Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: iMDP-3 cells were cultured in DM (DMEM supplemented with 10% FBS, antibiotics, 50 μg/mL ascorbic acid, 10 mM sodium β-glycerophosphate and 100 nM dexamethasone) for 0, 1, 3, 5, 7, 11 and 14 days. ( a ) Alizarin Red S (ARS) and ( b ) Alkaline phosphatase (ALP) staining of iMDP-3 cells on days 7 and 14 after differentiation induction by low and high magnifications. ( c ) Cell numbers on days 7 and 14 after differentiation induction. ( d ) Expression of Klf5 protein was detected by Western blot analysis using antibodies specific to Klf5 and β-actin. Protein expression of Klf5 was upregulated during odontoblastic differentiation of iMDP-3 cells. ( e ) Protein expression of Klf5 and β-actin was quantitated using image J software. Expression of Klf5 was normalized to β-actin expression. Expression level of Klf5 proteins on day 0 acts as one-fold increase. ( f ) Klf5, ( g ) Dspp and ( h ) Dmp1 mRNA expression was followed by qRT-PCR relative to Cyclo A. mRNA expression of Klf5, Dspp and Dmp1 is upregulated during odontoblastic differentiation of iMDP-3 cells. Expression level of Klf5, Dsp and Dmp1 at different time periods was divided by the Klf5, Dsp and Dmp1 expressions on day 0. * P < 0.05; ** P < 0.01.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Cell Culture, Staining, Expressing, Western Blot, Software, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a–e ) in iMDP-3 cells, ( f–j ) in MD10-F2 cells, the coexpression of Klf5 ( green , b and g) with Dsp ( red , c and h) was observed in these cells. ( k–o ) in molar at PN2, ( p–t ) in molar at PN6, Klf5 ( green , l and q) expression was observed in ameloblasts, SI, HERS, odontoblasts and dentin. Expression of Dsp ( red , m and r) was also overlapped with that of Klf5. a,f,k and p show bright images. ( d,i,n and s ) Cells were stained with Hoechst for nuclei. e , j , o and t are merged. Similar to the above description, (a′–t′) in incisors at PN3, coexpression of Klf5 ( green , e′–h′) and Dsp ( red , i′–l′) was visible in odontoblasts. a′–d′ show bright images. (m′–p′) Cell nuclei were stained with Hoechst. (q′–t′) The images were merged. Bars, 20 μm ( a–t ), 10 μm ( k–t , a′–t′).

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a–e ) in iMDP-3 cells, ( f–j ) in MD10-F2 cells, the coexpression of Klf5 ( green , b and g) with Dmp1 ( red , c and h) was observed in these cells. ( k–o ) in molar at PN2, ( p–t ) in molar at PN6, Klf5 ( green, l and q) expression was observed in ameloblasts, odontoblasts and dentin. Expression of Dmp1 ( red, m and r) was also overlapped with that of Klf5. a,f,k and p show bright images. ( e, j, o and t ) Images were merged. ( d , i , n and s ) Cells were stained with Hoechst for nuclei. Similar to the above description, (a′)–(t′) in incisors at PN3, coexpression of Klf5 ( green , e′–h′) and Dmp1 ( red , i′-l′) was visible in odontoblasts. a′–d′ show bright images. (m′–p′) Cell nuclei were stained with Hoechst. (q′–t′) The images were merged. Bars 20 μm ( a–t ), 10 μm ( a–t , a′–t′).

