Journal: Cell Death & Disease

Article Title: Disruption of the FasL/Fas axis protects against inflammation-derived tumorigenesis in chronic liver disease

doi: 10.1038/s41419-019-1391-x

Figure Lengend Snippet: a Representative TUNEL staining of 8-week-old NEMO f/f , NEMO Δhepa , Fas lpr , and NEMO Δhepa /Fas lpr livers. b TUNEL-positive cells were quantified and graphed. c Proliferation was determination by Ki-67-positive cells immunofluorescence. d Ki-67-positive cells were quantified and graphed. e Expression of PCNA, cleaved Caspase-3 (CC3), and RIPK1 was analysed by immunoblotting of whole liver extracts. GAPDH served as a loading control. Results are expressed as mean ± SEM ( n = 8 livers, p < 0.001–0.05)

Article Snippet: Isolated protein samples were probed with antibodies against RIPK1 (#5389) (Pro-Sci, Poway, CA, USA), Cleaved Caspase-3 (Asp175) (Cell Signaling Technology, Massachusetts, USA), PCNA (clone PC10) (Dianova GmbH, Hamburg, Germany), and GAPDH (MCA4739; 1:5000; AbD SeroTec, Düsseldorf, Germany).

Techniques: TUNEL Assay, Staining, Immunofluorescence, Expressing, Western Blot

Journal:

Article Title: T-cell factor 4 (Tcf7l2) maintains proliferative compartments in zebrafish intestine

doi: 10.1038/sj.embor.7401071

Figure Lengend Snippet: The absence of cycling cells in tcf4exI/exI at 6 weeks post-fertilization. (A) Haematoxylin and eosin (HE) staining of tcf4wt/wt zebrafish intestine with the proximal (Prox), middle (Mid) and distal (Dis) parts magnified respectively in lower panels. (B) PCNA staining on consecutive paraffin sections of wild-type (wt) fish. The arrows in the magnified lower panels indicate PCNA+ cells in the interfold pockets. (C) HE staining of tcf4exI/exI intestine. (D) PCNA staining on consecutive sections depicting absent proliferation in the middle and distal intestinal portions. Note that proliferation in the proximal intestine is maintained (arrows and arrowheads in the magnified proximal intestine in (D)). PCNA, proliferating cell nuclear antigen; wpf, weeks post-fertilization. Scale bars: top panels (A–D), 250 μm; middle and bottom panels (A–D), 50 μm.

Article Snippet: IHC staining with antibody for PCNA (PC10; Euro Diagnostica, Arnhem, The Netherlands) and BrdU (Becton Dickinson, Franklin Lakes, NJ, USA) was carried out as described previously ( van de Wetering et al , 2002 ).

Techniques: Staining

Journal:

Article Title: T-cell factor 4 (Tcf7l2) maintains proliferative compartments in zebrafish intestine

doi: 10.1038/sj.embor.7401071

Figure Lengend Snippet: Gut phenotype of homozygous Tcf4 mutant ‘escaper' fish. (A) Escaper compared with sibling is reduced in size. (B) Western blot analysis of Tcf4 protein from brain and fin tissues showing the absence of protein in escapers. (C) PCNA IHC in wild type (wt). The lower panel is a magnification of the boxed area; the arrows point to PCNA+ cells at the base of the folds. (D) PCNA IHC on Tcf4 escaper fish showing large areas of intestinal epithelium with loss of proliferation. The three lower panels are magnifications of the boxed areas. The arrows point to severely affected areas or to rare PCNA+ patches unequally distributed throughout the epithelial folds. The dashed line indicates the flat epithelial layer in this fish. IHC, immunohistochemistry; PCNA, proliferating cell nuclear antigen; Tcf-4, T-cell factor.

Article Snippet: IHC staining with antibody for PCNA (PC10; Euro Diagnostica, Arnhem, The Netherlands) and BrdU (Becton Dickinson, Franklin Lakes, NJ, USA) was carried out as described previously ( van de Wetering et al , 2002 ).

Techniques: Mutagenesis, Western Blot, Immunohistochemistry

Journal: Journal of Translational Medicine

Article Title: VEGF111b, a C-terminal splice variant of VEGF-A and induced by mitomycin C, inhibits ovarian cancer growth

doi: 10.1186/s12967-015-0522-0

Figure Lengend Snippet: VEGF111b overexpression inhibited growth of SKOV3 tumor xenografts. After transfection with empty lentivirus and VEGF111b lentivirus, SKOV3 cells were injected subcutaneously into the upper right flank region of nude mice. Tumor volume was calculated every 4 days for 20 days. ( a ) Tumor size of the 20th day tumor incubation was displayed. ( b ) Tumor volume was measured with a caliper rule every 4 days. Datas were presented as the mean tumor volumes of mice in both VEGF111b and empty lentivirus vector groups on the days incubation (*** p < 0.001). ( c ) Average tumor weight was shown at the end of the experiments (*** p < 0.001). ( d ) Tumor sections were stained positive for Ki67 and PCNA. The positive products localized in nucleus (magnification, 200×). ( e ) Tumor sections were stained positive for CD31 and VEGF. The positive products localized in cytoplasm (magnification, 200×). Bar heights represent average of data and bars represent the SEM of data. P value <0.05 was considered statisticall

Article Snippet: PCNA mAb (PC10, 1:100), Ki67 mAb (7B11, 1:100), VEGF pAb (ZA-0580, 1:100) and CD31 mAb (1A10, 1:75) were purchased from ZSGB-BIO (Beijing, China).

Techniques: Over Expression, Transfection, Injection, Incubation, Plasmid Preparation, Staining

Journal: The Journal of Cell Biology

Article Title: SET8 is degraded via PCNA-coupled CRL4(CDT2) ubiquitylation in S phase and after UV irradiation

doi: 10.1083/jcb.201009076

Figure Lengend Snippet: CRL-CDT2 targets SET8 for degradation in S phase. (A) SET8 protein level is low in S phase. U2-OS cells were treated with aphidicolin for 24 h and released for 4 h. The indicated cells were treated with MG132 1 h before harvest. Cells were processed for immunoblotting with the indicated antibodies. Vinculin was used as a loading control. Async, asynchronous control cells. Protein levels of SET8 are quantified and depicted below the figure. (B) SET8 contains a conserved PIP box degron. Cluster alignment of SET8 from different species show a highly conserved stretch of amino acids. When this stretch was aligned with CDT1 and p21, it revealed a conserved PIP box degron. (C) SET8 is mutually exclusive with PCNA and CDT2 on chromatin. U2-OS cells were synchronized with nocodazole, released into fresh medium, and collected at the indicated time points. Chromatin-bound fractions and whole cell extracts (WCE) were analyzed by immunoblotting with the indicated antibodies. (D) DDB1 and CDT2 depletion stabilizes SET8. U2-OS cells were treated with the indicated siRNAs 12 h before aphidicolin treatment. 24 h after aphidicolin treatment, cells were washed and released into fresh medium for 3 h and processed for immunoblotting with the indicated antibodies. Actin was used as a loading control. (E) CDT2 is important for the turnover of SET8 in S phase. U2-OS cells were treated with siRNA 12 h before aphidicolin treatment. 24 h after aphidicolin treatment, cells were washed and released into fresh medium for 3 h. Cells were then treated with cycloheximide (CHX) as indicated and processed for immunoblotting with the indicated antibodies. The blots were quantified using an image reader. Representative data from three similar experiments are shown. Data are presented as mean + SD. (F) SET8 is ubiquitylated in vivo mediated by CDT2. HEK293 cells were depleted for CDT2 using siRNA 12 h before transfection with Flag-HA-SET8 and HIS-ubiquitin (HIS-UBQ) as indicated. Cells were processed for immunoprecipitation using cobalt beads and immunoblotted with the indicated antibodies.

Article Snippet: PCNA (PC10) was purchased from Abnova, and CDT2 (NB-10-40840) was obtained from Novus Biologicals.

Techniques: Western Blot, Control, In Vivo, Transfection, Ubiquitin Proteomics, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: SET8 is degraded via PCNA-coupled CRL4(CDT2) ubiquitylation in S phase and after UV irradiation

doi: 10.1083/jcb.201009076

Figure Lengend Snippet: The interaction between PCNA and SET8 is important for CDT2-mediated degradation of SET8. (A) Schematic presentation of SET8. Two PIP boxes (PCNA-interacting domains) have been identified N terminally to the SET domain. (B) Mutation of PIP2 results in stabilization of SET8. HEK293 cells were transfected with plasmids expressing Flag-HA-SET8, Flag-HA-SET8-*PIP1, and Flag-HA-SET8-*PIP2. Cells were treated with cycloheximide and processed for immunoblotting with the indicated antibodies. MCM7 was used as a loading control. (C) Increased levels of PCNA result in degradation of WT SET8 but not SET8-*PIP2. HEK293 cells were transfected as illustrated and processed for immunoblotting with the indicated antibodies. (D) In vivo ubiquitylation of SET8-*PIP2 is reduced compared with SET8 WT. HEK293 cells were transfected with Flag-HA-SET8, Flag-HA-SET8-*PIP2, and HIS-ubiquitin as indicated. Cells were processed for immunoprecipitation using cobalt beads and immunoblotted with the indicated antibodies. (E) SET8 is ubiquitylated on chromatin. U2-OS cells were either treated with MG132 for 3 h or left untreated. Soluble and chromatin-bound fractions were analyzed by immunoblotting with SET8 antibody. *, nonspecific band. (F) SET8 WT and PCNA colocalize on chromatin after MG132 treatment. U2-OS cells expressing Flag-HA-SET8 or Flag-HA-SET8-*PIP2 were seeded on coverslips and treated with MG132 for 3 h before preextraction and fixation. Cells were stained with antibodies against HA and PCNA. Bars, 10 µm.

Article Snippet: PCNA (PC10) was purchased from Abnova, and CDT2 (NB-10-40840) was obtained from Novus Biologicals.

Techniques: Mutagenesis, Transfection, Expressing, Western Blot, Control, In Vivo, Ubiquitin Proteomics, Immunoprecipitation, Staining

Journal: The Journal of Cell Biology

Article Title: SET8 is degraded via PCNA-coupled CRL4(CDT2) ubiquitylation in S phase and after UV irradiation

doi: 10.1083/jcb.201009076

Figure Lengend Snippet: siRNA oligonucleotides used in this study

Article Snippet: PCNA (PC10) was purchased from Abnova, and CDT2 (NB-10-40840) was obtained from Novus Biologicals.

Techniques: