Journal: Nature Communications

Article Title: FTO-associated osteoclastogenesis promotes alveolar bone resorption in apical periodontitis male rat via the HK1/USP14/RANK pathway

doi: 10.1038/s41467-025-56615-1

Figure Lengend Snippet: a KEGG pathway enrichment analyses of differentially abundant proteins. b The differential proteins were enriched in the metabolic pathway. c The hexokinase 1 (HK1) mRNA in the alveolar bone (AB) tissues of apical periodontitis (AP) rats was determined ( n = 3 independent experiments; mean ± SD). d , e HK1 protein expression was analyzed in the AB tissues of rats with AP, the relative intensity of bolts ( n = 3 independent experiments; mean ± SD). f Immunohistochemistry analysis of HK1 protein expression in the AB tissues of rats with AP. Scale bar indicates 1 mm in the upper and indicates 100 μm in the bottom ( n = 3 independent experiments). g The HK1 mRNA level in the process of osteoclastogenesis were determined ( n = 3 independent experiments; mean ± SD). h The expression level of HK1 protein expression in the process of osteoclastogenesis were determined ( n = 3 independent experiments; mean ± SD). i The representative images of HK1 (green) and nuclear factor-κB receptor activator (RANK) (red) detection in the AB tissues of rats with AP ( n = 3 independent experiments). The yellow arrow indicated the colocalization between HK1 and RANK. j The representative images of phalloidin (green) and HK1 (red) detection in bone marrow macrophage (BMDM)-derived osteoclast (OC) ( n = 3 independent experiments; mean ± SD). k The levels of glucose, pyruvate, lactate and hexokinase activity were measured ( n = 3 independent experiments; mean ± SD). l – n Measuring the extracellular acidification rate (ECAR) in BMDM-derived OC ( n = 3 independent experiments with 3 technical replicates; mean ± SD). For the AP rat model, the contralateral teeth served as control. The major statistical procedures applied were: Shapro–Wiik test ( a , b , c , d , g , h – k , m ) , F-test ( a , b , c , d , g , h – k ), Kruskal–Wallis H test and Dunnett-t test ( c , d , g , h , k ), Student-t test ( a , b , i , j , m ). The statistical test used was two-sided. Source data are available online for this figure.

Article Snippet: The IgG antibody was served as a negative control ( n = 3 independent experiments; mean ± SD). g The relative protein in 293 T cells overexpressing RANK for 24 h was determined ( n = 3 independent experiments; mean ± SD). h The relative protein in 293T cells with hexokinase 1 (HK1) overexpression followed by PKM2-IN-1 (5 μM, MCE) was detected ( n = 3 independent experiments; mean ± SD). i The relative protein in OC was determined ( n = 3 independent experiments; mean ± SD). j The relative mRNA level in OC was determined ( n = 3 independent experiments; mean ± SD).

Techniques: Expressing, Immunohistochemistry, Derivative Assay, Activity Assay, Control

Journal: Nature Communications

Article Title: FTO-associated osteoclastogenesis promotes alveolar bone resorption in apical periodontitis male rat via the HK1/USP14/RANK pathway

doi: 10.1038/s41467-025-56615-1

Figure Lengend Snippet: a , b The level of nuclear factor of activated T cells (NFATc1), matrix metalloprotein 9 (MMP9), and cathepsin K (CTSK) protein in osteoclast (OC) after transfecting for 4 days with lenti-shhexokinase 1 (HK1) were determined ( n = 3 independent experiments; mean ± SD). c The cell viability of bone marrow macrophage (BMDM) after treatment with 2-DG was measured ( n = 3 independent experiments; mean ± SD). d The relative protein levels in OC were determined ( n = 3 independent experiments; mean ± SD). e The mRNA levels in OC after treatment with 2-DG for 4 days were determined ( n = 3 independent experiments; mean ± SD). f , g The representative TRAP staining images of OC after treatment ( n = 3 independent experiments). h , i The bone resorption pits on bone slices after treatment with were stained by toluidine blue. Arrowheads indicate the bone resorption ( n = 3 independent experiments). j The levels of glycolysis in OC were measured by Seahorse XF96 Extracellular Flux Analyzer after transfecting with Lenti-shHK1 for 4 days ( n = 3 independent experiments with 3 technical replicates; mean ± SD). k Seahorse XF96 Extracellular Flux Analyzer was used to measure the levels of glycolysis in OC after 2-DG treatment at concentrations ranging from 0.01 mM to 0.1 mM for 4 days ( n = 3 independent experiments with 3 technical replicates; mean ± SD). BMDM: Bone marrow macrophage, OC: osteoclast. The major statistical procedures applied were: Shapro–Wiik test ( b , c , d , e , j , k ) , F-test ( b , c , d , e , k ), One-way ANOVA ( b , c , d , e , k ), Dunnett-t test ( b , k ), SNK-q test ( c , d , e ), student-t test ( j ). The statistical test used was two-sided. Source data are available online for this figure.

Article Snippet: The IgG antibody was served as a negative control ( n = 3 independent experiments; mean ± SD). g The relative protein in 293 T cells overexpressing RANK for 24 h was determined ( n = 3 independent experiments; mean ± SD). h The relative protein in 293T cells with hexokinase 1 (HK1) overexpression followed by PKM2-IN-1 (5 μM, MCE) was detected ( n = 3 independent experiments; mean ± SD). i The relative protein in OC was determined ( n = 3 independent experiments; mean ± SD). j The relative mRNA level in OC was determined ( n = 3 independent experiments; mean ± SD).

Techniques: Staining

Journal: Nature Communications

Article Title: FTO-associated osteoclastogenesis promotes alveolar bone resorption in apical periodontitis male rat via the HK1/USP14/RANK pathway

doi: 10.1038/s41467-025-56615-1

Figure Lengend Snippet: a Flow diagram of the treatment of apical periodontitis (AP) with the macrophage-targeted AAV9-shHK1-GFP (4.67 ×10 11 v.g/rat, 10 μL/rat) or 2-DG (400 mg/kg) in rats ( n = 9 rats per group). b The representative images of hexokinase 1 (HK1) (red) and nuclear factor-κB receptor activator (RANK) (pink) detection in the alveolar bone (AB) tissues of rats ( n = 3 independent experiments). c The representative micro-CT images of AB tissues of the rats ( n = 3 independent experiments; mean ± SD). The bone resorption fraction was defined as bone resorption surface area (BRS)/bone resorption volume (BRV). d The representative micro-CT images of AB tissues of rats ( n = 3 independent experiments with 3 technical replicates; mean ± SD). The bone resorption fraction was defined as bone resorption surface area (BRS) (mm 2 )/bone resorption volume (BRV) (mm 3 ). e , f Morphometric imaging showed TRAP staining in the AB tissues of rats ( n = 3 independent experiments with 3 technical replicates; mean ± SD). Scale bar indicates 1 mm in the left and scale bar indicates 100 μm in the right. Arrowheads pointed the TRAP-positive surfaces. TRAP-positive multinucleated cells were counted as OC. g , h The mRNA and protein levels of HK1, nuclear factor of activated T cells (NFATc1), matrix metalloprotein 9 (MMP9), and cathepsin K (CTSK) in the AB tissues of rats were determined ( n = 3 independent experiments; mean ± SD). i The level of relevant protein in the AB tissues of rats were determined ( n = 3 independent experiments; mean ± SD). j The mRNA levels in the AB tissues of rats were determined ( n = 3 independent experiments; mean ± SD). The AAV9-shNC group AP rats were received the treatment of unedited plasmid carried by AAV9 vector; the PBS group AP rats were treated with PBS, a solvent for 2-DG. The major statistical procedures applied were: Shapro–Wiik test ( c , d , e – j ) , F-test ( i ), student-t test ( c , d , e – h , j ), Wilcoxon test ( g , h , j ), Kruskal–Wallis H test and Dunnett-t test ( i ). The statistical test used was two-sided. Source data are available online for this figure.