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a ) Klf5 gene plasmid tagged with green fluorescent protein (GFP) was overexpressed by transient transfection in iMDP-3 cells. After 48 h transfection, more than 50% of cells were positive to GFP. Klf5 overexpression in iMDP-3 cells was examined using RT-PCR ( b ) and Western blot analyses ( e and f ). The Dspp mRNA ( c ) and Dsp protein ( e and g ) levels were all increased on 48 h in Klf5 overexpression cells compared with control group. The mRNA ( d ) and protein ( e and h ) levels of Dmp1 were also increased on 48 h in Klf5 overexpression cells compared with control group. ( i and j ) Expression of ALP mRNA and protein was significantly increased after Klf5 overexpression in iMDP-3 cells. ARS ( k ) assay were used to monitor the progress of mineralization in Klf5 overexpression cells as compared to control groups. * P < 0.05; ** P < 0.01.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Plasmid Preparation, Transfection, Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: iMDP-3 cells were transfected with different Dspp and Dmp1 promoter constructs with either pcDNA3-Klf5 or pcDNA3 plasmid as control. Reporter activities were measured by a dual-luciferase assay in the presence or absence of pcDNA3-Klf5 co-transfection. ( a ) Activation of Dspp promoters containing pGL3-5.7 kb, pGL3-2.6 kb, pGL3-1.5 kb, pGL3-1,318 bp and pGL3-591bp was increased 5.8-, 3.0-, 1.8-, 1.9- and 1.1-folds, respectively. ( b ) Effect of different Klf5 concentrations on Dspp promoter activity containing pGL3-5.7 kb. ( c ) The transcription activities appeared to increase in a dosage-dependent manner for Dspp promoter-5.7 kb with Klf5 transfection. ( d ) Activation of Dmp1 promoters containing pGL3-2.6 kb, pGL3-1,656 bp, pGL3-1,187 bp, pGL3-656bp and pGL3-213bp were increased 5.4-, 4.0-, 3.2-, 2.2- and 2.0-folds, respectively. ( e ) Effect of different Klf5 concentrations on Dmp1 promoter activity containing pGL3-2.6 kb. ( f ) The transcription activities appeared to increase in a dosage-dependent manner for Dmp1 promoter-2.6 kb with Klf5 transfection. Luciferase (Luc) activity was normalized to the control group. * P < 0.05; ** P < 0.01.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Transfection, Construct, Plasmid Preparation, Luciferase, Cotransfection, Activation Assay, Activity Assay

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: Sequences of oligonucleotides of potential Klf5-binding sites.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a ) Scheme represents of 9 potential Klf5 binding sites in the mouse Dspp intron 1. ( b ) Conserved non-coding sequence islands are found by comparing the proximal promoters and the first introns of the mouse, rat and human Dspp genes. Many of the conserved non-coding sequences are in these regions. The scale on the y-axis goes to from 50 to 100% homology. The pink regions are peaks of conserved nucleotide sequences with a minimum of 70 homologies. ( c ) Nine 32 P-labeled double-stranded Dspp probes were generated for electrophoresis mobility shift assays (EMSAs). These included Dspp-site 1 to Dspp-site 9. EMSAs were carried out using nuclear extracts obtained from iMDP-3 cells. ( c , lane 1 ) free Dspp-site 1 probes only; (c, lane 2 ) binding of nuclear extracts to Dspp-site 1 probe; (c, lane 3 ) the labeled probe with nuclear extracts and 100× cold Dspp-site 1 oligo; (c, lane 4 ) the labeled probe with nuclear extracts and 100× cold Klf consensus sequence oligo; (c, lane 5 ) the labeled probe with nuclear extracts and 1 μg Klf5 antibody; (c, lane 6 ) the labeled probe with nuclear extracts and 2 μg Klf5 antibody; (c, lane 7 ) free Klf consensus sequence probe only; (c, lane 8 ) the binding of nuclear extracts to Klf probe; (c, lane 9 ) Klf probe with nuclear extracts and 100× cold Klf consensus sequence oligo; (c, lane 10 ) Klf probe with nuclear extracts and 100× cold Dspp-site 1 oligo; (c, lane 11 ) Klf probe with nuclear extracts and 1 μg Klf5 antibody; (c, lane 12 ) Klf probe with nuclear extracts and 2 μg Klf5 antibody. (d, lane 1–12 ) Dspp-site 2 and Klf probes; (e, lane 1–12 ) Dspp-site 3 and Klf probes; (f, lane 1–12 ) Dspp-site 4 and Klf probes; (g, lane 1–12 ) Dspp-site 5 and Klf probes; (h, lane 1–12 ) Dspp-site 6 and Klf probes; (i, lane 1–12 ) Dspp-site 7 and Klf probes; (j, lane 1–12 ) Dspp-site 8 and Klf probes; (k, lane 1–12 ) Dspp-site 9 and Klf probes.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Binding Assay, Sequencing, Labeling, Generated, Electrophoresis, Mobility Shift

