by SDS-PAGE than human RUNX2-WT or human RUNX2-SE, as is observed with mouse isoforms, yet the effects on target gene expression is consistent for both species).

Techniques: Recombinant, Membrane, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell reports

Article Title: Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning

doi: 10.1016/j.celrep.2021.109293

Figure Lengend Snippet: (A) Schematics of discovery approach identifying candidate drivers of mechanical memory-mediated metastasis. Gray dots, mechanically sensitive upstream regulators from RNA-seq analysis (141 genes); blue dots, Human Cancer Metastasis Database metastasis-associated genes (1,811 genes); black/red dots, intersecting genes (123 genes); red dots, intersecting genes that are known gene bookmarkers (5 genes); inset shows further characterization. See for in-depth description. (B) Heatmap showing clustering of ATAC-seq samples; scale shows Jaccard index. See for sample annotation. (C) Graphic representation of change in chromatin accessibility over time after changing the mechanical environment; inset shows RUNX and BACH family motifs as representative of the significantly enriched motifs in the delayed-closing and quick-closing sites, respectively (motifs analysis performed by HOMER). (D) The number of pairwise differentially decreased accessibility sites are shown consequent of RUNX2-knockdown (vertical arrows) and longitudinally upon transition to soft matrix (horizontal arrows). Data represent 3 biological replicates per condition. (E) Top enriched motifs are shown for the vertical comparisons in (D), where the first percentage is the number of differential peaks with the motif and the second is the number of GC-matched random genomic sequences with motif.

Article Snippet: To assay human RUNX2 isoform overexpression we subcloned human RUNX2-I (MRIPV isoform, GeneCopoeia #EX-I2457-Lv105) into pCIB (Addgene #119863), and the human-equivalent ERK-target sites were made using Quikchange and subcloning: wild-type pCIB-hsRUNX2, pCIB-hsRUNX2-S280A-S298A (RUNX2-SA) and hsRUNX2-S280E-S298E (RUNX2-SE) which were used for additional in vitro validations (Figures S9B and S9C; note that human RUNX2-SA does not run faster by SDS-PAGE than human RUNX2-WT or human RUNX2-SE, as is observed with mouse isoforms, yet the effects on target gene expression is consistent for both species).

Techniques: RNA Sequencing, Knockdown, Genomic Sequencing

Journal: Cell reports

Article Title: Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning

doi: 10.1016/j.celrep.2021.109293

Figure Lengend Snippet: (A) Immunoblot of RUNX2 in patient-derived xenograft (PDX) primary cells and breast cancer cell lines, preconditioned on soft and stiff hydrogels for 7 days (representative of n = 2 biological replicates). (B) Immunoblot of RUNX2 in SUM159 and T47D cells cultured for 7 days in 3D matrix consisting of soft 1.0 mg/mL rat-tail collagen-I, or stiff 1.0 mg/mL rat-tail collagen-I crosslinked with PEG-di(NHS) to stiffen the collagen lattice without changing ligand density (representative of n = 3 biological replicates). (C) qRT-PCR of 4 RUNX2 target genes in SUM159 cells preconditioned for 7 days on soft and stiff hydrogels with non-targeting small hairpin RNA (shRNA) (GIPZ), or on stiff hydrogels with 2 shRNAs targeting RUNX2 (n = 3 biological replicates). Data are means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001; 1-way ANOVA with Tukey’s multiple comparisons test. (D) qRT-PCR of RUNX2 in SUM159 cells preconditioned for 7 days on soft and stiff hydrogels with non-targeting shRNA (GIPZ), or on stiff hydrogels with 2 shRNAs targeting RUNX2 (n = 3 biological replicates). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; 1-way ANOVA with Sidak’s multiple comparisons test. (E) qRT-PCR of RUNX2 and 4 target genes, plus CTGF (YAP target) and PLIN1 (adipogenic biomarker) in SUM159 cells preconditioned, as indicated (n = 3 biological replicates). Data are means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001; 1-way ANOVA with Tukey’s multiple comparisons test. (F) qRT-PCR of the RUNX2 gene target OPN , and 3 YAP targets— CTGF , CYR61 , and ANKRD1 —in SUM159 cells preconditioned as indicated, and without media change for 48 h before sample collection (n = 3 biological replicates). Data are means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001; 1-way ANOVA with Tukey’s multiple comparisons test. (G) Immunoblot of RUNX2, ERK, and pERK in SUM159 cells stably expressing lentiviral shRUNX2 or GIPZ (non-targeting control), preconditioned for 7 days on soft or stiff hydrogels (representative of n = 3 biological replicates). (H) Immunoblot of pERK and ERK in SUM159 cells cultured on stiff hydrogels with 20 μM PD98059, 30 μM blebbistatin, 100 nM dasatinib, 1 μM Faki14, or DMSO for 1 h before lysis (representative of n = 3 biological replicates). (I) Immunofluorescence of pFAK, paxillin, and F-actin in SUM159 cells cultured on stiff or soft hydrogels for 7 days. Scale bar, 10 μm. (J) Immunoblot of OPN in SUM159 cells preconditioned for 7 days on soft and stiff hydrogels with non-targeting shRNA (GIPZ), on stiff hydrogels with 2 shRNAs targeting RUNX2, on soft hydrogels with constitutively active MEK-DD expression, or on stiff hydrogels with MEK inhibitor PD98059 (20 μM) (representative of n = 3 biological replicates). (K and L) qRT-PCR of OPN (K) and GM-CSF (L) in SUM159 cells preconditioned for 7 days on stiff hydrogels conjugated with either poly D-lysine (PDL) to reduce integrin binding, or collagen conjugated with DMSO (control), 30 μM blebbistatin, 100 nM dasatinib, or 1 μM Faki14 in media changed every other day (n = 3 biological replicates). Data are means ± SEMs and normalized to 7-day stiff controls. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; 1-way ANOVA with Sidak’s multiple comparisons test. (M) Immunoblot showing phospho-AKT levels in SUM159 cells treated with DMSO (control) or AKT inhibitor (1 μM MK-2206) (representative of n = 2 biological replicates). (N) qRT-PCR of RUNX2 target genes in SUM159 cells preconditioned for 7 days on stiff hydrogels and treated with DMSO or 1 μM AKT inhibitor MK-2206 (n = 3 biological replicates). Data are means ± SEMs. Multiple t test with Holm-Sidak multiple comparisons; adjusted p values are not significant (n.s.).

