Journal: Frontiers in Cell and Developmental Biology

Article Title: Upregulation of Parkin Accelerates Osteoblastic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells and Bone Regeneration by Enhancing Autophagy and β-Catenin Signaling

doi: 10.3389/fcell.2020.576104

Figure Lengend Snippet: Overexpression of Parkin enhances osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs). (A,B) Protein expression levels of Parkin were determined at 0, 6, 12, 24, 48, 72, and 96 h after induction of osteoblastic differentiation. (C) mRNA expression of Parkin was analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) at days 1 and 6 after infection. (D,E) Protein expression of Parkin was analyzed by western blot (WB) analysis at days 1 and 6 after infection. (F) Protein expression of Parkin was analyzed by immunofluorescence staining at day 6 after infection. (G) Proliferation was detected by Cell Counting Kit-8 in the Parkin-overexpressed and control group at days 1, 2, 3, 4, and 5. (H) Osteo-specific genes Alp, Runx2, and Col1 were analyzed by RT-qPCR at day 1 after osteogenic induction. (I) Osteo-specific genes Alp, Runx2, and Col1 were analyzed by RT-qPCR at day 3 after osteogenic induction. (J,K) Mineral deposits were determined by Alizarin Red staining at day 12 after osteogenic induction. (L) Alkaline phosphatase activity at day 3 of osteogenic differentiation of BMSCs. (M,N) Osteo-specific proteins Alp, Runx2, and Col1 were analyzed by WB at day 3 after osteogenic induction. Data are expressed as the mean ± standard deviation (SD) ( n = 3). NC, BMSCs transfected with negative control adenovirus vectors; OE, BMSCs transfected with Parkin-overexpressed adenovirus vectors; * P < 0.05 vs. BMSCs in the NC group.

Article Snippet: Parkin overexpression adenovirus vectors (OE; AV-CMV > Mouse Parkin/T2A/PolyA) and negative control adenovirus vectors (NC; AV-CMV > PolyA) were purchased from Cyagen Biosciences Inc., and the viral titers were l × 10 10 PFU/mL. mRNA and protein levels of Parkin were analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot (WB) analysis, respectively.

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Western Blot, Immunofluorescence, Staining, Cell Counting, Control, Activity Assay, Standard Deviation, Transfection, Negative Control

Journal: Nature communications

Article Title: Transcriptional and epigenetic rewiring by the NUP98::KDM5A fusion oncoprotein directly activates CDK12.

doi: 10.1038/s41467-025-59930-9

Figure Lengend Snippet: Fig. 7 | CDK12-controlled DNA repair is a vulnerability in NUP98::KDM5A-driven leukemia. A Cell viability assay of primary human NUP98-rearranged AML cells, healthy donor BM MNC and CD34+ progenitors treated with indicated con- centrations of THZ531 for 3 days (n = 3 technical replicates). B GI50 values from cell viability assays of murine AML cells treated with THZ531 for 3 days. C RT-qPCR analysis of dTAG-NUP98::KDM5A cells treated with THZ531 (2 µM, 24 h) showing log2FC values (n = 3 technical replicates). D Gene set enrichment analysis of RNA- seq data from a doxycycline-controlled NUP98::KDM5A cell line (Tet-Off) after 5 days of doxycycline-induced NUP98::KDM5A downregulation compared to DMSO. E Time series plot of Tet-Off RNA-seq data showing gene expression levels of DNA double strand break repair genes at day 3 and day 5 after NUP98::KDM5A downregulation normalized to NUP98::KDM5A-expressing cells, highlighting selected genes (left), with a density plot of normalized expression of all genes

Article Snippet: For the generation of the CDK12 wild type overexpression construct pCR8/GW_TOPO_Cdk12_Nterm_FlagHa (Addgene plasmid #127177) expressing the mouse Cdk12 transgene (NM_001109626.1 isoform) was cloned into pLX303-Gw (Addgene plasmid #25897) using Gateway LR Clonase II EnzymeMix (Invitrogen, USA) according the manufacturer’s protocol.

Techniques: Viability Assay, Quantitative RT-PCR, RNA Sequencing, Gene Expression, Expressing

Journal: Physiological Genomics

Article Title: FAR2 is associated with kidney disease in mice and humans

doi: 10.1152/physiolgenomics.00118.2017

Figure Lengend Snippet: The 9 bp sequence in the 5′-untranslated region (UTR) of Far2 binds NKX3-2. A: electrophoretic mobility shift assay (EMSA) performed with nuclear extract from mouse mesangial cells incubated with biotin-labeled oligonucleotide probes corresponding to the Far2 5′-UTR sequences with (ins-probe) and without (del-probe) the 9 bp sequence (lanes 2 and 4, respectively). B: EMSA performed with purified NKX3-2 protein (lane 2). A supershift assay was performed with an antibody against NKX3-2 (lane 3).