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a ) Scheme represents of 2 potential Klf5 binding sites in human Dmp1 promoter. ( b ) Conserved non-coding sequences of Dmp1 gene promoters in three mammalian species, mouse, rat and human are retrieved using Vista plots for comparative genomic analysis of gene promoters. The pink regions represent peaks of conserved nucleotide sequences with a minimum of 70 homologies. Many of the conserved non-coding sequences are in these regions. The scale on the y-axis goes to from 50 to 100% homology. Two 32 P-labeled double-stranded Dmp1 probes were generated for EMSAs. These included Dmp1-site 1 and Dmp1-site 2. EMSAs were carried out using nuclear extracts obtained from iMDP-3 cells. (c, lane 1 ) free Dmp1-site 1 probes only; (c, lane 2 ) the binding of nuclear extracts to Dmp1-site 1 probe; (c, lane 3 ) the probe with nuclear extracts and 100× cold Dmp1-site 1 oligo; (c, lane 4 ) the probe with nuclear extracts and 100× cold Klf consensus sequence oligo; (c, lane 5 ) the probe with nuclear extracts and 1 μg Klf5 antibody; (c, lane 6 ) the probe with nuclear extracts and 2 μg Klf5 antibody; (c, lane 7 ) free Klf consensus sequence probe only; (c, lane 8 ) the binding of nuclear extracts to the Klf probe; (c, lane 9 ) the probe with nuclear extracts and 100× cold Klf consensus sequence oligo; (c, lane 10 ) the probe with nuclear extracts and 100× cold Dmp1-site 1 oligo; (c, lane 11 ) the probe with nuclear extracts and 1 μg Klf5 antibody; (c, lane 12 ) the probe with nuclear extracts and 2 μg Klf5 antibody. (d, lane 1–12 ) Dmp1-site 2 and Klf probes.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Binding Assay, Labeling, Generated, Sequencing

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a ) The diagram shows that six pairs of primers were designed to amplify the Klf5 binding sites in the first intron 1 of mouse Dspp gene from Dspp-site 1 to Dspp-site 9 in vivo for ChIP assay. The position number was stated as Dspp-primer 1: 50 bp to 257 bp; Dspp-primer 2: 1,219 bp to 1,422 bp; Dspp-primer 3: 1,885 bp to 2,074 bp; Dspp-primer 4: 2,177 bp to 2,468 bp; Dspp-primer 5: 2,574 bp to 2,861 bp; Dspp-primer 6: 3,025 bp to 3,265 bp. ( b–g ) ChIP assay showed that endogenous Klf 5 interacted with its motifs in the Dspp regulatory regions while Klf 5 overexpression significantly increased binding to its motifs in Dspp regulatory regions from Dspp site 1 to Dspp site 9 in vivo . ( h ). Two pairs of primers were used for the Klf5 binding sites in human Dmp1 promoter from Dmp1-site 1 to Dmp1-site 2 for ChIP assay. ChIP assay was performed with chromatin from iMDP-3 cells with transfection of Dspp-5.7 kb reporter construct ( b–g ) and Dmp1–2.6 kb promoter construct (i and j) with either pcDNA3-Klf5 or pcDNA3 plasmid. The results revealed that Klf5 binds to the Dspp regulatory regions encompassing the CACCC/GGGTG boxes between +73 to +2.8 kb and the Dmp1 promoter between −2.6 kb to −1,656 bp in vivo and exhibited an increasing transcription in Klf5-stimulated groups in iMDP-3 cells.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Binding Assay, In Vivo, Over Expression, Transfection, Construct, Plasmid Preparation