Article Snippet: The IgG antibody was served as a negative control ( n = 3 independent experiments; mean ± SD). g The relative protein in 293 T cells overexpressing RANK for 24 h was determined ( n = 3 independent experiments; mean ± SD). h The relative protein in 293T cells with hexokinase 1 (HK1) overexpression followed by PKM2-IN-1 (5 μM, MCE) was detected ( n = 3 independent experiments; mean ± SD). i The relative protein in OC was determined ( n = 3 independent experiments; mean ± SD). j The relative mRNA level in OC was determined ( n = 3 independent experiments; mean ± SD).

Techniques: Micro-CT, Imaging, Staining, Plasmid Preparation, Solvent

Journal: Nature Communications

Article Title: FTO-associated osteoclastogenesis promotes alveolar bone resorption in apical periodontitis male rat via the HK1/USP14/RANK pathway

doi: 10.1038/s41467-025-56615-1

Figure Lengend Snippet: a m 6 A enrichment on hexokinase 1 ( HK1 ) mRNAs in the alveolar bone (AB) tissues of rats. The IgG antibody was served as a negative control ( n = 3 independent experiments; mean ± SD). b The interplay between obesity-associated protein (FTO) and HK1 was measured in the AB tissues of rats. The IgG antibody was served as a negative control ( n = 3 independent experiments; mean ± SD). c The interplay between FTO and HK1 was measured in bone marrow macrophage (BMDM)-derived osteoclast (OC). The IgG antibody was served as a negative control ( n = 3 independent experiments; mean ± SD). d The relative protein levels in 293T cells were determined ( n = 3 independent experiments; mean ± SD). e The relative protein levels of HK1 in OC were determined ( n = 3 independent experiments; mean ± SD). f The HK1 protein levels in OC after treatment with Dac51 were determined ( n = 3 independent experiments; mean ± SD). g The mRNA stability of HK1 after FTO overexpression in 293 T cells was detected ( n = 3 independent experiments with 3 technical replicates; mean ± SD). h Measuring the levels of glycolysis in OC ( n = 3 independent experiments with 3 technical replicates; mean ± SD). i The levels of the mitochondrial activity in OC by oxygen consumption rate (OCR) ( n = 3 independent experiments with 3 technical replicates; mean ± SD). j The levels of glycolysis in OC after Dac51 treatment by extracellular acidification rate (ECAR) ( n = 3 independent experiments with 3 technical replicates; mean ± SD). For the AP rat model, the contralateral teeth served as control. The Lenti-shNC group BMDM cells were received the treatment of unedited plasmid carried by lentiviral vector. The major statistical procedures applied were: Shapro–Wiik test ( a – j ) , F-test ( b , d , e , f , h , j ), paired t test ( a ) , Student-t test ( c , g , i ), One-way ANOVA and Dunnett-t test ( d , e , f , h , j ). The statistical test used was two-sided. Source data are available online for this figure.

Article Snippet: The IgG antibody was served as a negative control ( n = 3 independent experiments; mean ± SD). g The relative protein in 293 T cells overexpressing RANK for 24 h was determined ( n = 3 independent experiments; mean ± SD). h The relative protein in 293T cells with hexokinase 1 (HK1) overexpression followed by PKM2-IN-1 (5 μM, MCE) was detected ( n = 3 independent experiments; mean ± SD). i The relative protein in OC was determined ( n = 3 independent experiments; mean ± SD). j The relative mRNA level in OC was determined ( n = 3 independent experiments; mean ± SD).

Techniques: Negative Control, Derivative Assay, Over Expression, Activity Assay, Control, Plasmid Preparation

Journal: Nature Communications

Article Title: FTO-associated osteoclastogenesis promotes alveolar bone resorption in apical periodontitis male rat via the HK1/USP14/RANK pathway

doi: 10.1038/s41467-025-56615-1

Figure Lengend Snippet: a The relative protein in bone marrow macrophage (BMDM)-derived osteoclast (OC) was determined ( n = 3 independent experiments; mean ± SD). b The relative mRNA in OC was determined ( n = 3 independent experiments; mean ± SD). c The protein expressed in OC cells ( n = 3 independent experiments; mean ± SD). d The relative protein expression in the AB tissues of apical periodontitis (AP) rats ( n = 3 independent experiments). e The intensity of the blots were analyzed ( n = 3 independent experiments; mean ± SD). f The interplay between obesity-associated protein (FTO) and nuclear factor-κB receptor activator (RANK) was measured by RIP assay. The IgG antibody was served as a negative control ( n = 3 independent experiments; mean ± SD). g The relative protein in 293 T cells overexpressing RANK for 24 h was determined ( n = 3 independent experiments; mean ± SD). h The relative protein in 293T cells with hexokinase 1 (HK1) overexpression followed by PKM2-IN-1 (5 μM, MCE) was detected ( n = 3 independent experiments; mean ± SD). i The relative protein in OC was determined ( n = 3 independent experiments; mean ± SD). j The relative mRNA level in OC was determined ( n = 3 independent experiments; mean ± SD). k The relative protein in OC after treatment with 2-DG was determined ( n = 3 independent experiments; mean ± SD). l The relative mRNA in OC after treatment with 2-DG were determined ( n = 3 independent experiments; mean ± SD). m The relative protein in the AB tissues were determined ( n = 3 independent experiments; mean ± SD). The Lenti-shNC group BMDM cells and the AAV9-shNC group AP rats were received the treatment of unedited plasmid carried by lentiviral vector and AAV9 vector, respectively. The major statistical procedures applied were: Shapro–Wiik test ( a – m ) , F-test ( a , b , f – l ), Student-t test ( c , e , m ), Repeated measures ANOVA and Dunnett-t test ( a ), One-way ANOVA and Dunnett-t test ( f – k ). The statistical test used was two-sided. Source data are available online for this figure.

Article Snippet: The IgG antibody was served as a negative control ( n = 3 independent experiments; mean ± SD). g The relative protein in 293 T cells overexpressing RANK for 24 h was determined ( n = 3 independent experiments; mean ± SD). h The relative protein in 293T cells with hexokinase 1 (HK1) overexpression followed by PKM2-IN-1 (5 μM, MCE) was detected ( n = 3 independent experiments; mean ± SD). i The relative protein in OC was determined ( n = 3 independent experiments; mean ± SD). j The relative mRNA level in OC was determined ( n = 3 independent experiments; mean ± SD).

Techniques: Derivative Assay, Expressing, Negative Control, Over Expression, Plasmid Preparation

Journal: Nature Communications

Article Title: FTO-associated osteoclastogenesis promotes alveolar bone resorption in apical periodontitis male rat via the HK1/USP14/RANK pathway

doi: 10.1038/s41467-025-56615-1

Figure Lengend Snippet: a The relative ubiquitination level in OC after treatment with 0.1 mM 2-DG for 4 days was determined by western blotting ( n = 3 independent experiments). b The relative protein levels of nuclear factor-κB receptor activator (RANK), nuclear factor of activated T cells (NFATc1), matrix metalloprotein 9 (MMP9), and cathepsin K (CTSK) in 2-DG-treated OC after treatment with MG132 (0.5 μM) were determined by western blotting ( n = 3 independent experiments; mean ± SD). c The representative images of RANK (green) and ubiquitin-specific protease 14 (USP14) (red) detection by immunofluorescence in the process of bone marrow macrophage (BMDM)-derived osteoclast (OC). Scale bar indicates 20 μm ( n = 3 independent experiments). d Co-IP assay to examine endogenous interaction between USP14 and RANK in BMDM, and BMDM-derived OC at 5 days ( n = 3 independent experiments; mean ± SD). e Co-IP assay to examine endogenous USP14 and RANK interaction in HEK-293T cells overexpressing RANK after USP14 and hexokinase 1 (HK1) overexpression for 24 h ( n = 3 independent experiments; mean ± SD). The major statistical procedures applied were: Shapro–Wiik test ( b , d , e ) , F-test ( b , e ), Student-t test ( d ), Kruskal–Wallis H test and Dunnett-t test ( b , e ). The statistical test used was two-sided. Source data are available online for this figure.

Article Snippet: The IgG antibody was served as a negative control ( n = 3 independent experiments; mean ± SD). g The relative protein in 293 T cells overexpressing RANK for 24 h was determined ( n = 3 independent experiments; mean ± SD). h The relative protein in 293T cells with hexokinase 1 (HK1) overexpression followed by PKM2-IN-1 (5 μM, MCE) was detected ( n = 3 independent experiments; mean ± SD). i The relative protein in OC was determined ( n = 3 independent experiments; mean ± SD). j The relative mRNA level in OC was determined ( n = 3 independent experiments; mean ± SD).

Techniques: Ubiquitin Proteomics, Western Blot, Immunofluorescence, Derivative Assay, Co-Immunoprecipitation Assay, Over Expression

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 2. Expression of mouse CST gene in various tissues. (A) Northern blot of CST mRNAs. First, 20 mg total RNA from indicated organs was electrophoresed, blotted, and hybridized with a digoxigenin-labeled (±) strand RNA probe made from the full-length mouse CST cDNA. Lane 1, kidney; lane 2, brain; lane 3, testis; lane 4, heart; lane 5, skeletal muscle; lane 6, small intestine; lane 7, stomach; lane 8, liver; lane 9, lung; lane 10, spleen. (B) RT-PCR analysis of CST and actin mRNAs. Next, 1 mg total RNA from indicated organs was reverse-transcribed with a random primer. The resulting cDNAs were separately amplified by PCR using a set of CST primers, 50-GGGTTTCCTGAGATGAC-30 (nucleotides 1±17 in Fig. 1) and 50-TAGTGCGCGTTGTAGCT-30 (nucleotides 634±650) and actin primers, 50-TTACCAACTGGGACGACATG-30 (nucleotides 149±168 in [16]) and 50-AGGAGCCAGAGCAGTAATCT-30 (nucleotides 869±888). The PCR products were separately electrophoresed, blotted, and hybridized with CST and actin probes. The observed PCR products showed the predicted sizes of 650 (CST) and 740 bases (actin). Lanes: 1, brain; 2, heart; 3, skeletal muscle; 4, kidney; 5, stomach; 6, small intestine; 7, lung; 8, liver; 9, spleen; 10, testis. (C) CST activity. Whole homogenates from indicated organs were assayed for CST activity and protein concentration as described in Materials and methods. 1, kidney; 2, brain; 3, testis; 4, heart; 5, skeletal muscle; 6, small intestine; 7, stomach; 8, liver; 9, lung; 10, spleen.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Expressing, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Amplification, Activity Assay, Protein Concentration

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 3. Multiple forms of mouse CST cDNA with 50-UTR unique sequences. Seven types of unique DNA sequences on the 50 end of mouse CST cDNAs are represented by boldface letters. The 50-terminus of the homologous sequence is indicated by arrows. The translation-initiation codon ATG is boxed and indicated by arrowheads. In forms A, B2, and C, type a, b, and c sequences are underlined with continuous lines and type d sequence is underlined with broken lines.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Sequencing

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 5. RT-PCR analysis of tissue-specific use of alternative exon 1. (A) Position of each primer and probe. Boxes represent exons. Symbols for exons are the same as in Fig. 4. Black boxes are coding exons. Total RNAs from various tissues were reverse transcribed using GSP1 primer. After isolation of the first cDNA strand, PCR was performed using a combination of a common antisense-primer 2A, corresponding to exon 2, and seven sense-primers relating to the seven exons 1, aS, bS, cS, dS, eS, fS, or gS. The PCR products were subjected to Southern hybridization with a probe EX2, corresponding to exon 2. (B) RNA from indicated organs was examined. PCR was performed using sense-primer aS (lane a), bS (lane b), cS (lane c), dS (lane d), eS (lane e), fS (lane f ), or gS (lane g). Minor bands in lanes a and b are represented by asterisks in small intestine.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Isolation, Hybridization

Journal: Cell

Article Title: Fibrinogen-like protein 1 is a major immune inhibitory ligand of LAG3

doi: 10.1016/j.cell.2018.11.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Mouse Ly6C (clone HK1.4), 162-Dy , Fluidigm , Cat#: 3162014B.

Techniques: Functional Assay, Virus, Recombinant, Mass Cytometry, Conjugation Assay, Enzyme-linked Immunosorbent Assay, Diagnostic Assay, cDNA Library Assay, Software

Journal: Cell Death & Disease

Article Title: The dual mechanism of m 6 A demethylase ALKBH5 in regulating energy metabolism during exposure to MC-LR

doi: 10.1038/s41419-025-07791-x

Figure Lengend Snippet: A RNA-seq data analysis of glycolytic pathway enzyme genes in liver tissue from MC-LR exposed mice. B Heatmap of RNA expression of the glycolytic pathway enzyme genes. C , D The effect of ALKBH5 knockdown on the expression of PIK3R1 and glycolytic pathway enzymes in THLE-3 cells, as detected by Western blotting and quantified. E , F The effect of MC-LR exposure on the expression of MC-LR, ALKBH5, PIK3R1 and glycolytic pathway enzymes in mouse liver tissues, as detected by Western blotting and quantified. G , H The effect of overexpression of ALKBH5 on the expression levels of MC-LR, ALKBH5, PIK3R1 and glycolytic pathway enzymes proteins in THLE-3 cells treated with MC-LR, as detected by Western blotting and quantified. I m 6 A modifications of HK1, HK2, PKM and LDHA were not affected by MC-LR exposure and ALKBH5 overexpression. J , K The effect of PIK3R1 knockdown on the expression of glycolytic pathway enzymes in THLE-3 cells, as detected by Western blotting and quantified. Data are means ± SD from three independent experiments. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: The detection antibodies were as follows: ALKBH5 antibody (Proteintech, 16837-1-AP); PIK3R1 antibody (Proteintech, 30092-1-AP); MC-LR antibody(Alexis Corporation); β-actin antibody(Cell Signalling Technology, 4967); HK1 antibody (Proteintech, 15656-1-AP); HK2 antibody (Proteintech, 22029-1-AP); PKM antibody (Proteintech, 25659-1-AP); LDHA antibody (Proteintech, 21799-1-AP); ETFDH antibody (Proteintech, 11109-1-AP);NDUFAF4 antibody (Proteintech, 26003-1-AP); Microcystin-LR monoclonal antibody (Enzo Life Sciences, ALX-804-320-C200).

Techniques: RNA Sequencing, RNA Expression, Knockdown, Expressing, Western Blot, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: SHP2 Positively Regulates TGFβ1-induced Epithelial-Mesenchymal Transition Modulated by Its Novel Interacting Protein Hook1 *

doi: 10.1074/jbc.M113.546077

Figure Lengend Snippet: Hook1 was a new binding protein of SHP2. A , co-immunoprecipitation assay to verify the interaction of overexpressed full-length SHP2 (MYC-SHP2) or PTP domain of SHP2 (MYC-SHP2-PTP) with FLAG-Hook1 in HEK293T cells. B , co-immunoprecipitation assay to verify the endogenous interaction of SHP2 with Hook1 in HEK293T cells.

Article Snippet: The Hook1 (NM_015888) human cDNA clone (cat. no. SC114574) was from OriGene (Rockville, MD).

Techniques: Binding Assay, Co-Immunoprecipitation Assay

Journal: The Journal of Biological Chemistry

Article Title: SHP2 Positively Regulates TGFβ1-induced Epithelial-Mesenchymal Transition Modulated by Its Novel Interacting Protein Hook1 *

doi: 10.1074/jbc.M113.546077

Figure Lengend Snippet: Hook1 negatively regulated EMT. A , quantification of the mRNA levels of CDH1 , Hook1, Hook2 , and Hook3 in A549 cells with TGFβ1 treatment (5 ng/ml) for 0, 3, 6, and 12 h (**, p < 0.01). B , Western blot analysis of the expression levels of Hook1 after treatment with TGFβ1 (5 ng/ml) for 48 h. C , Hook1 was knockdown after Hook1siRNA transfection in A549 cells. D , cell adhesion in control siRNA and Hook1siRNA-treated A549 cells. E , Western blot analysis of the effects of Hook1 overexpression on EMT markers (E-Cad and VIM) and ERK, Smad2, Smad3 activation in A549 cells with or without TGFβ1 treatment (5 ng/ml) for 24 h. F , QPCR analysis of EMT markers ( CDH1 , CDH2 , VIM , MMP9 , and COL1A1 ) under the same conditions as E . ***, p < 0.005 between with and without TGFβ1 treatment; &, p < 0.01 between vector and Hook1. G , transwell assays to assess the migration of control and Hook1-transfected A549 cells, with or without TGFβ1 treatment (5 ng/ml) for 16 h. Scale bar, 100 μm. ***, p < 0.005 between with and without TGFβ1 treatment; &, p < 0.01 between vector and Hook1. H , QPCR analysis of EMT-related markers ( CDH1 , FN1 , ZEB1 , and COL1A1 ) in control siRNA and Hook1siRNA-transfected A549 cells, with or without TGFβ1 treatment (5 ng/ml) for 24 h. ***, p < 0.005 between with and without TGFβ1 treatment; &, p < 0.05 between CtrlsiRNA and Hook1siRNA. I, transwell assays to assess the migration of control and Hook1siRNA-treated A549 cells, with or without TGFβ1 treatment (5 ng/ml) for 16 h. Scale bar, 100 μm. ***, p < 0.005 between with and without TGFβ1 treatment; &, p < 0.05 between CtrlsiRNA and Hook1siRNA.

Article Snippet: The Hook1 (NM_015888) human cDNA clone (cat. no. SC114574) was from OriGene (Rockville, MD).

Techniques: Western Blot, Expressing, Knockdown, Transfection, Control, Over Expression, Activation Assay, Plasmid Preparation, Migration

Journal: The Journal of Biological Chemistry

Article Title: SHP2 Positively Regulates TGFβ1-induced Epithelial-Mesenchymal Transition Modulated by Its Novel Interacting Protein Hook1 *

doi: 10.1074/jbc.M113.546077

Figure Lengend Snippet: Interaction of SHP2-NSH2 and PTP domains with Hook1. A , co-immunoprecipitation assay to verify the direct interaction of FLAG-Hook1C with either MYC-SHP2-NSH2 or MYC-SHP2-PTP in HEK293T cells. B , PTP activity assay to test SHP2 activity in immunocomplex from control siRNA or Hook1siRNA-transfected HEK293T cells, with or without EGF (50 ng/ml) stimulation for 15 min. ***, p < 0.005 between Ctrl and EGF; &, p < 0.05 between CtrlsiRNA and Hook1siRNA. C, PTP activity assay to test SHP2 activity in immunocomplex from A549 cells with or without TGFβ1 (5 ng/ml) stimulation for 24 h (***, p < 0.005). D , QPCR analysis of mRNA levels of EMT markers ( Snail1 , COL1A1 , FN1 ) in vector, Hook1, SHP2 E76V -transfected, Hook1 and E76V co-transfected A549 cells, with or without TGFβ1 treatment (5 ng/ml) for 24 h. ***, p < 0.005 between with and without TGFβ1 treatment; &, p < 0.05 between SHP2E76V only and SHP2E76V plus Hook1. E , schematic model of the interaction between SHP2 and Hook1 in EMT. Hook1 locks the auto-inhibition caused by the intramolecular interaction between N-SH2 and PTP. Upon TGFβ1 treatment, Hook1 is reduced, and this weakens the interaction between N-SH2 and PTP, leading to the full activation of SHP2 by other molecules, including substrates.

Article Snippet: The Hook1 (NM_015888) human cDNA clone (cat. no. SC114574) was from OriGene (Rockville, MD).

Techniques: Co-Immunoprecipitation Assay, Activity Assay, Control, Transfection, Plasmid Preparation, Inhibition, Activation Assay