Journal: Molecular Medicine Reports

Article Title: Analysis of different components in the peritumoral tissue microenvironment of colorectal cancer: A potential prospect in tumorigenesis

doi: 10.3892/mmr.2016.5584

Figure Lengend Snippet: Expression levels of CD1, CD133, CK18, α-SMA and vimentin indicated by immunohistochemical staining. Expression levels of (A) CD1, (B) CD133, (C) CK18, (D) α-SMA and (E) vimentin, in samples no. 1, 2 and 3 (left to right). CD1, cyclin D1; CD133, cluster of differentiation 133; CK18, cytokeratin 18; α-SMA, α-smooth muscle actin.

Article Snippet: The primary antibodies used in the IHC staining in the present study were as follows: Rabbit polyclonal against E-cadherin (Abcam, Cambridge, MA, USA; cat. no. ab15148), rat monoclonal against CRB3 (Abcam; cat. no. ab180835), rabbit monoclonal against α-SMA (Abcam; cat. no. 124964), rabbit polyclonal against PAR-3 (Bioss, Inc., Woburn, MA, USA; cat. no. bs-9510R), rabbit polyclonal against Hyal-1 (Abcam; cat. no. ab103977), mouse monoclonal against Col-I (Abcam; cat. no. ab6308), rabbit polyclonal against cytokeratin 18 (CK18; Bioss, Inc.; cat. no. bs-1339R), rabbit polyclonal against vimentin (Bioss, Inc.; cat. no. bs-8533R), rabbit polyclonal against CD1 (Bioss, Inc.; cat. no. bs-0623R) and rabbit polyclonal against CD133 (Bioss, Inc.; cat. no. bs-0395R).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Molecular Medicine Reports

Article Title: Analysis of different components in the peritumoral tissue microenvironment of colorectal cancer: A potential prospect in tumorigenesis

doi: 10.3892/mmr.2016.5584

Figure Lengend Snippet: CCA value of ten biomarkers in samples No. 1, No. 2 and No. 3. Demonstrated by the CCA value, the expression levels of E-cadherin, CK18, Crumbs-3 and Par-3 in sample No. 3 were lower compared with sample No. 1 (P<0.05). The expression of CK18 in sample No. 2 was lower than in sample No. 3 (P<0.0001). The expression levels of Hyal-1, Col-I, vimentin, CD1, CD133 and α-SMA in sample No. 3 were higher than in sample No. 1 (P<0.05), however, the expression levels of Hyal-1, Col-I, α-SMA in sample No. 3 were lower than that in sample No. 2, the result for Hyal-1 was not significant. CCA, corrected absorbance value; CK18, cytokeratin 18; Par-3, proteinase activated receptor 3; Hyal-1, hyaluronidase 1; Col-I, collagen type I; CD1, cyclin D1; CD133, cluster of differentiation 133; α-SMA, α-smooth muscle actin.

Article Snippet: The primary antibodies used in the IHC staining in the present study were as follows: Rabbit polyclonal against E-cadherin (Abcam, Cambridge, MA, USA; cat. no. ab15148), rat monoclonal against CRB3 (Abcam; cat. no. ab180835), rabbit monoclonal against α-SMA (Abcam; cat. no. 124964), rabbit polyclonal against PAR-3 (Bioss, Inc., Woburn, MA, USA; cat. no. bs-9510R), rabbit polyclonal against Hyal-1 (Abcam; cat. no. ab103977), mouse monoclonal against Col-I (Abcam; cat. no. ab6308), rabbit polyclonal against cytokeratin 18 (CK18; Bioss, Inc.; cat. no. bs-1339R), rabbit polyclonal against vimentin (Bioss, Inc.; cat. no. bs-8533R), rabbit polyclonal against CD1 (Bioss, Inc.; cat. no. bs-0623R) and rabbit polyclonal against CD133 (Bioss, Inc.; cat. no. bs-0395R).

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Analysis of different components in the peritumoral tissue microenvironment of colorectal cancer: A potential prospect in tumorigenesis

doi: 10.3892/mmr.2016.5584

Figure Lengend Snippet: Heatmap of biomarker expression in the present study according to the CCA value of samples No. 1, No. 2 and No. 3. The heatmap diagram indicates the result of the two-way hierarchical clustering of markers and samples. Each row represents a marker and each column represents a sample. The marker clustering tree is presented on the right, and the sample clustering tree at the top. Cluster analysis arranges samples and markers into groups based on their CCA values, which allows formation of hypotheses regarding the association between markers and samples. The color of each pattern indicates the CCA value, green to red is −0.8 to 1.0. CCA, corrected absorbance value; α-SMA, α-smooth muscle actin; CD1, cyclin D1; CD133, cluster of differentiation 133; PAR-3, proteinase activated receptor 3; Col-I, collagen type I; Hyal-1, hyaluronidase 1.

Article Snippet: The primary antibodies used in the IHC staining in the present study were as follows: Rabbit polyclonal against E-cadherin (Abcam, Cambridge, MA, USA; cat. no. ab15148), rat monoclonal against CRB3 (Abcam; cat. no. ab180835), rabbit monoclonal against α-SMA (Abcam; cat. no. 124964), rabbit polyclonal against PAR-3 (Bioss, Inc., Woburn, MA, USA; cat. no. bs-9510R), rabbit polyclonal against Hyal-1 (Abcam; cat. no. ab103977), mouse monoclonal against Col-I (Abcam; cat. no. ab6308), rabbit polyclonal against cytokeratin 18 (CK18; Bioss, Inc.; cat. no. bs-1339R), rabbit polyclonal against vimentin (Bioss, Inc.; cat. no. bs-8533R), rabbit polyclonal against CD1 (Bioss, Inc.; cat. no. bs-0623R) and rabbit polyclonal against CD133 (Bioss, Inc.; cat. no. bs-0395R).

Techniques: Biomarker Assay, Expressing, Marker

Journal: Nature Communications

Article Title: LncRNA DILA1 inhibits Cyclin D1 degradation and contributes to tamoxifen resistance in breast cancer

doi: 10.1038/s41467-020-19349-w

Figure Lengend Snippet: a Western blotting showing the levels of Cyclin D1, phosphorylated Cyclin D1 (p-D1) (Thr286), p-D1 (Ser-90), and GSK3β in MCF7-Pa and MCF-Re cells. b – d Western blotting showing the levels of Cyclin D1, p-D1(Thr286), and GSK3β in DILA1-silenced MCF-Re cells ( b ), in DILA1-overexpressed MCF7-Pa cells ( c ), and in DILA1 and constitutively activated GSK3β (GSK3β-CA) both overexpressing MCF7-Pa cells ( d ). GAPDH was used as a loading control. e – g Western blotting showing the levels of nuclear and cytoplasmic Cyclin D1 in MCF7-Pa and MCF-Re cells ( e ), in DILA1-silenced MCF-Re cells ( f ), and in DILA1-overexpressed MCF7-Pa cells ( g ). GAPDH and Lamin A/C was used as cytoplasmic and nuclear controls, respectively. h RNA pull-down showing the interaction between Cyclin D1 and full-length or serial truncation mutants of DILA1. i eCLIP-qPCR assay indicating the exact DILA1 region responsible for Cyclin D1 binding in MCF7-Re cells (bottom). A schematic diagram of DILA1 primers (P1–P11) designed for eCLIP-qPCR, covering the full length of DILA1 (top). j RNA pull-down showing the interaction between Cyclin D1 and DILA1 or DILA mutant with deletion of hairpin A (DILA1-mA). k RNA pull-down showing the interaction between DILA1 and HA-tagged full length or truncation mutants (FL (1–295), T1 (20–295), T2 (91–295), and T3 (1–256)) of Cyclin D1 proteins in MCF-7 cells. AS (antisense) was used as a negative control. l In vitro kinase assay showing the phosphorylation of Cyclin D1 at Thr286 by active GSK3β-CA or inactive GSK3β-KD in the absence or presence of DILA1 or DILA1-mA. m RIP-qPCR of DILA1 immunoprecipitated with anti-HA antibody or IgG in MCF7-Re cells with ectopically expressed HA-Cyclin D1 or HA-Cyclin D1(T286A). n eCLIP-qPCR assay in MCF7-Re cells with HA-Cyclin D1 or HA-Cyclin D1(T286A) overexpressed. For i , m , n , n = 3 biologically independent experiments, means ± s.d. are shown, and p values were determined by two-tailed Student’s test. o Western blotting showing the levels of Cyclin D1 and p-D1(Thr286) in MCF7-Re cells transfected with control vector or vector expressing Cyclin D1(T286A) and then transfected with NC or DILA1-ASOs. For a – h , j – l , o , representative images of three biologically independent experiments are shown.

Article Snippet: Tissues were incubated with goat serum for 30 min at room temperature and then with anti-CyclinD1 (NBP2-32840, Novus, dilution 1:100), anti-ki67 (ZM0166, ZSGB-BIO, ready to use), anti-phospho-Cyclin D1 (Thr286) (STJ90457, St John’s Laboratory, dilution 1:50), and anti-phospho-Rb (Ser780) (ab47763, Abcam, dilution 1:100) antibodies at 4 °C overnight, respectively.

Techniques: Western Blot, Control, Binding Assay, Mutagenesis, Negative Control, In Vitro, Kinase Assay, Phospho-proteomics, Immunoprecipitation, Two Tailed Test, Transfection, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: LncRNA DILA1 inhibits Cyclin D1 degradation and contributes to tamoxifen resistance in breast cancer

doi: 10.1038/s41467-020-19349-w

Figure Lengend Snippet: MCF7-Re cells were inoculated into the mammary fat pads of NOD/SCID mice. When tumors became palpable, MCF7-Re tumors were injected with NC or DILA1-ASOs intratumorally. The tumor picture ( a ) and the tumor growth curves ( b ) are shown and compared among the groups. c Immunohistochemistry (IHC) staining of Ki67, Cyclin D1, p-D1(Thr286), and p-Rb(Ser780) and in situ hybridization (ISH) staining of DILA1 in the tumors. Representative images of six xenografts from each group are shown. d – h The quantification of IHC scores of Ki67( d ), Cyclin D1( e ), p-D1(Thr286) ( f ), and p-Rb(Ser780) ( g ) and ISH scores of DILA1 ( h ). For b , d – h , n = 6 mice per group, means ± s.d. are shown, and p values were determined by two-tailed Student’s test. Scale bars represents 50 µm.

Article Snippet: Tissues were incubated with goat serum for 30 min at room temperature and then with anti-CyclinD1 (NBP2-32840, Novus, dilution 1:100), anti-ki67 (ZM0166, ZSGB-BIO, ready to use), anti-phospho-Cyclin D1 (Thr286) (STJ90457, St John’s Laboratory, dilution 1:50), and anti-phospho-Rb (Ser780) (ab47763, Abcam, dilution 1:100) antibodies at 4 °C overnight, respectively.

Techniques: Injection, Immunohistochemistry, In Situ Hybridization, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: LncRNA DILA1 inhibits Cyclin D1 degradation and contributes to tamoxifen resistance in breast cancer

doi: 10.1038/s41467-020-19349-w

Figure Lengend Snippet: a ISH staining for DILA1 and IHC staining for Cyclin D1, p-D1(Thr286), p-Rb(Ser780), and Ki67 in breast cancer specimen from ER-positive patients with or without relapse. Representative images of tumor specimens from ER-positive patients with ( n = 36) or without relapse ( n = 154) are shown. Scale bars, 50 μm. b – e The distribution of Cyclin D1, p-D1(Thr286), p-Rb(Ser780), and Ki67 determined by the staining index in the high ( n = 109) or low ( n = 81) DILA1-expressing groups. n = 190, p values were determined by two-tailed Chi-square test. f , g The distribution of DILA1 ( f ) and Cyclin D1 ( g ) by the staining index in the samples of ER-positive patients with ( n = 36) or without relapse ( n = 154). n = 190, means ± s.d. are shown, p values were determined by two-tailed Student’s test. h , i Relapse-free survival of ER-positive breast cancer patients with low and high DILA1 ( h ) or Cyclin D1 ( i ) was analyzed by Kaplan–Meier plots. n = 190, p values were determined by two-tailed log-rank test.