Article Snippet: A stable Far2 -overexpressing mesangial cell line was established by transfecting MES13 cells with the pCMV6 plasmid (OriGene) containing the mouse Far2 cDNA clone {"type":"entrez-nucleotide","attrs":{"text":"NM_178797","term_id":"1331036860","term_text":"NM_178797"}} NM_178797 with a Myc/DDK-tag (MR225671, OriGene) using 350 ng FuGENE and 100 ng of plasmid DNA.

Techniques: Sequencing, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Purification

Journal: Physiological Genomics

Article Title: FAR2 is associated with kidney disease in mice and humans

doi: 10.1152/physiolgenomics.00118.2017

Figure Lengend Snippet: Far2 is an enzyme involved in de novo platelet-activating factor (PAF) production. A: PAF concentration measured in both culture media and cell lysates between Far2-overexpressing (gray, n = 3) and wild-type (black, n = 3) mesangial cells. Labeled fatty alcohol (hexadecanol) (B) and PAF (C) were detected through mass spectrometry after a mixture of 13C- and 12C-palmitate was added to Far2-overexpressing cells. D: proposed pathway highlighting the role of FAR2 in de novo synthesis of PAF.

Article Snippet: A stable Far2 -overexpressing mesangial cell line was established by transfecting MES13 cells with the pCMV6 plasmid (OriGene) containing the mouse Far2 cDNA clone {"type":"entrez-nucleotide","attrs":{"text":"NM_178797","term_id":"1331036860","term_text":"NM_178797"}} NM_178797 with a Myc/DDK-tag (MR225671, OriGene) using 350 ng FuGENE and 100 ng of plasmid DNA.

Techniques: Concentration Assay, Labeling, Mass Spectrometry

Journal: Physiological Genomics

Article Title: FAR2 is associated with kidney disease in mice and humans

doi: 10.1152/physiolgenomics.00118.2017

Figure Lengend Snippet: Generation and characterization of B6N(Cg)-Far2tm2a(KOMP)Wtsi/2J. A: a simplified scheme showing the wild-type Far2 allele and knock-in allele Far2tm2a(KOMP)Wtsi. The lacZ and neomycin expression cassette was inserted between exon 4 and exon 5 and is flanked by FRT (Flippase Recognition Target) sites. Exons 5–8 are flanked by loxP sites. B: quantitative PCR for Far2 on brain and eyelid of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) knockout mice (n = 3/genotype) levels were calculated as relative fold change (RFC) compared with wild type. *P < 0.01 compared with +/+; #P < 0.01 compared with +/−. C: quantification of mesangial matrix expansion (MME) in knockout mice vs. wild-type mice at 12 and 18 mo of age relative to 6 mo measurements. MME is reported as percentage of the glomerular area occupied by matrix for 50 glomeruli per mouse and 10 mice per group. P = 1.27 × 10−6. D: glomerular filtration rate (GFR) data in knockout mice (gray) vs. wild-type mice (black) at 6, 12, and 18 mo of age. n = 10 per group. *P = 0.0118.

Article Snippet: A stable Far2 -overexpressing mesangial cell line was established by transfecting MES13 cells with the pCMV6 plasmid (OriGene) containing the mouse Far2 cDNA clone {"type":"entrez-nucleotide","attrs":{"text":"NM_178797","term_id":"1331036860","term_text":"NM_178797"}} NM_178797 with a Myc/DDK-tag (MR225671, OriGene) using 350 ng FuGENE and 100 ng of plasmid DNA.