Journal: Science Advances

Article Title: A programmable genetic platform for engineering noninvasive biosensors

doi: 10.1126/sciadv.aec1211

Figure Lengend Snippet: ( A ) Schematic representation of the proposed proteolytic activation mechanism. ( B ) Workflow for constructing and evaluating protease-activatable aquaporins. ( C ) Diffusivities of CHO cells expressing hAqp1 fused with TEVP-cleavable DD(s). ( D ) Representative Western blot of membrane extracts from CHO cells engineered to express hAqp1 fused at its C terminus to the TEVP-cleavable FKBP12-DD. Membrane lysates from cells treated with shield-1 are included as a positive control. ( E ) Representative confocal images of CHO cells expressing hAqp1 fused at its C terminus to the TEVP-cleavable FKBP12-DD. Scale bars, 10 μm. ( F ) Diffusivities of CHO cells expressing hAqp1 tagged at the C terminus with FKBP12-DD with Gly-Ser spacers flanking the TEVP cleavage site (cs). ( G ) Diffusivities of CHO cells expressing hAqp1 with multiple TEVP cleavage sites inserted before FKBP12-DD. The optimized DD-MAPPER circuit (three cut sites) exhibits the largest fold change. ( H ) Modularity of DD-MAPPER facilitates adaptation for multiple proteases. ( I ) Representative diffusion maps of CHO cells expressing DD-MAPPER. The minimum and maximum diffusivity values (μm 2 /ms) are specified at the edges of the color bars. ( J ) Diffusivities of CHO cells expressing HCVP- and TVMVP-activatable DD-MAPPER circuits. ( K ) Polycistronic constructs for implementing DD-MAPPER in different cell lines. ( L ) Representative diffusion maps of various cell types expressing DD-MAPPER. ( M ) Diffusivities of various cell types expressing DD-MAPPER. ( N ) Schematic illustrating the putative degradation pathways of DD-MAPPER and their inhibition by small-molecule modulators. ( O ) Representative Western blot of lysates prepared from cells expressing DD-MAPPER in the presence of chloroquine or MG132. Error bars represent SD from n ≥ 3 measurements. *** P < 0.001; and n.s., P ≥ 0.05 (two-sided t test). Panels (B) and (N) created (in part) using BioRender. A. Mukherjee (2025); https://BioRender.com/xgnoc5l . IRES, internal ribosomal entry site.

Article Snippet: Plasmid containing the FlipGFP TEVP sensor was obtained from Addgene (#124429).

Techniques: Activation Assay, Expressing, Western Blot, Membrane, Positive Control, Diffusion-based Assay, Construct, Inhibition

Journal: Science Advances

Article Title: A programmable genetic platform for engineering noninvasive biosensors

doi: 10.1126/sciadv.aec1211

Figure Lengend Snippet: ( A ) Schematic representation illustrating the integration of DD-MAPPER with destabilized proteases for the detection of trimethoprim (TMP). ( B ) Quantification of diffusivities in CHO cells engineered to express the TMP-sensing DD-MAPPER circuit as depicted in (A), acquired in the presence and absence of TMP treatment for 24 hours. ( C ) Dose-response analysis of the TMP-sensing DD-MAPPER expressed in CHO cells incubated with varying concentrations of TMP for 24 hours. ( D ) Schematic depiction of the application of DD-MAPPER for the detection of small-molecule protease inhibitors. ( E ) Diffusivities of CHO cells engineered to express HCVP-modulated DD-MAPPER, with and without treatment using antiviral HCVP inhibitors: boceprevir (BV) or telaprevir (TV) for 24 hours. ( F ) Dose-response analysis of the HCVP-based DD-MAPPER in CHO cells exposed to varying concentrations of boceprevir for 24 hours. ( G ) Schematic illustrating the detection of a protease-based two-input biological AND gate using DD-MAPPER. The corresponding genetic constructs for introducing the two-input AND gates and DD-MAPPER are shown alongside. ( H ) Diffusivities of CHO cells engineered to express the protease-based AND gate and corresponding DD-MAPPER detector. ( I ) Integration of DD-MAPPER with split protease technology to image protein-protein interactions. The corresponding genetic constructs are shown alongside. ( J ) Diffusivities of CHO cells expressing the split TEVP-based DD-MAPPER for detecting protein-protein interaction. Control measurements are obtained in cells expressing split TEVP fragments without the fused coiled-coil peptides. ( K ) Schematic illustrating the engineering of split TEVP-based DD-MAPPER for calcium imaging and the corresponding genetic constructs. ( L ) Diffusivities of CHO cells expressing the calcium-sensing DD-MAPPER circuit. Control measurements are obtained in wild-type cells lacking the sensor. Error bars represent SD from n ≥ 3 measurements. ** P < 0.01; *** P < 0.001; and n.s., P ≥ 0.05 (two-sided t test). HA, hemagglutinin; WT, wild type; EBFP, enhanced blue fluorescent protein.

Article Snippet: Plasmid containing the FlipGFP TEVP sensor was obtained from Addgene (#124429).

Techniques: Incubation, Construct, Protein-Protein interactions, Expressing, Control, Imaging

Journal: Science Advances

Article Title: A programmable genetic platform for engineering noninvasive biosensors

doi: 10.1126/sciadv.aec1211

Figure Lengend Snippet: ( A ) Schematic representation of the proposed proteolytic activation mechanism. Genetic constructs encoding ER-MAPPER are depicted alongside. ( B ) Representative confocal images of CHO cells transduced with ER-MAPPER. ( C ) Representative diffusion maps of various cell types expressing ER-MAPPER. The minimum and maximum diffusivity values (μm 2 /ms) are specified at the edges of the color bars. ( D ) Diffusivities of various cell types expressing ER-MAPPER. ( E ) Representative diffusion maps CHO cell pellets expressing ER-MAPPER with and without induction of the corresponding protease. ( F ) Diffusivities of CHO cells expressing ER-MAPPER with and without induction of the corresponding protease. ( G ) Diffusivities of CHO cells engineered to express HCVP-modulated ER-MAPPER, with and without treatment using antiviral HCVP inhibitors: boceprevir (BV) or telaprevir (TV) for 24 hours. CCs, coiled-coils. ( H ) Diffusivities of CHO cells expressing the split TEVP-based ER-MAPPER sensor for detecting protein-protein interaction. Control measurements are obtained in cells expressing split TEVP fragments without the fused p3/p4 coiled-coils. ( I ) Schematic illustrating the adaptation of split TEVP-based ER-MAPPER to detect rapamycin-induced dimerization of FKBP and Frb. ( J ) Diffusivities of CHO cells expressing split TEVP-based sensor depicted in ( J ). Control measurements are obtained in cells expressing split TEVP without the fused FKBP and Frb domains. ( K ) Time-dependent activation of ER-MAPPER in CHO cells engineered to express FKBP and Frb-fused split TEVP fragments upon rapamycin induction. Control measurements are obtained in cells expressing split TEVP without the fused FKBP or Frb. Error bars represent SD from n ≥ 3 measurements. Statistical significance is denoted by ** P < 0.01; *** P < 0.001; n.s., P ≥ 0.05 (two-sided t test). CID, chemically induced dimerization.

Article Snippet: Plasmid containing the FlipGFP TEVP sensor was obtained from Addgene (#124429).

Techniques: Activation Assay, Construct, Transduction, Diffusion-based Assay, Expressing, Control