Journal: The Journal of Experimental Medicine

Article Title: Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

doi: 10.1084/jem.20141479

Figure Lengend Snippet: Impact of Eβ on topology, structural protein deposition, and transcription of Vβ segments. (A and B) Schematics and histograms of 3C data for the Trbv5 (A) and Trbv23 (B) viewpoints (anchors) in RAG-deficient DN thymocytes or pro-B cells (see for details). (C) Published ChIP-seq profile for CTCF in RAG-deficient DN thymocytes (top; ). (C and D) ChIP-qPCR for CTCF (C) and RAD21 (D) binding at the indicated sites in WT or mEβ thymocytes versus RAG-deficient pro-B cells. Data are presented as mean values for percent input signal from at least three independent experiments (±SEM). (E) Germline transcription of Trbv segments as monitored by RT-qPCR assays in the indicated cell types. Mean values from three independent experiments after normalization to signals for Actb are shown (±SEM). Thymocytes were pooled from 5–10 mice per experiment. Significant differences between WT and mEβ samples are denoted as *, P < 0.05 (Student’s t test).

Article Snippet: The following antibodies were used: CTCF (Rockland), Rad21 (Abcam), H3ac (EMD Millipore), H3K4me2 (Abcam), H3K4me3 (Abcam), H3K9me2 (Abcam), H3K27me3 (Abcam), and IgG (Santa Cruz Biotechnology, Inc.).

Techniques: ChIP-sequencing, ChIP-qPCR, Binding Assay, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

doi: 10.1084/jem.20141479

Figure Lengend Snippet: Deletion of the 5′RC flank resolves two Trbv interaction domains. (A and B) 3C analysis of RAG-deficient thymocytes (WT, ΔPDβ1, or mEβ alleles) and pro-B cells using the Dβ2 (A, top), Trbv5 (A, bottom), and Eβ (B) viewpoints (anchors). Individual HindIII fragments are represented by alternating white and gray bars. Bold black bars indicate viewpoint locations. Schematics of Tcrb are shown on top and below primary 3C data, which are presented as mean values (±SEM) from at least three independent experiments. Thymocytes were pooled from 5–10 mice per 3C experiment. Significant differences between WT and ΔPDβ1 samples are denoted as *, P < 0.05 (Student’s t test). See for details of cartoon data summaries. Here, red shading indicates that Trbv -Dβ2 cross-linking in ΔPDb1 relative to WT alleles was unchanged (darkest red) or reduced to background levels in pro-B cells (white). (C and D) ChIP-qPCR assay for CTCF (C) and RAD21 (D) binding at sites near the indicated Trbv segments. Refer to for details. Data are presented as mean percent input (±SEM) with thymocytes pooled from at least 5–10 mice per experiment. (E) Trbv germline transcription was quantified relative to Actb by qRT-PCR from at least three independent experiments (involving one to three mice per experiment). Data are presented as mean relative expression (±SEM). Statistically significant differences are denoted as *, P < 0.05 (Student’s t test). (F) 3C assays were performed with the Trbv23 viewpoint (anchor). Schematic of Tcrb is shown on top. Data are presented as mean relative cross-linking (±SEM). Statistically significant differences between WT and ΔPDβ1 are denoted as *, P < 0.05 (Student’s t test).

Article Snippet: The following antibodies were used: CTCF (Rockland), Rad21 (Abcam), H3ac (EMD Millipore), H3K4me2 (Abcam), H3K4me3 (Abcam), H3K9me2 (Abcam), H3K27me3 (Abcam), and IgG (Santa Cruz Biotechnology, Inc.).

Techniques: ChIP-qPCR, Binding Assay, Quantitative RT-PCR, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

doi: 10.1084/jem.20141479

Figure Lengend Snippet: Identification of a Trbv tethering point in the RC flank. (A–E) 3C data for Trbv5 (A; the bottom shows ChIP-seq track for CTCF in DN thymocytes as well as locations of repetitive elements), 5′PC (B; schematic shown on top for 5′PC viewpoint; see ), Trbv3 (C), Trbv12-2 (D), and Trbv23 (E) viewpoints (anchors) in RAG-deficient DN thymocytes (WT, ΔPDβ1, or ΔminPDβ1 mice) or pro-B cells (see for details). (F) ChIP-qPCR for CTCF and RAD21 at 5′PC in the indicated cell types. All data are represented as means (±SEM) of three independent experiments. Thymocytes were pooled from 5–10 mice for each 3C or ChIP assay. Significant differences are denoted as *, P ≤ 0.05 (Student’s t test between WT and ΔPDβ1 genotypes).

Article Snippet: The following antibodies were used: CTCF (Rockland), Rad21 (Abcam), H3ac (EMD Millipore), H3K4me2 (Abcam), H3K4me3 (Abcam), H3K9me2 (Abcam), H3K27me3 (Abcam), and IgG (Santa Cruz Biotechnology, Inc.).

Techniques: ChIP-sequencing, ChIP-qPCR

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Coexpression Analysis of the EZH2 Gene Using The Cancer Genome Atlas and Oncomine Databases Identifies Coexpressed Genes Involved in Biological Networks in Breast Cancer, Glioblastoma, and Prostate Cancer

doi: 10.12659/MSM.922346

Figure Lengend Snippet: Arrays used in coexpression analysis.

Article Snippet: The C4-2 human prostate cancer cell line was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% streptomycin-penicillin (Invitrogen, Carlsbad, CA, USA), and 1% L-glutamine (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% carbon dioxide.

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Coexpression Analysis of the EZH2 Gene Using The Cancer Genome Atlas and Oncomine Databases Identifies Coexpressed Genes Involved in Biological Networks in Breast Cancer, Glioblastoma, and Prostate Cancer

doi: 10.12659/MSM.922346

Figure Lengend Snippet: The effects of siEZH2 in the C4-2 human prostate cancer cell line. ( A ) The results of cell cycle analysis showed that knockdown of EZH2 resulted in G2/M arrest ( B ) 23.98% for the control vs. 28.12% for shEZH2 (P<0.05). ( C ) Single-cell agarose gel electrophoresis for the cell transfected with control or siEZH2. ( D ) The results showed the tail length of cells transfected with shEZH2 was significantly longer than the cell in the control group (9.9 8±1.51 for the control group vs. 16.31±2.6 for the siEZH2 group). ( E ) Colony formation assay shows that knockdown of EZH2 inhibited cell proliferation when irradiated, and the results are shown in panel F . ( G ) Based on The Cancer Genome Atlas (TCGA) prostate dataset, the increased expression of EZH2, MCM4, KIAA0101, MKI67, RRM2, and CDC25A were associated with recurrence or progression after radiation. (* P<0.05; ** P<0.01)

Article Snippet: The C4-2 human prostate cancer cell line was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% streptomycin-penicillin (Invitrogen, Carlsbad, CA, USA), and 1% L-glutamine (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% carbon dioxide.

Techniques: Cell Cycle Assay, Knockdown, Control, Agarose Gel Electrophoresis, Transfection, Colony Assay, Irradiation, Expressing

Journal: Nucleic Acids Research

Article Title: The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA

doi: 10.1093/nar/gks240

Figure Lengend Snippet: Subcelluar distribution of CDK5RAP1 variants. ( a ) Representation of the two investigated CDK5RAP1 variants with mitochondrial import sequence and catalytic domain. ( b ) Live-cell images of HeLa cells transfected with C-terminally GFP-tagged CDK5RAP1-v1 and CDK5RAP1-v2. For the counterstain of mitochondria, cells were incubated with MitoTracker Red dye. The merge shows that CDK5RAP1-v1 colocalizes with mitochondria, while CDK5RAP1-v2 is distributed in both cytoplasm and nucleus. ( c ) Formaldehyde-fixed HeLa cells were stained with an antibody against the N-terminal region of CDK5RAP1-v2 (Alexa-488, green) and against CDK5 (Alexa-555, red). The localization of CDK5RAP1 is predominantly nuclear with little overlap to CDK5. ( d ) Again, fixed HeLa cells were stained with anti-CDK5RAP1 (green). For localization of nucleic acids, propidium iodide (red) was used. CDK5RAP1 shows an association with the mitotic spindle.

Article Snippet: GFP-reporter plasmids for the subcellular localization of the human CDK5RAP1 variants were obtained from Origene (RG216600 and RG208724).