Article Snippet: To assay human RUNX2 isoform overexpression we subcloned human RUNX2-I (MRIPV isoform, GeneCopoeia #EX-I2457-Lv105) into pCIB (Addgene #119863), and the human-equivalent ERK-target sites were made using Quikchange and subcloning: wild-type pCIB-hsRUNX2, pCIB-hsRUNX2-S280A-S298A (RUNX2-SA) and hsRUNX2-S280E-S298E (RUNX2-SE) which were used for additional in vitro validations (Figures S9B and S9C; note that human RUNX2-SA does not run faster by SDS-PAGE than human RUNX2-WT or human RUNX2-SE, as is observed with mouse isoforms, yet the effects on target gene expression is consistent for both species).

Techniques: Western Blot, Derivative Assay, Cell Culture, Quantitative RT-PCR, shRNA, Biomarker Discovery, Stable Transfection, Expressing, Control, Lysis, Immunofluorescence, Binding Assay

Journal: Cell reports

Article Title: Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning

doi: 10.1016/j.celrep.2021.109293

Figure Lengend Snippet: (A) Immunofluorescence staining of RUNX2 in SUM159 cells on soft and stiff hydrogels. Scale bars, 10 μm. (B) Immunofluorescence corresponding to . Cell boundaries are delineated in blue. Scale bars, 10 μm. (C) Quantification of (B) (n = 40 cells in each condition from n = 3 biological replicates). ****p < 0.0001; 2-tailed unpaired Student’s t test. (D and E) Immunofluorescence staining (D) and quantification of nuclear localization (E) of RUNX2 in PC3 prostate cancer cells (n = 50 cells each from n = 3 biological replicates). (F and G) Immunofluorescence staining of RUNX2 (F) and quantification of nuclear intensity of RUNX2 in CK + cells (G) in supraphysiological stiffness-naive patient-derived xenograft HCI-005 tumor cells (n = 60 cells each from n = 3 biological replicates). (H) Immunofluorescence staining in SUM159 cells, preconditioned on soft and stiff hydrogels for 7 days before transferring to collagen-coated glass for 3 h. (I and J) Quantification of nuclear RUNX2 (I) and cell area (J) from cells in (H). Data are means ± SEMs. ****p < 0.0001; 2-tailed unpaired Student’s t test. (K) Correlation analysis of (I) and (J), showing no positive intracellular correlation between cell spreading and nuclear RUNX2 in either soft- or stiff-preconditioned cells spreading on glass (n = 40 cells each from n = 3 biological replicates). Soft-to-glass Pearson’s r = −0.31 (not significant [n.s.]); stiff-to-glass Pearson’s r = −0.30 (n.s.). (L) Immunofluorescence staining of RUNX2 in SUM159 cells preconditioned for 7 days on stiff hydrogels, treated with DMSO (control), 1 μM taxol, 50 nM jas-plakinolide, 30 μM blebbistatin, or 20 μM Y27632, added 3 h before fixation. Scale bars, 10 μm. (M) Immunofluorescence staining of RUNX2 in SUM159 cells preconditioned for 7 days on soft hydrogels, with DMSO (control), 1 μg/mL lysophosphatidic acid (LPA), 10 μg/mL Rho Activator II, or 10 μM nocodazole, added 3 h before fixation. Scale bars, 10 μm. (N) Quantification of (L) (n R 40 cells each condition from n = 3 biological replicates). (O) Quantification of (M) (n R 40 cells each condition from n = 3 biological replicates). Data are means ± SEMs. ****p < 0.0001; 1-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: To assay human RUNX2 isoform overexpression we subcloned human RUNX2-I (MRIPV isoform, GeneCopoeia #EX-I2457-Lv105) into pCIB (Addgene #119863), and the human-equivalent ERK-target sites were made using Quikchange and subcloning: wild-type pCIB-hsRUNX2, pCIB-hsRUNX2-S280A-S298A (RUNX2-SA) and hsRUNX2-S280E-S298E (RUNX2-SE) which were used for additional in vitro validations (Figures S9B and S9C; note that human RUNX2-SA does not run faster by SDS-PAGE than human RUNX2-WT or human RUNX2-SE, as is observed with mouse isoforms, yet the effects on target gene expression is consistent for both species).

Techniques: Immunofluorescence, Staining, Derivative Assay, Transferring, Control

Journal: Cell reports

Article Title: Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning

doi: 10.1016/j.celrep.2021.109293

Figure Lengend Snippet: (A) qRT-PCR of RUNX2 target genes in SUM159 cells overexpressing RUNX2-WT, RUNX2-SE, or RUNX2-SA, preconditioned for 7 days on soft or stiff hydrogels (n = 3 biological replicates). (B) Quantification of invasion of SUM159 cells preconditioned as indicated in (A) (n = 3 biological replicates with n = 3 technical replicates). See – and . (C) Micro-CT 3D reconstructions of proximal tibia from mice 4 weeks after intracardiac injection of SUM159 cells preconditioned as in (A) or no cancer cells (control). (D) Micro-CT analysis of bone volume from mice in (C) ( n , mice; soft RUNX2-WT 5; soft RUNX2-SE 6; stiff RUNX2-WT 5; stiff RUNX2-SA 5; control 3). (E) Time course of SUM159 cells spreading on synthetic bone matrix, preconditioned as in (A) (n = 36 cells in each condition from n = 3 biological replicates). See . Shaded regions are means ± SEMs. ****p < 0.0001; 2-way ANOVA with Tukey’s multiple comparisons test. (F) Tartrate-resistant acid phosphatase (TRAP) staining of RAW264.7 cells after 7 days’ incubation: 4 days with 50 ng/mL RANKL in growth media, and then 3 days with 50% SUM159-conditioned media (CM) + 50% growth media. CM was collected 24 h after addition to hydrogels with equal SUM159 cell counts in each experimental group. Scale bar, 100 μm. (G and H) Quantification of (F) (n = 3 biological replicates with n = 3 technical replicates). Two-tailed unpaired Student’s t test. Data are means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; 1-way ANOVA with Holm-Sidak’s multiple comparisons test, except for (E) and (G).

Article Snippet: To assay human RUNX2 isoform overexpression we subcloned human RUNX2-I (MRIPV isoform, GeneCopoeia #EX-I2457-Lv105) into pCIB (Addgene #119863), and the human-equivalent ERK-target sites were made using Quikchange and subcloning: wild-type pCIB-hsRUNX2, pCIB-hsRUNX2-S280A-S298A (RUNX2-SA) and hsRUNX2-S280E-S298E (RUNX2-SE) which were used for additional in vitro validations (Figures S9B and S9C; note that human RUNX2-SA does not run faster by SDS-PAGE than human RUNX2-WT or human RUNX2-SE, as is observed with mouse isoforms, yet the effects on target gene expression is consistent for both species).

Techniques: Quantitative RT-PCR, Micro-CT, Injection, Control, Staining, Incubation, Two Tailed Test