Techniques: Knock-In, Expressing, Real-time Polymerase Chain Reaction, Knock-Out, Filtration

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) Coomassie blue–stained gel showing the Wnt5a protein purification scheme. (B) The 24-h Wnt3a protein treatment activates the STF luciferase reporter in a dose-responsive manner. (C) Concomitant Wnt5a protein treatment inhibits Wnt3a-induced STF reporter activation. (D) Time-course analysis of Wnt5a-mediated inhibition of Wnt3a. At approximately 40 h post-transfection, cells were treated with Wnt3a protein for 0, 2, 4, 6, or 8 h alone or in conjunction with Wnt5a at t = 0, t = 1 h, or t = 3 h. (Filled diamonds indicate Wnt3a treatment alone; open diamonds indicate Wnt3a treatment at t = 0 plus Wnt5a protein treatment at t = 0; filled triangles indicate Wnt3a treatment at t = 0 plus Wnt5a protein treatment at t = 1; open circles indicate Wnt3a treatment at t = 0 plus Wnt5a protein treatment at t = 3.) (E) Wnt5a pretreatment does not enhance its inhibitory activity. At 24 h post-transfection, cells were pretreated with vehicle or Wnt5a protein (200 ng/ml) for 8 h. Cells were then treated with vehicle, Wnt3a alone, or Wnt3a concomitantly with Wnt5a for an additional 24 h, and luciferase assay was performed. (Filled squares indicate cells that were pretreated with vehicle for 8 h prior to Wnt addition; open squares indicate cells pretreated with Wnt5a protein for 8 h prior to Wnt addition.) Unless stated in text, luciferase activity was measured 48 h after transfection, approximately 24 h after Wnt treatment, and results are shown as the average fold change ± SD in luciferase activity over transfected, untreated negative controls.

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Staining, Protein Purification, Luciferase, Activation Assay, Inhibition, Transfection, Activity Assay

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) Wnt5a protein treatment does not affect cytosolic β-catenin protein levels. The 293 cells were treated for 3 h with the indicated concentrations of Wnt proteins and then hypotonically lysed for Western blot analysis of cytosolic fractions. (B) Wnt5a protein treatment does not alter β-catenin cellular localization. Cells grown on coverslips were treated with the indicated concentrations of Wnt proteins for 6 h and then fixed and stained as described in the text. Membrane localized β-catenin is indicated by arrowheads; membrane and cytosolic staining is indicated by arrows.

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Western Blot, Staining, Membrane

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) Wnt5a-mediated inhibition is PTX insensitive. At 24 h post-transfection, 293 cells were pretreated with vehicle or 100 ng/ml PTX for 24 h. Cells were then treated with indicated Wnt proteins concomitantly with vehicle or PTX for an additional 24 h, and luciferase assay was performed. (B) PTX is active in 293 cells as PTX pretreatment of cells inhibits LPA-induced Erk1,2 activation. The 293 cells were pretreated with 100 ng/ml PTX for 24 h and then treated with 10 μM LPA or vehicle for 10 min. Western blot analysis was then performed on total cell lysates using anti–phospho-Erk1,2 antibody. Membrane was stripped and reprobed for total Erk1,2 as a loading control. (C) Wnt5a protein does not directly stimulate intracellular Ca 2+ flux. The 293 and 293Fz4 cells were loaded with Fura-2-dextran and then monitored for changes in intracellular Ca 2+ concentration as determined by the change in 340/380 excitation wavelength ratio following Wnt5a (500 ng/ml) and subsequent ionomycin treatment. Data represent the average 340/380 excitation wavelength ratio of three or more independent cells within the same field ± SD. Wnt5a has little effect on the ratio over the 15-min time course, whereas ionomycin treatment rapidly and robustly induces Ca 2+ flux in these cells. Prolonged treatment with Wnt5a (up to 1 h) did not result in changes in intracellular Ca 2+ levels (unpublished data).

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Inhibition, Transfection, Luciferase, Activation Assay, Western Blot, Membrane, Control, Concentration Assay

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) Wnt5a treatment leads to β-catenin stabilization specifically in cells expressing mFz4. The 293 and 293Fz4 cells were treated with Wnt proteins and then assayed for cytosolic β-catenin protein accumulation via Western blot analysis. (B) Wnt5a treatment activates the STF reporter when LRP5 is coexpressed. At 24 h post-transfection with STF reporter and LRP5 or empty vector, 293Fz4 cells were treated with increasing concentrations of Wnt5a protein for an additional 24 h, and luciferase assay was performed as described in . (C) Wnt5a does not inhibit Wnt3a-mediated reporter activation in 293Fz4 cells. The 293 and 293Fz4 cells were treated with Wnts as indicated 24 h post-transfection with reporters, and luciferase assay was performed. (D) Loss of Wnt5a-mediated reporter inhibition is specific to mFz4 overexpression. The 293, 293Fz4, and 293Fz8 cells were treated with the indicated Wnts for 24 h, 24 h post-transfection, and then luciferase assay was performed. Wnt5a maintains inhibitory activity when mFz8, but not mFz4, is overexpressed.

