Journal: iScience

Article Title: Quantitative increase in T regulatory cells enhances bone remodeling in osteogenesis imperfecta

doi: 10.1016/j.isci.2022.104818

Figure Lengend Snippet:

Article Snippet: TaqMan Gene Expression Assay Runx-2 , Thermo Fisher , Cat#4453320; Mm00501584_m1.

Techniques: Recombinant, Magnetic Beads, Diagnostic Assay, cDNA Synthesis, Gene Expression, Enzyme-linked Immunosorbent Assay, Staining, Software, Micro-CT, Irradiation

Journal: Journal of Cellular Physiology

Article Title: Long non‐coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR‐204‐5p and miR‐125a‐3p

doi: 10.1002/jcp.26424

Figure Lengend Snippet: Functional roles of TCONS_00041960 in adipogenesis and osteogenesis. (a) BMSCs were transfected with Ex‐lnc or Ex‐NC and analyzed by qRT‐PCR. GAPDH served as an internal control. (b) Osteogenesis‐associated genes (Runx2, osterix, and osteocalcin) and adipogenesis‐associated genes (PPARγ, C/EBPα, and GILZ) were detected by qRT‐PCR. Data were normalized to GAPDH mRNA levels. (c) Western blot analysis to verify osteogenesis‐associated (Runx2, osterix, and osteocalcin) and adipogenesisassociated (PPARγ, C/EBPα, and GILZ) protein levels. GAPDH was used as a reference. (d and e) TG contents and ALP activity were measured at day 14. (f) Oil red O staining was performed at day 14. * p < 0.05, ** p < 0.01 versus Dex or Ex‐NC groups

Article Snippet: Primary antibodies, anti‐runt‐related transcription factor 2 (Runx2) (1:500, Abcam, Cambridge, MA), anti‐osterix (1:500, Abcam), anti‐osteocalcin (1:300, Santa Cruz Biotechnology, Dallas, TX), anti‐PPARγ (1:1,000, Abcam), anti‐C/EBPα (1:500, Santa Cruz Biotechnology), and anti‐glucocorticoid‐induced leucine zipper (GILZ) (1:500, Santa Cruz Biotechnology), were applied overnight at 4 °C.

Techniques: Functional Assay, Transfection, Quantitative RT-PCR, Western Blot, Activity Assay, Staining

Journal: Journal of Cellular Physiology

Article Title: Long non‐coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR‐204‐5p and miR‐125a‐3p

doi: 10.1002/jcp.26424

Figure Lengend Snippet: Runx2 and GILZ are direct targets of miR‐204‐5p and miR‐125a‐5p, respectively. (a and b) Putative binding sites between miR‐204‐5p and Runx2, and between miR‐125a‐5p and GILZ. (c and d) Western blot analysis of Runx2 and GILZ expression in transfected cells. GAPDH was used as an internal reference. (e and f) Dual luciferase assays were performed in cells co‐transfected with wild‐type or mutant pmirGLO‐Runx2 and wild‐type or mutant pmirGLO‐GILZ with miR‐204‐5p and miR‐125a‐3p. * p < 0.05

Article Snippet: Primary antibodies, anti‐runt‐related transcription factor 2 (Runx2) (1:500, Abcam, Cambridge, MA), anti‐osterix (1:500, Abcam), anti‐osteocalcin (1:300, Santa Cruz Biotechnology, Dallas, TX), anti‐PPARγ (1:1,000, Abcam), anti‐C/EBPα (1:500, Santa Cruz Biotechnology), and anti‐glucocorticoid‐induced leucine zipper (GILZ) (1:500, Santa Cruz Biotechnology), were applied overnight at 4 °C.

Techniques: Binding Assay, Western Blot, Expressing, Transfection, Luciferase, Mutagenesis

Journal: Journal of Cellular Physiology

Article Title: Long non‐coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR‐204‐5p and miR‐125a‐3p

doi: 10.1002/jcp.26424

Figure Lengend Snippet: Down‐regulation of miR‐204‐5p and miR‐125a‐3p enhances osteogenesis and suppresses adipogenesis of glucocorticoid‐treated rat BMSCs. Blank group, non‐transfected and untreated BMSCs; Dex group, dexamethasone‐treated BMSCs; NC group, transfected with the negative control and treated with dexamethasone; miR‐204‐5p inhibitor group, transfected with miR‐204‐5p inhibitor and treated with dexamethasone; miR‐125a‐3p inhibitor group, transfected with miR‐125a‐3p inhibitor and treated with dexamethasone. (a and b) Dexamethasone‐treated BMSCs transfected with miR‐204‐5p inhibitor or miR‐125a‐3p inhibitor were analyzed by qRT‐PCR. U6 served as an internal control. (c–f) qRT‐PCR analysis and western blotting were used to detect expression of osteogenesis‐associated genes Runx2, osterix, and osteocalcin, and adipogenesis‐associated genes PPARγ, C/EBPα, and GILZ. Data were normalized to GAPDH mRNA levels. (g and h) TG contents and ALP activity were measured at day 14. (i) Oil red O staining was performed at day 14. * p < 0.05

Article Snippet: Primary antibodies, anti‐runt‐related transcription factor 2 (Runx2) (1:500, Abcam, Cambridge, MA), anti‐osterix (1:500, Abcam), anti‐osteocalcin (1:300, Santa Cruz Biotechnology, Dallas, TX), anti‐PPARγ (1:1,000, Abcam), anti‐C/EBPα (1:500, Santa Cruz Biotechnology), and anti‐glucocorticoid‐induced leucine zipper (GILZ) (1:500, Santa Cruz Biotechnology), were applied overnight at 4 °C.

Techniques: Transfection, Negative Control, Quantitative RT-PCR, Western Blot, Expressing, Activity Assay, Staining

Journal: Journal of Cellular Physiology

Article Title: Long non‐coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR‐204‐5p and miR‐125a‐3p

doi: 10.1002/jcp.26424

Figure Lengend Snippet: TCONS_0004196 regulates osteogenic and adipogenic differentiation of glucocorticoid‐treated rat BMSCs by targeting miR‐204‐5p and miR‐125a‐3p. (a and b) BMSCs were transfected with recombinant lentiviruses carrying TCONS_00041960, miR‐204‐5p inhibitor, or miR‐125a‐3p inhibitor and then analyzed by qRT‐PCR. U6 served as an internal control. (c) Expression of osteogenesis‐associated genes (Runx2, osterix, and osteocalcin) and adipogenesis‐associated genes (PPARγ, C/EBPα, and GILZ) was detected by qRT‐PCR. Data were normalized to GAPDH mRNA levels. (d) TG contents were measured in rat BMSCs transfected with recombinant lentiviruses carrying TCONS_00041960 or miR‐125a‐3p mimics and cotransfected with recombinant lentiviruses carrying TCONS_00041960 and miR‐125a‐3p mimics. (e) ALP activity was detected in rat BMSCs transfected with recombinant lentiviruses carrying TCONS_00041960 or miR‐204‐5p mimics and co‐transfected with recombinant lentiviruses carrying TCONS_00041960 and miR‐204‐5p mimics. * p < 0.05

Article Snippet: Primary antibodies, anti‐runt‐related transcription factor 2 (Runx2) (1:500, Abcam, Cambridge, MA), anti‐osterix (1:500, Abcam), anti‐osteocalcin (1:300, Santa Cruz Biotechnology, Dallas, TX), anti‐PPARγ (1:1,000, Abcam), anti‐C/EBPα (1:500, Santa Cruz Biotechnology), and anti‐glucocorticoid‐induced leucine zipper (GILZ) (1:500, Santa Cruz Biotechnology), were applied overnight at 4 °C.

Techniques: Transfection, Recombinant, Quantitative RT-PCR, Expressing, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: LRP4 and Agrin Are Modulated by Cartilage Degeneration and Involved in β-Catenin Signaling in Human Articular Chondrocytes

doi: 10.3390/ijms26031007

Figure Lengend Snippet: Effect of recombinant human (rh) Agrin treatment on gene expression in human articular chondrocytes. ( A ) LRP4 expression was downregulated at both 48 h and 72 h after rhAgrin treatment. LRP5/6 expression was upregulated at 24 h after rhAgrin treatment. ( B ) SRY-box transcription factor 9 (SOX9) expression seemed to be upregulated gradually, but did not show significant differences from the control group. ACAN expression was downregulated. Expression of Runt-related transcription factor 2 (RUNX2) and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) was upregulated, whereas matrix metalloproteinases (MMPs) did not show significant differences. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001.

Article Snippet: RNA samples (500 ng) were reverse-transcribed with Primescript RT master mix (Takara Bio Inc., Shiga, Japan), and the resulting cDNAs were used for real-time PCR using the QuantStudioTM 1 real-time PCR system (Thermo Fisher Scientific) with TaqMan Gene Expression Assays for human LRP4 (Hs00391006_m1), LRP5 (Hs01124561_m1), LRP6 (Hs00233945_m1), SOX9 (Hs00165814_m1), ACAN (Hs00165814_m1), AGRN (Hs00233992_m1), RUNX2 (Hs00231692_m1), ADAMTS4 (Hs00192708_m1), MMP3 (Hs00968305_m1), MMP13 (Hs00233992_m1), and GAPDH (Hs02786624_g1) (Thermo Fisher Scientific).

Techniques: Recombinant, Gene Expression, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: LRP4 and Agrin Are Modulated by Cartilage Degeneration and Involved in β-Catenin Signaling in Human Articular Chondrocytes

doi: 10.3390/ijms26031007

Figure Lengend Snippet: Western blot analysis of the effect of rhAgrin treatment on human articular chondrocytes. ( A ) At the whole protein level, the expression of β-catenin, RUNX2, and SOX9 were increased after rhAgrin treatment. ( B ) Analysis of nuclear extraction protein also showed that the expression of both β-catenin and SOX9 were increased after rhAgrin treatment. In contrast, analysis of nuclear extraction protein showed no significant difference in the expression of NF-κB p65. * p < 0.05, ** p < 0.01, ns: not significant.

Article Snippet: RNA samples (500 ng) were reverse-transcribed with Primescript RT master mix (Takara Bio Inc., Shiga, Japan), and the resulting cDNAs were used for real-time PCR using the QuantStudioTM 1 real-time PCR system (Thermo Fisher Scientific) with TaqMan Gene Expression Assays for human LRP4 (Hs00391006_m1), LRP5 (Hs01124561_m1), LRP6 (Hs00233945_m1), SOX9 (Hs00165814_m1), ACAN (Hs00165814_m1), AGRN (Hs00233992_m1), RUNX2 (Hs00231692_m1), ADAMTS4 (Hs00192708_m1), MMP3 (Hs00968305_m1), MMP13 (Hs00233992_m1), and GAPDH (Hs02786624_g1) (Thermo Fisher Scientific).

Techniques: Western Blot, Expressing, Extraction

Journal: International Journal of Molecular Sciences

Article Title: LRP4 and Agrin Are Modulated by Cartilage Degeneration and Involved in β-Catenin Signaling in Human Articular Chondrocytes

doi: 10.3390/ijms26031007

Figure Lengend Snippet: Effect of LRP4 knockdown on gene expression in human articular chondrocytes. ( A ) LRP4 expression was suppressed effectively at 24 h, 48 h, and 72 h after si LRP4 transfection. LRP5 expression was significantly upregulated at both 48 h and 72 h. LRP6 expression was significantly upregulated at 48 h. ( B ) Agrin expression was significantly upregulated at 72 h. Expression of SOX9 and ACAN were significantly downregulated at 24 h, and then SOX9 expression was significantly upregulated, whereas ACAN was not upregulated at 72 h. Expression of RUNX2 was significantly upregulated at 24 h and then ADAMTS-4 was upregulated at 48 h. * p < 0.05, ** p < 0.01, *** p < 0.0001, ns: not significant.

Article Snippet: RNA samples (500 ng) were reverse-transcribed with Primescript RT master mix (Takara Bio Inc., Shiga, Japan), and the resulting cDNAs were used for real-time PCR using the QuantStudioTM 1 real-time PCR system (Thermo Fisher Scientific) with TaqMan Gene Expression Assays for human LRP4 (Hs00391006_m1), LRP5 (Hs01124561_m1), LRP6 (Hs00233945_m1), SOX9 (Hs00165814_m1), ACAN (Hs00165814_m1), AGRN (Hs00233992_m1), RUNX2 (Hs00231692_m1), ADAMTS4 (Hs00192708_m1), MMP3 (Hs00968305_m1), MMP13 (Hs00233992_m1), and GAPDH (Hs02786624_g1) (Thermo Fisher Scientific).

Techniques: Knockdown, Gene Expression, Expressing, Transfection

Journal: International Journal of Molecular Sciences

Article Title: LRP4 and Agrin Are Modulated by Cartilage Degeneration and Involved in β-Catenin Signaling in Human Articular Chondrocytes

doi: 10.3390/ijms26031007

Figure Lengend Snippet: The combined effect of LRP4 knockdown and rhAgrin treatment on gene expression in human articular chondrocytes. On the expression of LRP4/5/6, Agrin, SOX9, ACAN, RUNX2, ADAMTS-4, MMP-3, and MMP-13, there were no significant differences between the si LRP4 group and the si LRP4 + Agrin group. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: RNA samples (500 ng) were reverse-transcribed with Primescript RT master mix (Takara Bio Inc., Shiga, Japan), and the resulting cDNAs were used for real-time PCR using the QuantStudioTM 1 real-time PCR system (Thermo Fisher Scientific) with TaqMan Gene Expression Assays for human LRP4 (Hs00391006_m1), LRP5 (Hs01124561_m1), LRP6 (Hs00233945_m1), SOX9 (Hs00165814_m1), ACAN (Hs00165814_m1), AGRN (Hs00233992_m1), RUNX2 (Hs00231692_m1), ADAMTS4 (Hs00192708_m1), MMP3 (Hs00968305_m1), MMP13 (Hs00233992_m1), and GAPDH (Hs02786624_g1) (Thermo Fisher Scientific).

Techniques: Knockdown, Gene Expression, Expressing

Journal: Biomacromolecules

Article Title: Cationic Hyperbranched Polymers with Biocompatible Shells for siRNA Delivery

doi: 10.1021/acs.biomac.8b00902

Figure Lengend Snippet: Effects of HBP polymers and core–shell structures on osteoblast proliferation and differentiation at 2.5 μg/mL (a,c,e,g) and 10 μg/mL (b,d,f,h). (a,b) The rate of cell growth by polymer treatments after 5 days in culture. (c,d) Effect of polymers on both Runx2 and Osx gene expressions compared to untreated groups at 30 h. (e,f) The gene expression levels after polymer treatment at day seven. (g,h) The effect of treatment of polymers on osteoblast mineralization after 14 d in culture in alizarin red positive colonies. Data expressed as mean ± SD of three replicate determinations. Significant differences between PBS-treated cells vs polymer-treated cells. **p < 0.01, *p < 0.05.

Article Snippet: Taqman probes for Runx2 (Mm 00501578_m1), Osterix/Sp7( Osx ) (Mm 04209856_m1), alkaline phosphatase ( Alp ) (Mm00475831_m1), and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1) were used for quantification.

Techniques: Expressing

Journal: Biomacromolecules

Article Title: Cationic Hyperbranched Polymers with Biocompatible Shells for siRNA Delivery

doi: 10.1021/acs.biomac.8b00902

Figure Lengend Snippet: Cationic hyperbranched polymer-based RNAi against Runx2 in primary osteoblasts by delivery of Runx2 siRNAs at 20 pM doses. (a) Schematic of experimental time course. The siRNA polyplexes were delivered 24 h prior to delivery of recombinant human bone morphogenetic protein 2 (rhBMP-2, 100 ng/mL). Analysis of mRNA expression was conducted after 6 h of treatment by rhBMP-2. (b) Gene silencing effects on Runx2 mRNA expressions by polymer/scramble siRNAs polyplexes compared to rhBMP-2 treated cells without siRNA. (c) Gene silencing effects on Runx2 mRNA expressions by polymer/siRNAs polyplexes. Data expressed as mean ± SD of three replicate determinations. **p < 0.01, *p < 0.05, versus rhBMP-2 treated cells without siRNA.

Article Snippet: Taqman probes for Runx2 (Mm 00501578_m1), Osterix/Sp7( Osx ) (Mm 04209856_m1), alkaline phosphatase ( Alp ) (Mm00475831_m1), and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1) were used for quantification.

Techniques: Recombinant, Expressing

Journal: Biomacromolecules

Article Title: Cationic Hyperbranched Polymers with Biocompatible Shells for siRNA Delivery

doi: 10.1021/acs.biomac.8b00902

Figure Lengend Snippet: Alizarin red staining was performed after siRNA against Runx2 transfections. (a) Schematic of experimental time course. RNAi treatments against Runx2 were delivered 24 h prior to delivery of rhBMP-2. Cell culture media were refreshed in conjunction with RNAi treatments and rhBMP-2 every 2 days for the duration of the study. After 14 d in culture, mineral deposition in osteoblasts was assessed by Alizarin red staining. (b) The scramble siRNAs delivered by polyOEGMA–HBP and polyDMSO–HBP, a polymer, siRNA weight ratio at 20/1, had no significant effect on mineral deposition compared to rhBMP-2 treated cells without siRNA. RNAi treatments against Runx2 with polyOEGMA–HBP and polyDMSO–HBP, a polymer/siRNA weight ratio at 20/1, resulted in significant reductions in mineral deposition in osteoblasts compared to cells receiving rhBMP-2 treatments without siRNA. (c) The percentage of nodule area was measured using ImageJ. Data expressed as mean ± SD of three replicate determinations. **p < 0.01, versus rhBMP-2 treated cells without siRNA.

Article Snippet: Taqman probes for Runx2 (Mm 00501578_m1), Osterix/Sp7( Osx ) (Mm 04209856_m1), alkaline phosphatase ( Alp ) (Mm00475831_m1), and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1) were used for quantification.

Techniques: Staining, Transfection, Cell Culture

Journal: Frontiers in Endocrinology

Article Title: Investigating the role of ASCC1 in the causation of bone fragility

doi: 10.3389/fendo.2023.1137573

Figure Lengend Snippet: Gene expression in patient-derived skin fibroblasts. (A) Transcriptomics and (B) proteomics analyses showing significance (-log10(P), y-axis) versus fold change (log2(fold change), x-axis). Blue genes are significantly downregulated, and red genes are upregulated. (C) Quantitative PCR showing the mRNA expression ratio of ASCC1 , SERPINF1 , and RUNX2 in age-, gender-, and passage-matched patient and control fibroblasts. Target genes were normalized to HPRT1 . Columns represent the mean of at least three independent experiments, and error bars show the standard error of the mean (SEM). The p values were calculated using a two-tailed t test.

Article Snippet: Analysis of ASCC1 , RUNX2 , SERPINF1 , MMAA , CACFD1 , RABEPK , and HPRT1 gene expression levels was performed on the LightCycler ® 480 System (Roche Diagnostics, Vienna, Austria) using LightCycler ® FastStart DNA Master HybProbe (Roche Diagnostics, #12239272001) and the following TaqMan ® probes: Hs00418608_m1 (ASCC1), Hs01051148_g1 (RUNX2), Hs00369340_m1 (SERPINF1), Hs02800695_m1 (HPRT1).

Techniques: Gene Expression, Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Control, Two Tailed Test

Journal: Frontiers in Endocrinology

Article Title: Investigating the role of ASCC1 in the causation of bone fragility

doi: 10.3389/fendo.2023.1137573

Figure Lengend Snippet: Gene expression analyses in the osteogenic and adipogenic differentiation of ASCC1 knockdown hMSCs. (A) ASCC1 and the osteoblast specific markers ALPL , RUNX2 , and CTNNB1 are substantially downregulated in ASCC1 KD hMSC-derived osteoblasts. (B) ASCC1 is downregulated and the adipocyte-specific markers FASN , PPARG , and CEBPA are upregulated in ASCC1 KD hMSC-derived adipocytes. The relative mRNA expression ratio was calculated with the delta–delta Ct method using 36B4 as a housekeeping gene. Columns represent the mean of four biological replicates and show standard error of the mean (SEM). The p values were calculated using a two-tailed t test.

Article Snippet: Analysis of ASCC1 , RUNX2 , SERPINF1 , MMAA , CACFD1 , RABEPK , and HPRT1 gene expression levels was performed on the LightCycler ® 480 System (Roche Diagnostics, Vienna, Austria) using LightCycler ® FastStart DNA Master HybProbe (Roche Diagnostics, #12239272001) and the following TaqMan ® probes: Hs00418608_m1 (ASCC1), Hs01051148_g1 (RUNX2), Hs00369340_m1 (SERPINF1), Hs02800695_m1 (HPRT1).

Techniques: Gene Expression, Knockdown, Derivative Assay, Expressing, Two Tailed Test

Journal: Frontiers in Endocrinology

Article Title: Investigating the role of ASCC1 in the causation of bone fragility

doi: 10.3389/fendo.2023.1137573

Figure Lengend Snippet: Mutant ASCC1 directs MSC fate toward adipocytes by increasing adipogenic markers PPARG and FASN and away from osteoblasts by decreasing osteogenic markers RUNX2 , ALPL , and CTNNB1 . Mutant ASCC1 inhibits TGF-β/SMAD signaling when stimulated with recombinant human TGF-β1, through downregulating the expression ratio of pSMAD3/SMAD3 in patient fibroblasts. Figure created with BioRender.com.

Article Snippet: Analysis of ASCC1 , RUNX2 , SERPINF1 , MMAA , CACFD1 , RABEPK , and HPRT1 gene expression levels was performed on the LightCycler ® 480 System (Roche Diagnostics, Vienna, Austria) using LightCycler ® FastStart DNA Master HybProbe (Roche Diagnostics, #12239272001) and the following TaqMan ® probes: Hs00418608_m1 (ASCC1), Hs01051148_g1 (RUNX2), Hs00369340_m1 (SERPINF1), Hs02800695_m1 (HPRT1).

Techniques: Mutagenesis, Recombinant, Expressing

Journal: World Journal of Stem Cells

Article Title: Constitutive aryl hydrocarbon receptor facilitates the regenerative potential of mouse bone marrow mesenchymal stromal cells

doi: 10.4252/wjsc.v15.i8.807

Figure Lengend Snippet: Primer sequences for quantitative polymerase chain reaction (5’-3’)

Article Snippet: Anti-AhR (NB300-515, 1:200, Novus), anti-biomineralization associated [tissue-nonspecific alkaline phosphatase (ALPL)] (11187-1-AP, 1:1000, Proteintech), anti-runt-related transcription factor 2 (RUNX2) (PB0171, 1:2000, Boster), anti-phosphorylated STAT3 (Tyr705) (9145, 1:1000, CST), anti-STAT3 (9139, 1:1000, CST) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (BM3876, 1:5000, Boster) primary antibodies were used.

Techniques: Real-time Polymerase Chain Reaction

Journal: World Journal of Stem Cells

Article Title: Constitutive aryl hydrocarbon receptor facilitates the regenerative potential of mouse bone marrow mesenchymal stromal cells

doi: 10.4252/wjsc.v15.i8.807

Figure Lengend Snippet: The effect of aryl hydrocarbon receptor overexpression or knockdown on the osteogenic differentiation in mouse bone marrow mesenchymal stromal cells. A: Alkaline phosphatase (ALP) staining (upper) at 7 th day and Alizarin red staining staining (lower) at 14 th day of osteogenic induction; B: Relative mRNA expression of ALP, biomineralization associated [tissue-nonspecific alkaline phosphatase (ALPL)] and runt-related transcription factor 2 (RUNX2) of overexpression-negative control and overexpression-aryl hydrocarbon receptor (AhR) mouse bone marrow mesenchymal stromal cells (mBMSCs); C: Relative ALPL and RUNX2 mRNA expression of knockdown-negative control and knockdown-AhR mBMSCs at 7 th day of osteogenic induction; D: Representative images of western blot of ALPL and RUNX2 in mBMSCs of different groups at 7 th day of osteogenic induction. oe-NC: Overexpression-negative control; oe-AhR: Overexpression-aryl hydrocarbon receptor; sh-NC: Knockdown-negative control; sh-AhR: Knockdown-aryl hydrocarbon receptor; ALPL: Tissue-nonspecific alkaline phosphatase; RUNX2: Runt-related transcription factor 2; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Anti-AhR (NB300-515, 1:200, Novus), anti-biomineralization associated [tissue-nonspecific alkaline phosphatase (ALPL)] (11187-1-AP, 1:1000, Proteintech), anti-runt-related transcription factor 2 (RUNX2) (PB0171, 1:2000, Boster), anti-phosphorylated STAT3 (Tyr705) (9145, 1:1000, CST), anti-STAT3 (9139, 1:1000, CST) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (BM3876, 1:5000, Boster) primary antibodies were used.

Techniques: Over Expression, Staining, Expressing, Negative Control, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Molecular Interaction of Soluble Klotho with FGF23 in the Pathobiology of Aortic Valve Lesions Induced by Chronic Kidney Disease

doi: 10.7150/ijbs.92447

Figure Lengend Snippet: FGF23 induces AVIC fibrosis and calcification. (A) Human AVICs from normal valves were treated with recombinant FGF23 in different concentrations for 72 hours. Representative immunoblots (left) and densitometric data (right) show that FGF23 upregulates the expression of inflammatory (ICAM-1 and VCAM-1), fibrogenic (collagen I and collagen IV) and osteogenic (RUNX2 and ALP) mediators in AVICs. (B) Representative images of Picrosirius Red (PSR) staining (upper) and Alizarin Red S staining (lower), along with corresponding spectrophotometric data, show that prolonged treatment with recombinant FGF23 (10 days or 14 days) induces collagen and calcium deposition in AVICs. Images were taken using a 10x objective. Scale bar = 100 µm. Quantitative data are expressed as mean ± SEM, n = 4 cell isolates from distinct donor valves in each group. * P < 0.05 versus untreated control.

Article Snippet: Antibodies against runt-related transcription factor 2 (RUNX2, DIH7) and vascular cell adhesion molecule 1 (VCAM-1, EIE8X) were obtained from Cell Signaling Technology, Danvers, MA.

Techniques: Recombinant, Western Blot, Expressing, Staining, Control