Techniques: Sequencing, Transfection, Incubation, Staining

Journal: Nucleic Acids Research

Article Title: The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA

doi: 10.1093/nar/gks240

Figure Lengend Snippet: CDK5RAP1 is the human homolog of the bacterial protein MiaB. ( a ) Complementation assay in E. coli. Quantification of m 6 A, i 6 A and ms 2 i 6 A in tRNA of wild-type E. coli , a MiaB-deficient strain and a MiaB-deficient strain complemented with CDK5RAP1. m 6 A was used as internal standard and levels did not change during the experiment. i 6 A level increased in ΔMiaB and compared to WT, because of lack of conversion to ms 2 i 6 A. ΔMiaB + CDK5RAP1 shows a decreased i 6 A level in comparison to ΔMiaB, generated by the complementation. By the expression of CDK5RAP1, the ms 2 i 6 A level increases 15-fold in comparison to ΔMiaB. ( b ) siRNA knockdown in HeLa cells. Cells were transfected twice with esiRNA against CDK5RAP1. Non-coding esiRNA against EGFP served as a control. After 72 h, total RNA was extracted and the modification content was determined by LC-MS. m 6 A and i 6 A remain constant, while ms 2 i 6 A decreased ∼78%.

Article Snippet: GFP-reporter plasmids for the subcellular localization of the human CDK5RAP1 variants were obtained from Origene (RG216600 and RG208724).

Techniques: Comparison, Generated, Expressing, Knockdown, Transfection, esiRNA, Control, Modification, Liquid Chromatography with Mass Spectroscopy

Journal: Chemical biology & drug design

Article Title: TFP5 attenuates cyclin-dependent kinase 5-mediated islet β-cell damage in diabetes.

doi: 10.1111/cbdd.14235

Figure Lengend Snippet: FIGURE 1 HG conditions induced the overexpression of CDK5 and reduced insulin secretion in islet β-cells. (a) The protein expression levels of CDK5, P35/25 in MIN6 cells induced by 25 mM glucose treatment at different times were analyzed via western blotting. (b–d) Statistical analysis of CDK5 and P35/25 expression levels in MIN6 cells presented in panel A. (e) Cdk5 mRNA levels were analyzed via Rt- RCR. (f) The insulin concentration in the cell culture supernatant was detected via ELISA. *p < .05 versus HG 0 h group, #p < .05 versus LG group. MIN6 cells, mouse islet β-cells; LG, low glucose concentration; HG, high glucose concentration.

Article Snippet: Anti- CDK5 (A15844) and anti- β- tubulin (AC008) antibodies were obtained from ABclonal (Wuhan, China).

Techniques: Over Expression, Expressing, Western Blot, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Chemical biology & drug design

Article Title: TFP5 attenuates cyclin-dependent kinase 5-mediated islet β-cell damage in diabetes.

doi: 10.1111/cbdd.14235

Figure Lengend Snippet: FIGURE 3 TFP5 inhibited HG-induced CDK5 overexpression and restored insulin secretion from islet β-cells. Protein expression levels (a) and the statistical analysis of CDK5, P35/25 expression levels in MIN6 cells (b–d). (e, f) CDK5 and P35/25 in MIN6 cells were detected via immunofluorescence. (g) The insulin concentration in the cell culture supernatant was detected via ELISA. The data represent the mean ± standard deviation (n = 3). *p < .05 versus LG group, #p < .05 versus HG group. Magnification: 400×. R, roscovitine.

Article Snippet: Anti- CDK5 (A15844) and anti- β- tubulin (AC008) antibodies were obtained from ABclonal (Wuhan, China).

Techniques: Over Expression, Expressing, Immunofluorescence, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Chemical biology & drug design

Article Title: TFP5 attenuates cyclin-dependent kinase 5-mediated islet β-cell damage in diabetes.

doi: 10.1111/cbdd.14235

Figure Lengend Snippet: FIGURE 5 TFP5 inhibited CDK5 overexpression and restored insulin secretion in db/db mice. (a) Western bloting (a) and the statistical analysis of CDK5 and P35/25 in mouse islets (b–d). (e) ELISA analysis of insulin concentration in the culture supernatant of mouse islet cells. (f) Immunohistochemical staining of expression of CDK5 and P35/25 in mouse pancreatic tissues. (g, h) Quantification of CDK5 and P35/25 levels in panel f. The data represent the mean ± standard deviation (n = 3). *p < .05 versus C57BL/6J group, #p < .05 versus db/ db + SCB group. Magnification: 400×.

Article Snippet: Anti- CDK5 (A15844) and anti- β- tubulin (AC008) antibodies were obtained from ABclonal (Wuhan, China).

Techniques: Over Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Immunohistochemical staining, Staining, Expressing, Standard Deviation