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activation Assay, Inhibition, Over Expression, Activity Assay

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) Wnt5a synergizes with mRor2 to inhibit Wnt3a-mediated reporter activation. The 293 cells stably expressing exogenous mRor2 (293Ror2) were treated with Wnts as indicated, 24 h post-transfection with reporters, and luciferase assay was performed as outlined in . (B) Wnt5a binds directly to the Fz4 and mRor2 CRD domains. Wnt5a protein specifically binds to the purified mFz4 and mRor2 CRDs; only background binding to the Smoothened CRD negative control is observed. While it may appear that Wnt5a binds to a similar extent to the Ror2 CRDs and Smo CRD, the input levels of the Smo CRD-IgG exceeded the Ror2 CRD-IgG levels, accounting for the increased background binding Smo CRD-IgG. (C) The mRor2 CRD domain is required for mediating Wnt5a inhibitory activity. The 293 cells were transiently transfected with reporters along with empty vector, mRor2, or mRor2ΔCRD constructs, treated with Wnts as described in text 24 h post-transfection for an additional 24 h, and then luciferase assay was performed as outlined in .

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Activation Assay, Stable Transfection, Expressing, Transfection, Luciferase, Purification, Binding Assay, Negative Control, Activity Assay, Plasmid Preparation, Construct

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: (A) In 293 cells, mRor2 overexpression inhibits Wnt5a-mediated activation of the STF reporter. The 293 and 293Fz4 cells were transiently transfected with the reporters and LRP5 with or without mRor2 and then treated with Wnt proteins for 24 h, 24 h post-transfection. Overexpression of wild-type mRor2 inhibits Wnt5a-mediated activation of the STF reporter as well as allowing Wnt5a to inhibit Wnt3a-mediated STF reporter activation in 293Fz4 cells. (B) mRor2 overexpression inhibits Wnt5a-mediated activation of the STF reporter and allows Wnt5a to block Wnt3a-induced STF reporter activation in mouse L cells. L and LFz4 cells were transiently transfected with the reporters and LRP5 with or without mRor2 and then treated with Wnt proteins for 24 h, 24 h post-transfection. (C) Quantitative real-time PCR analysis of Ror2 expression in 293 and L cells. Reverse transcription followed by 45 cycles of quantitative real-time amplification of 293 cell RNA reveals a robust Ror2 product that is over 700 times more abundant than that produced from RNA derived from mouse L cells. Hprt quantification serves as an internal normalization control and RNA derived from 293Ror2 and LRor2 cells serves as a positive control for the reaction. The products from the PCR analysis are displayed below the charted quantitative data. (D) The cytoplasmic domain of mRor2 is required for its inhibitory function. Expression of a membrane-tethered variant of mRor2 possessing the extracellular domain of mRor2 fused to a GPI linkage (mRor2-GPI) reduces Wnt5a's ability to inhibit Wnt3a induced canonical Wnt signaling as well as inhibits wild type mRor2′s ability to enhance Wnt5a-mediated inhibition. (E) Expression of a membrane-tethered variant of mRor2 (mRor2-TM) that contains the extracellular and transmembrane domains of mRor2, but lacks the cytoplasmic domain, does not enhance Wnt5a mediated inhibition of canonical Wnt signaling. Expression of mRor2-TM reduces Wnt5a's ability to inhibit Wnt3a-induced canonical Wnt signaling when wild-type mRor2 is overexpressed. Western blot analysis shows that wild-type mRor2 levels do not change when the truncated mRor2 variants are coexpressed.

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: Over Expression, Activation Assay, Transfection, Blocking Assay, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription, Amplification, Produced, Derivative Assay, Control, Positive Control, Membrane, Variant Assay, Inhibition, Western Blot

Journal: PLoS Biology

Article Title: Purified Wnt5a Protein Activates or Inhibits β-Catenin–TCF Signaling Depending on Receptor Context

doi: 10.1371/journal.pbio.0040115

Figure Lengend Snippet: In the presence of Fz4 and LRP5, Wnt5a can activate β-catenin/TCF signaling. However, when Ror2 is expressed, Wnt5a inhibits canonical Wnt signaling downstream of β-catenin stabilization, at the level of TCF-mediated transcription.

Article Snippet: Wnt5a protein was purified from 6 L of media conditioned by mouse L cells stably overexpressing mouse Wnt5a (CRL-2814; American Type Culture Collection [ ATCC], Manassas, Virginia, United States) created in the laboratory as previously described [ ] with the addition of a fourth purification step.

Techniques: