Journal: eLife

Article Title: Beta-catenin signaling regulates barrier-specific gene expression in circumventricular organ and ocular vasculatures

doi: 10.7554/eLife.43257

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rabbit anti-GLUT1 (Thermo Fisher Scientific RB-9052-P1); rat anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); mouse anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); rabbit anti-GFP, Alexa Fluor 488 conjugate (Thermo Fisher Scientific A21311); rabbit anti-6xMyc (JH6204), rabbit mAb anti-LEF1 (Cell Signaling Technologies C12A5), rabbit anti-ZO-1 (Invitrogen 40–2200), rabbit anti-Occludin (Invitrogen 406100), rabbit mAb anti-MDR1 (E1Y7S; Cell Signaling Technology 13978S), chicken anti-Vimentin (EMD Millipore Corp AB5733), and rabbit anti-MFSD2A (a kind gift of David Silver, Duke-NUS Medical School).

Techniques: Recombinant, Software, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Melatonin antagonizes bone loss induced by mechanical unloading via IGF2BP1-dependent m 6 A regulation

doi: 10.1007/s00018-025-05588-x

Figure Lengend Snippet: IGF2BP1 could regulate LEF1 expression by directly binding to the m 6 A modification site in Lef1 mRNA. a mRNA expression of Lef1 in MC3T3-E1 cells by qRT-PCR after transfection of si- Igf2bp1 , pEX- Igf2bp1 or the corresponding control (n = 3). b Protein levels of LEF1 in MC3T3-E1 cells after transfection (n = 3). c mRNA expression of Lef1 after MC3T3-E1 cells were transfected with pEX- Igf2bp1 and treated with clinostat unloading for 48 h (n = 3). d Protein expression of Lef1 (n = 3). e IF staining of OCN (red) and LEF1 (green) on the bone surface for distal femurs of mice in each group (n = 5). Scale bars, 50 μm. f The online SRAMP database revealed the several possible m 6 A modified sites in Lef1 . g RNA decay analysis was performed to detect the Lef1 mRNA level with IGF2BP1 silencing (n = 3). h The RIP-PCR assay was performed using anti-IGF2BP1 antibody to detect the binding within IGF2BP1 and Lef1 mRNA (n = 3). * P < 0.05, ** P < 0.01

Article Snippet: The membrane was blocked with 5% skim milk for 2 h, followed by overnight incubation at 4 °C with primary antibodies against as follows: GAPDH (1:1000; Cell Signaling Technology, USA), IGF2BP1 (1:10,000; Proteintech, China), LEF1 (1:1000; Proteintech, China), PCNA (1:1000; Cell Signaling Technology, USA), and Lamin B (1:10,000; Proteintech, China), c-Myc (1:1000; Cell Signaling Technology, USA), Cyclin D1(1:10,000; Abcam, UK).

Techniques: Expressing, Binding Assay, Modification, Quantitative RT-PCR, Transfection, Control, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Melatonin antagonizes bone loss induced by mechanical unloading via IGF2BP1-dependent m 6 A regulation

doi: 10.1007/s00018-025-05588-x

Figure Lengend Snippet: LEF1 could promote osteoblast proliferation. a, b Protein level of LEF1 in MC3T3-E1 cells after transfecting with si- Lef1 −1, si- Lef1 −2, pEX- Lef1 or the corresponding control (n = 3). c Protein level of PCNA after transfecting with si- Lef1 or pEX- Lef1 (n = 3). d Cell proliferation was evaluated by CCK-8 assay (n = 3). The symbol * (green) represents the si-NC vs. si- Lef1 −1 group; the symbol * (orange) represents the si-NC vs. si- Lef1 −2 group, the symbol * (red) represents the pEX vs. pEX- Lef1 group. e The EdU incorporation assay was analyzed by confocal microscopy (n = 3). Scale bar, 50 µm. f Protein levels of c-Myc and Cyclin D1 after transfecting with si- Lef1 , pEX- Lef1 or the corresponding control (n = 3). * P < 0.05, ** P < 0.01

Article Snippet: The membrane was blocked with 5% skim milk for 2 h, followed by overnight incubation at 4 °C with primary antibodies against as follows: GAPDH (1:1000; Cell Signaling Technology, USA), IGF2BP1 (1:10,000; Proteintech, China), LEF1 (1:1000; Proteintech, China), PCNA (1:1000; Cell Signaling Technology, USA), and Lamin B (1:10,000; Proteintech, China), c-Myc (1:1000; Cell Signaling Technology, USA), Cyclin D1(1:10,000; Abcam, UK).

Techniques: Control, CCK-8 Assay, Confocal Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Melatonin antagonizes bone loss induced by mechanical unloading via IGF2BP1-dependent m 6 A regulation

doi: 10.1007/s00018-025-05588-x

Figure Lengend Snippet: LEF1 is responsible for IGF2BP1-mediated osteoblast proliferation. a Protein levels of PCNA, c-Myc and Cyclin D1 after co-transfected with si- Igf2bp1 and pEX- Lef1 (n = 3). b Cell proliferation was evaluated by CCK-8 assay (n = 3). The symbol * (black) represents the si-NC vs. si- Igf2bp1 group; the symbol * (blue) represents the si- Igf2bp1 + pEX vs. si- Igf2bp1 + pEX- Lef1 group. c The EdU incorporation assay was analyzed by confocal microscopy (n = 3). Scale bar, 50 µm. d Protein level of PCNA after MC3T3-E1 cells were transfected with pEX- Igf2bp1 and si- Lef1 and treated with clinostat unloading for 48 h (n = 3). e Cell proliferation was evaluated by CCK-8 assay (n = 3). The symbol * (black) represents the CON vs. Clino group; the symbol * (green) represents the Clino vs. Clino + pEX- Igf2bp1 group. The symbol * (blue) represents the Clino + pEX- Igf2bp1 + si-NC vs. Clino + pEX- Igf2bp1 + si- Lef1 group. f The EdU incorporation assay was analyzed by confocal microscopy (n = 3). Scale bar, 50 µm. * P < 0.05, ** P < 0.01

Article Snippet: The membrane was blocked with 5% skim milk for 2 h, followed by overnight incubation at 4 °C with primary antibodies against as follows: GAPDH (1:1000; Cell Signaling Technology, USA), IGF2BP1 (1:10,000; Proteintech, China), LEF1 (1:1000; Proteintech, China), PCNA (1:1000; Cell Signaling Technology, USA), and Lamin B (1:10,000; Proteintech, China), c-Myc (1:1000; Cell Signaling Technology, USA), Cyclin D1(1:10,000; Abcam, UK).

Techniques: Transfection, CCK-8 Assay, Confocal Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Melatonin antagonizes bone loss induced by mechanical unloading via IGF2BP1-dependent m 6 A regulation

doi: 10.1007/s00018-025-05588-x

Figure Lengend Snippet: MT promotes osteoblast proliferation and bone formation through modulation of IGF2BP1/LEF1 pathway. a mRNA expression of Igf2bp1 in bone tissue from each group (n = 5). b Representative IHC images of IGF2BP1 in the bone tissue of HLU mice hind limbs (n = 5). Scale bars, 50 μm. c qRT-PCR analysis of Igf2bp1 mRNA levels after MT treatment (n = 3). d The protein expression of IGF2BP1, c-Myc, CyclinD1 and PCNA after MT treatment by western blotting (n = 3). e western blotting analysis of LEF1, PCNA, c-Myc, and Cyclin D1 protein levels after MC3T3-E1 cells were transfected with si- Igf2bp1 and MT treatment (n = 3). f The EdU incorporation assay was analyzed by confocal microscopy (n = 3). Scale bar, 50 µm. g Cell proliferation was evaluated by CCK-8 assay (n = 3). The symbol * (red) represents the si-NC vs. si- Igf2bp1 group; the symbol * (blue) represents the si- Igf2bp1 vs. si- Igf2bp1 + MT group. * P < 0.05, ** P < 0.01

