Journal: Cell reports

Article Title: PPARγ Interaction with UBR5/ATMIN Promotes DNA Repairto Maintain Endothelial Homeostasis

doi: 10.1016/j.celrep.2019.01.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat Anti-ORC2 Monoclonal Antibody, Unconjugated, Clone 3G6 , Cell Signaling Technology , Cat#4736, RRID:AB_2157716.

Techniques: Control, Recombinant, In Vitro, Transfection, Flow Cytometry, Single Cell Gel Electrophoresis, Mass Spectrometry, Sequencing, Real-time Polymerase Chain Reaction, Cloning, Plasmid Preparation, Software

Journal:

Article Title: Functional interactions of DNA topoisomerases with a human replication origin

doi: 10.1038/sj.emboj.7601578

Figure Lengend Snippet: Topo I interacts with the lamin B2 origin in a cell-cycle-dependent manner, and is a member of the origin binding complex. (A) Localization and orientation of the primer sets in the analyzed region; the positions of the detected topo I–DNA complexes are indicated by vertical arrows. (B) LM-PCR-mediated detection of CPT-induced topo I cleavage complexes on the lower and upper strands in different moments of the cell cycle; G, in vitro DMS-treated genomic DNA. (C) Identification of the presence of topo I on the CPT-induced cleavage sites and interaction of the enzyme with Orc2p; in the upper portion are shown the positions of the primers utilized (arrows) relative to topo I cutting sites; HeLa cells subjected to CPT treatment were crosslinked or not with DSP, lysed and the DNA was immunopurified with anti-topo I, anti-Orc2p or unrelated antibodies; the lower portion shows the PCR analysis of untreated genomic DNA (lanes 1 and 17), of the DNA immunopurified with anti-topo I antibodies (lanes 2–4), with anti-Orc2p antibodies (lanes 13–16) or with unrelated antibodies (lanes 5–12). (D) Topo I co-immunoprecipitates with Orc2p in HeLa nuclear extract: Western blot of proteins immunoprecipitated with anti-Orc2p antibody and assayed with anti-topo I or anti-Orc2p antibodies. (E) Formaldehyde crosslinking shows that both topo I and Orc2p associate with the lamin B2 origin in late G1; the DNA from formaldehyde crosslinked HeLa cells was immunopurified using anti-topo I or anti-Orc2 antibodies or pre-immune serum and subjected to competitive PCR analysis; B48, origin region; B13 non-origin region.

Article Snippet: Co-immunoprecipitation HeLa nuclear extract (Cilbiotech) was used for immunoprecipitation with anti-Orc2p antibodies (Stressgen) and the ProFound co-immunoprecipitation kit (Pierce), as described by the manufacturer.

Techniques: Binding Assay, In Vitro, Western Blot, Immunoprecipitation

Journal:

Article Title: Functional interactions of DNA topoisomerases with a human replication origin

doi: 10.1038/sj.emboj.7601578

Figure Lengend Snippet: Topo II interacts with the lamin B2 origin in a cell-cycle-dependent manner, and is a member of the origin binding complex. (A) TD-PCR-mediated detection of VP16-induced topo II cleavages on the upper strand; G, in vitro DMS-treated genomic DNA. (B) Localization and orientation of the primer sets in the region analyzed; the positions of the detected topo II–DNA complexes are indicated by vertical arrows. (C) TD-PCR-mediated detection of VP16-induced topo II cleavages on the lower strand; G, in vitro DMS-treated genomic DNA. (D) TD-PCR analysis of DNA immunopurified with anti-Orc2p (lanes 3–6) or unrelated antibodies (lanes 1 and 2) from HeLa cells synchronized in the middle of G1, treated with VP16, DSP or both; topo II binding sites at or outside of the origin are indicated with one or two asterisks respectively; G, in vitro DMS-treated genomic DNA.

Article Snippet: Co-immunoprecipitation HeLa nuclear extract (Cilbiotech) was used for immunoprecipitation with anti-Orc2p antibodies (Stressgen) and the ProFound co-immunoprecipitation kit (Pierce), as described by the manufacturer.

Techniques: Binding Assay, In Vitro

Journal:

Article Title: Functional interactions of DNA topoisomerases with a human replication origin

doi: 10.1038/sj.emboj.7601578

Figure Lengend Snippet: Summary of the mapped protein–DNA interactions at the lamin B2 origin. The cartoon summarizes the interaction of active topo I and topo II molecules with the origin sequence along the cell cycle demonstrated in the present work and reports also the data previously obtained for the interactions of Orc1p, Orc2p and Cdc6p with the same sequence (Abdurashidova et al, 2003). The interaction of the two enzymes with the ORC complex is also shown.

Article Snippet: Co-immunoprecipitation HeLa nuclear extract (Cilbiotech) was used for immunoprecipitation with anti-Orc2p antibodies (Stressgen) and the ProFound co-immunoprecipitation kit (Pierce), as described by the manufacturer.

Techniques: Sequencing

Journal: Cell & Bioscience

Article Title: Conditional ablation of MAPK7 expression in chondrocytes impairs endochondral bone formation in limbs and adaptation of chondrocytes to hypoxia

doi: 10.1186/s13578-020-00462-8

Figure Lengend Snippet: Loss of MAPK7 in chondrocytes caused growth restriction and short limbs in postnatal mice. a Phenotype of Mapk7 CKO and CON littermates at P21. Scale bar, 1 cm. b Analysis of body length of Mapk7 CKO and CON mice at P21 ( n = 3). * P < 0.05 (Student’s t test). Data are presented as mean ± SD. c Comparison of body weight between CON and Mapk7 CKO mice at various time points. Male and female mice were combined ( n = 4). * P < 0.05 (paired-sample t test). Data are presented as mean ± SD. d Representative image and ( e, f ) quantification analysis of limb bones from 60-day-old CON and Mapk7 CKO mice ( n = 6). Scale bar, 5 mm. * P < 0.05 (Student’s t test). N.S., not significant. Data are presented as mean ± SD

Article Snippet: Mapk7 floxed mice which carried the targeted allele with a couple of loxP sites flanking exons 4, 5, 6, and 7 of Mapk7 gene were generated by Cyagen Biosciences (Suzhou, China), and schematic diagram of loxp sites was shown in Additional file : Fig. S1a.

Techniques: Comparison

Journal: Cell & Bioscience

Article Title: Conditional ablation of MAPK7 expression in chondrocytes impairs endochondral bone formation in limbs and adaptation of chondrocytes to hypoxia

doi: 10.1186/s13578-020-00462-8

Figure Lengend Snippet: Mice with MAPK7 deficiency in chondrocytes displayed decreased cortical thickness and bone mass loss. a HE staining of distal femur sections from P60 mice. Boxed areas are magnified in the images at the bottom. Scale bar, 200 μm. b Three-dimensional reconstruction μCT images ( n = 5) of (upper images) femur trabecular and (lower images) cortical bone in CON and Mapk7 CKO mice at P60. c–i Quantitative μCT analysis of CON and Mapk7 CKO femurs, including ( c ) cortical thickness, ( d ) bone mineral density (BMD), ( e ) bone volume/tissue volume ratio (BV/TV), ( f ) trabecular thickness, ( g ) trabecular number, ( h ) trabecular separation, and ( i ) the trabecular pattern factor. * P < 0.05 (Student’s t test). Data are presented as mean ± SD. j – l Immunofluorescence staining of ( j ) OSX, ( k ) OPN and ( l ) OCN on representative sections of proximal tibial growth plates from P7 mice

Article Snippet: Mapk7 floxed mice which carried the targeted allele with a couple of loxP sites flanking exons 4, 5, 6, and 7 of Mapk7 gene were generated by Cyagen Biosciences (Suzhou, China), and schematic diagram of loxp sites was shown in Additional file : Fig. S1a.

Techniques: Staining, Immunofluorescence

Journal: Cell & Bioscience

Article Title: Conditional ablation of MAPK7 expression in chondrocytes impairs endochondral bone formation in limbs and adaptation of chondrocytes to hypoxia

doi: 10.1186/s13578-020-00462-8

Figure Lengend Snippet: MAPK7 deficiency reduced survival and proliferation of chondrocytes in the central proliferative layer. a HE- and ( b ) TUNEL-stained sections of the distal femoral growth plate at P7. Boxed areas in the center of the growth plate are magnified in the images at the bottom. c HE- and ( d ) TUNEL-stained sections of the distal femoral growth plates of P1 mice. Boxed areas in the center of the proliferative layer are magnified in the images at the bottom. Scale bar, 200 μm. e mRNA levels of Pcna , Ki67 , Cyclin B1 , and Cyclin D1 in tibial and femoral growth plates isolated from CON and Mapk7 CKO mice at P1 were assayed by real-time quantitative PCR ( n = 3). * P < 0.05 (Student’s t test). Data are presented as mean ± SD. f Protein levels of MAPK7 and PCNA in tibial and femoral growth plates isolated from CON and Mapk7 CKO mice at P1 were assayed by western blot. g EdU labeling of chondrocytes in tibiae from P1 mice. Boxed areas in the top images, which represent the counted regions in the quantification of EdU-positive cells, are magnified in the images at the bottom. Scale bar, 200 μm. h EdU-positive cells were counted in the center and the periphery of the proliferation layer ( n = 3) * P < 0.05 (Student’s t test). N.S., not significant. Data are presented as mean ± SD

Article Snippet: Mapk7 floxed mice which carried the targeted allele with a couple of loxP sites flanking exons 4, 5, 6, and 7 of Mapk7 gene were generated by Cyagen Biosciences (Suzhou, China), and schematic diagram of loxp sites was shown in Additional file : Fig. S1a.

Techniques: TUNEL Assay, Staining, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Labeling

Journal: Cell & Bioscience

Article Title: Conditional ablation of MAPK7 expression in chondrocytes impairs endochondral bone formation in limbs and adaptation of chondrocytes to hypoxia

doi: 10.1186/s13578-020-00462-8

Figure Lengend Snippet: Differentiation of hypertrophic chondrocytes within the central region of growth plates was impaired in Mapk7 CKO mice. a EdU labeling-chasing assay and corresponding HE staining of tibial sections from P4 mice. Dotted lines areas represent the proliferative zone and hypertrophic zone. PZ: proliferative zone; HZ: hypertrophic zone. b Quantification of EdU-positive cells in proliferative and hypertrophic zone. The percentage means EdU-labeled in hypertrophic zone relative to total number of EdU-positive chondrocytes in the proliferative and hypertrophic zone. HCs, hypertrophic chondrocytes. ( n = 5) * P < 0.05 (Student’s t test). Data are presented as mean ± SD. c Real-time quantitative PCR was performed to measure Col10a1 , Mmp13 , Runx2 , Opn , and Ihh mRNA expression levels in growth plates from P1 mice ( n = 3). * P < 0.05 (Student’s t test). Data are presented as mean ± SD. d Protein levels of MAPK7, COL10A1, MMP13, RUNX2, and IHH in growth plates isolated from CON and Mapk7 CKO mice at P1 were assayed by western blot. e – g , i Immunofluorescence staining of ( e ) RUNX2, ( f ) COL10A1, ( g ) MMP13, and ( i ) OSX on representative sections of proximal tibial growth plates from P1 mice. Boxed areas are magnified in the images at the bottom. h Von Kossa staining of proximal tibial growth plates. Boxed areas are magnified in the images at the bottom. Scale bar, 200 μm

Article Snippet: Mapk7 floxed mice which carried the targeted allele with a couple of loxP sites flanking exons 4, 5, 6, and 7 of Mapk7 gene were generated by Cyagen Biosciences (Suzhou, China), and schematic diagram of loxp sites was shown in Additional file : Fig. S1a.

Techniques: Labeling, Staining, Real-time Polymerase Chain Reaction, Expressing, Isolation, Western Blot, Immunofluorescence

Journal: Cell & Bioscience

Article Title: Conditional ablation of MAPK7 expression in chondrocytes impairs endochondral bone formation in limbs and adaptation of chondrocytes to hypoxia

doi: 10.1186/s13578-020-00462-8

Figure Lengend Snippet: Activation of MAPK7 in chondrocytes is essential for hypoxic adaptation and enhancement of HIF1α signaling under hypoxia. a , b Immunofluorescence staining of ( a ) MAPK7 ( b ) P-MAPK7 on representative sections of distal femoral growth plates at P1. Scale bar, 200 μm. RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone. c Western blot analysis of MAPK7 and p- MAPK7 in primary chondrocytes cultured under hypoxic conditions for 0, 1, 4, 12, or 24 h. d Immunofluorescence staining of SOX9 on representative sections of distal femoral growth plates at P1. Scale bar, 200 μm. e Western blot analysis of SOX9, COL2A1, MAPK7, HIF1α, and VEGFA in CON and Mapk7 CKO chondrocytes cultured under normoxic or hypoxic conditions for 48 h. f Western blot analysis of SOX9, COL2A1, MAPK7, p-MAPK7, HIF1α, and VEGFA protein levels in XMD8-92- or DMSO-treated primary chondrocytes cultured under normoxic or hypoxic conditions. Primary chondrocytes harvested from wild-type mouse knees were cultured in the presence of 5 μM XMD8-92 or an equal amount of DMSO under normoxia for 3 h, followed by normoxia or hypoxia for 48 h. XMD, XMD8-92. g , h mRNA levels of ( g ) Sox9 and ( h ) Col2a1 in CON and Mapk7 CKO chondrocytes cultured under normoxia or hypoxia were measured by real-time quantitative PCR (n = 3). *P < 0.05 (one-way ANOVA followed by Dunnett’s post hoc test). Data are presented as mean ± SD. i Immunofluorescence staining of HIF1α on representative proximal tibial growth plate sections from P1 mice. Scale bar, 200 μm. j mRNA levels of Hif1a and its known target genes were measured in growth plate cartilage from P1 Mapk7 CKO mice by real-time quantitative PCR. k HIF1α activity in CON and Mapk7 CKO chondrocytes cultured under normoxia or hypoxia, as evaluated by HIF1α-responsive element luciferase reporter (HRE-luc) assay ( n = 5). * P < 0.05 (one-way ANOVA followed by Dunnett’s post hoc test). Data are presented as mean ± SD. l mRNA level of hif1a in CON and Mapk7 CKO chondrocytes cultured under normoxia or hypoxia were measured by real-time quantitative PCR (n = 3). *P < 0.05 (one-way ANOVA followed by Dunnett’s post hoc test). Data are presented as mean ± SD. m Free ATP levels in CON and Mapk7 CKO chondrocytes cultured under normoxic or hypoxic conditions ( n = 4). * P < 0.05 (one-way ANOVA followed by Dunnett’s post hoc test). Data are presented as mean ± SD. n Primary chondrocytes were transfected with WT-MAPK7 or DN-MAPK7 expression vector in the presence of CA-MEK5 expression vector. Cells were harvested 48 h after transfection and protein levels of HIF1α, MAPK7 and p-MAPK7 were assayed by western blot

Article Snippet: Mapk7 floxed mice which carried the targeted allele with a couple of loxP sites flanking exons 4, 5, 6, and 7 of Mapk7 gene were generated by Cyagen Biosciences (Suzhou, China), and schematic diagram of loxp sites was shown in Additional file : Fig. S1a.

Techniques: Activation Assay, Immunofluorescence, Staining, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Transfection, Expressing, Plasmid Preparation

Journal: Cell & Bioscience

Article Title: Conditional ablation of MAPK7 expression in chondrocytes impairs endochondral bone formation in limbs and adaptation of chondrocytes to hypoxia

doi: 10.1186/s13578-020-00462-8

Figure Lengend Snippet: Targeted disruption of Mapk7 in chondrocytes inhibited vascular invasion into epiphyseal cartilage and delayed secondary ossification center (SOC) formation. a HE staining of proximal tibia and distal femur sections from P14 mice. Black dotted lines indicate the contour of the SOC. Scale bar, 200 μm. b Quantitative analysis of the areas of the tibial and femoral SOCs from P14 mice ( n = 4). * P < 0.05 (Student’s t test). Data are presented as mean ± SD. c , d Safranin O staining of proximal tibia and distal femur sections from P7 mice. Boxed areas are magnified in the images at the bottom. Scale bar, 200 μm. e , f Immunofluorescence staining of ( e ) CD31 and ( f ) COL10A1 on representative sections of distal femurs from P7 mice. Boxed areas are magnified in the images at the bottom. Scale bar, 200 μm

Article Snippet: Mapk7 floxed mice which carried the targeted allele with a couple of loxP sites flanking exons 4, 5, 6, and 7 of Mapk7 gene were generated by Cyagen Biosciences (Suzhou, China), and schematic diagram of loxp sites was shown in Additional file : Fig. S1a.

Techniques: Disruption, Staining, Immunofluorescence

Journal: Cell & Bioscience

Article Title: Conditional ablation of MAPK7 expression in chondrocytes impairs endochondral bone formation in limbs and adaptation of chondrocytes to hypoxia

doi: 10.1186/s13578-020-00462-8

Figure Lengend Snippet: Proposed role of MAPK7 in growth plate development. a The expression patterns of MAPK7 and p-MAPK7 in the growth plate. The depth of color represents the expression levels of corresponding markers. RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone. b A schematic model summarizing the role of MAPK7 in the regulation of growth plate development and modulating HIF1α signaling for hypoxic adaptation

Article Snippet: Mapk7 floxed mice which carried the targeted allele with a couple of loxP sites flanking exons 4, 5, 6, and 7 of Mapk7 gene were generated by Cyagen Biosciences (Suzhou, China), and schematic diagram of loxp sites was shown in Additional file : Fig. S1a.

Techniques: Expressing