Article Snippet: The membrane was blocked with 5% skim milk for 2 h, followed by overnight incubation at 4 °C with primary antibodies against as follows: GAPDH (1:1000; Cell Signaling Technology, USA), IGF2BP1 (1:10,000; Proteintech, China), LEF1 (1:1000; Proteintech, China), PCNA (1:1000; Cell Signaling Technology, USA), and Lamin B (1:10,000; Proteintech, China), c-Myc (1:1000; Cell Signaling Technology, USA), Cyclin D1(1:10,000; Abcam, UK).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Confocal Microscopy, CCK-8 Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Melatonin antagonizes bone loss induced by mechanical unloading via IGF2BP1-dependent m 6 A regulation

doi: 10.1007/s00018-025-05588-x

Figure Lengend Snippet: The schema of the role and mechanism of MT/IGF2BP1/LEF1 pathway in bone loss induced by mechanical unloading. MT could enhance IGF2BP1 expression and IGF2BP1 significantly promoted the expression of LEF1 via directly binding to Lef1 mRNA and recognizing m 6 A modification, which resulted in enhancing osteoblast proliferation and bone formation during mechanical unloading condition

Article Snippet: The membrane was blocked with 5% skim milk for 2 h, followed by overnight incubation at 4 °C with primary antibodies against as follows: GAPDH (1:1000; Cell Signaling Technology, USA), IGF2BP1 (1:10,000; Proteintech, China), LEF1 (1:1000; Proteintech, China), PCNA (1:1000; Cell Signaling Technology, USA), and Lamin B (1:10,000; Proteintech, China), c-Myc (1:1000; Cell Signaling Technology, USA), Cyclin D1(1:10,000; Abcam, UK).

Techniques: Expressing, Binding Assay, Modification

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Association between LEF1 mRNA expression with survival status of MM patients. A, The overall survival curve exhibited the overall survival of MM patients with low or high expression levels of LEF1 mRNA using GenomicScape software ( www.genomicscape.com ). B, The progression-free survival (Left) and overall survival (Right) status of MM patients with low or high expression levels of LEF1 mRNA using GenomicScape software ( www.genomicscape.com ). Red line: high expression group, blue line: low expression group. P values were denoted on the plot.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Expressing, Software

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Knockdown of LEF1 suppressed growth of U266 and RPMI8226 myeloma cells. A, The mRNA expression level of LEF1 in U266 and RPMI8226 cells infected with LEF1 shRNA (shLEF1) or scramble shRNA as the control (shCon) for 48 h was assessed by quantitative real-time RT-PCR (qRT-PCR). B, The protein expression level of LEF1 in U266 and RPMI8226 cells infected with shLEF1 or shCon for 72 h was detected by Westernblot analysis. C, The cell viability of U266 and RPMI8226 cells following transduction with shLEF1 or shCon was determined by CCK-8 assay. D, Brdu incorporation was determined in U266 and RPMI8226 cells transduced with shLEF1 or shCon. * P < 0.05 and ** P < 0.01 versus shcon group.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Knockdown, Expressing, Infection, shRNA, Control, Quantitative RT-PCR, Transduction, CCK-8 Assay, BrdU Incorporation Assay

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Depletion of LEF1 induced apoptosis of U266 and RPMI8226 myeloma cells. A-B, The cellular apoptosis was detected in U266 and RPMI8226 cells transduced with shLEF1 or shCon for 5 days by flow cytometry analysis following Annexin V/7AAD staining. C, The protein levels of PARP and cleaved caspase 3 was detected in U266 and RPMI8226 cells transduced with shLEF1 or shCon for 5 days by Westernblot analysis. D, Intracellular ROS production was determined by CM-H2DCFDA staining in U266 and RPMI8226 cells following transduction with shLEF1 or shCon for 72 h. * P < 0.05 and ** P < 0.01 versus shCon group.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Transduction, Flow Cytometry, Staining

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: LEF1 inhibited transcription of CYLD gene through promoter binding. A, The protein levels of CYLD in U266 and RPMI8226 cells following transduction with shLEF1 or shCon for 72 h was measured by Westernblot analysis. B, The CYLD mRNA expression level in U266 and RPMI8226 cells transduced with shLEF1 or shCon for 72 h was determined using qRT-PCR. C, Schematic representation of potential LEF1-binding sites in CYLD promoter and the location of the primers used for ChIP assay. D, U266 cells transduced with shLEF1 or shCon were co-transfected with either wild-type CYLD luciferase reporter construct (pGL3-CYLD-wt) or the plasmid containing the mutation in the putative LEF1 binding site (pGL3-CYLD-mt) together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined with dual-luciferase reporter assay kit. E, ChIP assay was performed to show binding of LEF1 on CYLD promoter in U266 cells. Anti-LEF1 antibody or control IgG was used to precipitate sonicated chromatin from U266 cells. The purified DNA was analyzed by real-time PCR with primers flanking potential LEF1 binding site. Data showed fold enrichment relative to control IgG. * P < 0.05 and ** P < 0.01 versus shCon group.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Binding Assay, Transduction, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Construct, Plasmid Preparation, Mutagenesis, Activity Assay, Reporter Assay, Control, Sonication, Purification, Real-time Polymerase Chain Reaction

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Depletion of LEF1 repressed NF-κB signaling pathway through modulation of CYLD in U266 cells. A, The protein levels of p65 and p-p65 in U266 cells following LEF1 knockdown for 72 h was measured by Westernblot analysis. B, U266 cells transduced with shLEF1 or shCon were co-transfected with NF-κB reporter construct (p-NF-κB-luc) together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined using dual-luciferase reporter assay kit. C, The mRNA expression levels of Cyclin D1, Bcl-2 and Bcl-xL in U266 cells transduced with shLEF1 or shCon for 72 h were determined using qRT-PCR. * P < 0.05 and ** P < 0.01 versus shCon group. D, U266 cells transduced with shLEF1 or shCon were co-transfected with NF-κB reporter construct (p-NF-κB-luc) in the presence of CYLD siRNA or the control siRNA together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined using dual-luciferase reporter assay kit. * P < 0.05 and ** P < 0.01.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Knockdown, Transduction, Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Expressing, Quantitative RT-PCR, Control

Journal: Molecular and Cellular Biology

Article Title: The Microphthalmia-Associated Transcription Factor Mitf Interacts with β-Catenin To Determine Target Gene Expression

doi: 10.1128/mcb.02299-05

Figure Lengend Snippet: FIG. 1. Interactions between -catenin and Mitf. (A) Schematic representation of the different -catenin constructs used in the GST pull-down. Armadillo repeats are shown as numbered boxes. The binding domains of known -catenin interacting partners, members of the Tcf/Lef1 transcription factor family, p300, and the C-terminal part of -catenin are indicated below the full-length -catenin protein. NT/CT: N-terminal and C-terminal tails of -catenin, respectively. (B and C) Mapping the Mitf interaction domain in -catenin. GST and GST--catenin fusion proteins were immobilized on glutathione-Sepharose beads, as indicated, and incubated with [35S]methionine-labeled Mitf. After extensive washing, material retained on the beads was analyzed by SDS-PAGE and fluorography, together with 10% of the input material. (D) Mapping the MITF interaction domain in -catenin using the yeast two-hybrid assay. The different -catenin constructs were tested for interactions with Mitf in a yeast two-hybrid assay. The different fragments used are outlined on the left, and their binding capacities are indicated on the right, as monitored by -galactosidase activity: , high binding capacity; , good binding capacity; , weak binding capacity; , no binding capacity. -cat, -catenin; M, marker.

Article Snippet: Chromatin immunoprecipitation assays were performed as described previously (13) with mouse monoclonal anti-Mitf antibody (C5), goat polyclonal anti- -catenin antibody (C-18; Santa Cruz), rabbit polyclonal anti-LEF1 antibody (Santa Cruz), or 10 l of nonspecific immunoglobulin G (Bio-Rad).

Techniques: Construct, Binding Assay, Incubation, Labeling, SDS Page, Y2H Assay, Activity Assay, Marker

Journal: Molecular and Cellular Biology

Article Title: The Microphthalmia-Associated Transcription Factor Mitf Interacts with β-Catenin To Determine Target Gene Expression

doi: 10.1128/mcb.02299-05

Figure Lengend Snippet: FIG. 4. Helix 1 of the Mitf bHLH domain provides critical contact points for the interaction with -catenin. (A) Sequence comparison of the -catenin binding domain of different Mitf homologues with known -catenin binding domains of members of the Tcf/Lef1 transcription factor family. Protein alignments were carried out using ClustalW at the San Diego Supercomputer Center workbench (http://workbench.sdsc.edu/) and then further adjusted by eye. (B) To better visualize their potential functional importance, the positions of the conserved amino acids, helix 1, and the loop were also depicted in a helical wheel structure, using the sequence analysis program BIOEDIT. (C) To confirm the sequence analysis results, coimmunoprecipi- tation assays were performed. Cell lysates from HEK293 cells, transfected with an HA-tagged -catenin construct, were incubated with the different in vitro translated and 35S-labeled Mitf deletion constructs, precipitated with an HA-specific antibody, and analyzed for the presence of the Mitf deletion construct using SDS-PAGE and radiography. -Catenin expression and immunoprecipitation were confirmed by Western blot analysis using a -catenin- specific antibody. (D) To analyze the importance of the D222 and D236 amino acids for the interaction with -catenin, colocalization studies with the different Mitf mutant constructs were performed. COS7 cells were treated as before, and -catenin was labeled with a goat anti--catenin antibody (Santa Cruz) and a secondary anti-goat Alexa 488 antibody (Molecular Probes). The nuclear localization of the different Mitf constructs was analyzed with a mouse anti-Mitf antibody and a secondary anti-mouse Alexa 568 antibody. IP, immunoprecipitation; wt, wild type.

Article Snippet: Chromatin immunoprecipitation assays were performed as described previously (13) with mouse monoclonal anti-Mitf antibody (C5), goat polyclonal anti- -catenin antibody (C-18; Santa Cruz), rabbit polyclonal anti-LEF1 antibody (Santa Cruz), or 10 l of nonspecific immunoglobulin G (Bio-Rad).

Techniques: Sequencing, Comparison, Binding Assay, Functional Assay, Transfection, Construct, Incubation, In Vitro, Labeling, SDS Page, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: The Microphthalmia-Associated Transcription Factor Mitf Interacts with β-Catenin To Determine Target Gene Expression

doi: 10.1128/mcb.02299-05

Figure Lengend Snippet: FIG. 6. Mitf recruits -catenin to specific target genes. (A) Association of -catenin with Mitf-regulated genes was analyzed by ChIP assays. 501mel melanoma cells were fixed in paraformaldehyde and harvested, and DNA was sheared by sonication. Cell lysates were precipitated with either a nonspecific antibody or a polyclonal -catenin antibody. After precipitation, cross-links were reverted, and precipitated DNA was purified and analyzed via PCR for known Mitf binding sequences or for control sequences. (B) To confirm that the presence of -catenin at the tyrosinase and Tyrp-1 promoters depends on Mitf–-catenin interactions, Mitf was depleted using specific siRNAs prior to ChIP. (C) To confirm that the observed results were not dependent on a scaffold effect of Lef1, control ChIP assays with an anti-Lef1 antibody were performed on both control-depleted and Mitf-siRNA-depleted 501 cells. Cells were treated as above. IgG, immunoglobulin G; AB, antibody.

Article Snippet: Chromatin immunoprecipitation assays were performed as described previously (13) with mouse monoclonal anti-Mitf antibody (C5), goat polyclonal anti- -catenin antibody (C-18; Santa Cruz), rabbit polyclonal anti-LEF1 antibody (Santa Cruz), or 10 l of nonspecific immunoglobulin G (Bio-Rad).

Techniques: Sonication, Binding Assay, Control

Journal: Molecular and Cellular Biology

Article Title: The Microphthalmia-Associated Transcription Factor Mitf Interacts with β-Catenin To Determine Target Gene Expression

doi: 10.1128/mcb.02299-05

Figure Lengend Snippet: FIG. 7. Model for the dynamic interactions between -catenin and Mitf transcriptional activity. Mitf plays a key role at different stages of melanoblast and melanocyte development, probably by regulating dif- ferent sets of target genes. To achieve this diversity, the activity of Mitf as a transcriptional activator is modulated either by regulating its protein level or by its engagement in changing and dynamic interac- tions with other factors in a highly regulated and promoter-specific manner. Moreover, signaling through the canonical Wnt signaling pathway provides several levels of regulation. Signaling through -catenin activates Mitf expression. Depending on the protein levels of Mitf, Mitf can interact and cooperate either with Lef1 or -catenin alone or in a complex with both to activate downstream targets.

Article Snippet: Chromatin immunoprecipitation assays were performed as described previously (13) with mouse monoclonal anti-Mitf antibody (C5), goat polyclonal anti- -catenin antibody (C-18; Santa Cruz), rabbit polyclonal anti-LEF1 antibody (Santa Cruz), or 10 l of nonspecific immunoglobulin G (Bio-Rad).

Techniques: Activity Assay, Expressing