Article Snippet: Tissues were incubated with goat serum for 30 min at room temperature and then with anti-CyclinD1 (NBP2-32840, Novus, dilution 1:100), anti-ki67 (ZM0166, ZSGB-BIO, ready to use), anti-phospho-Cyclin D1 (Thr286) (STJ90457, St John’s Laboratory, dilution 1:50), and anti-phospho-Rb (Ser780) (ab47763, Abcam, dilution 1:100) antibodies at 4 °C overnight, respectively.

Techniques: Staining, Immunohistochemistry, Expressing, Two Tailed Test

Journal: FEBS letters

Article Title: Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

doi: 10.1016/j.febslet.2004.09.040

Figure Lengend Snippet: Fig. 1. Synthetic DNA sequences. Capital letters in bold indicate sequences from human tRNAtyr gene (A and B). The intron sequence of tRNAtyr (plain capital letters in B) was substituted with a 10 bp oligonucleotide (plain capital letters in A), generating two restriction sites (StuI and BamHI) to ease insertion of any antisenses, not limited to cyclin D1 as in this work. Letters in lower case represent the promoter sequence of U6 SnRNA gene (A), which enhances the transcription efficiency of subsequent pre-tRNA. The U6 SnRNA promoter and the tRNA gene were spaced by two copies of tet operator 2 sequence that serve as the binding sites for two molecules of the Tet repressor protein, so as to block the transcription of pre-tRNA from both U6 SnRNA and tRNA promoters unless with tetracycline inducement.

Article Snippet: For cyclin D1 detection, the sections were hydrated, and probed with an anti-cyclin D1 monoclonal antibody as mentioned above, and then a biotinized anti-mouse antibody and SABC complex (streptavidin–biotin-peroxidase, Boster Biotech, Wuhan, China) in sequence.

Techniques: Sequencing, Binding Assay, Blocking Assay

Journal: FEBS letters

Article Title: Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

doi: 10.1016/j.febslet.2004.09.040

Figure Lengend Snippet: Fig. 2. In vitro transcription and splicing of pre-tRNA. With U6 SnRNA promoter, pre-tRNAs in pUT-tyr and pUT-ccnd1 were transcribed more efficiently than pUC-tyr control. The 20 bp anti-cy- clin D1 insert replacing the intron sequence of tRNAtyr gene was correctly spliced out (pUT-ccnd1) as well as the native intron of tRNAtyr gene (pUT-tyr).

Article Snippet: For cyclin D1 detection, the sections were hydrated, and probed with an anti-cyclin D1 monoclonal antibody as mentioned above, and then a biotinized anti-mouse antibody and SABC complex (streptavidin–biotin-peroxidase, Boster Biotech, Wuhan, China) in sequence.

Techniques: In Vitro, Control, Sequencing

Journal: FEBS letters

Article Title: Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

doi: 10.1016/j.febslet.2004.09.040

Figure Lengend Snippet: Fig. 3. Western blot analysis of cyclin D1 expression. Stable blastici- din-resistant H22 cell lines were created by transfection with pTRUT- tyr or pTRUT-ccnd1. Both of the two cell lines together with un-transfected H22 control cells were induced with tetracycline for 24 h. Then, cyclin D1 expression was analyzed by Western blot. b-Actin expression was also analyzed as a control. In the figure, cyclin D1 expression was significantly reduced by cyclin D1 antisense RNA. Whereas intron of pre-tRNAtyr generated in the same way had no effect on cyclin D1 expression.

Article Snippet: For cyclin D1 detection, the sections were hydrated, and probed with an anti-cyclin D1 monoclonal antibody as mentioned above, and then a biotinized anti-mouse antibody and SABC complex (streptavidin–biotin-peroxidase, Boster Biotech, Wuhan, China) in sequence.

Techniques: Western Blot, Expressing, Transfection, Control, Generated

Journal: FEBS letters

Article Title: Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

doi: 10.1016/j.febslet.2004.09.040

Figure Lengend Snippet: Fig. 6. Immunohistochemical analysis of cyclin D1 expression and apoptosis in s.c. tumor xenografts with and without plasmid pUT-ccnd1 injection. Preformed tumor xenografts were harvested at day 17 after 6 times of plasmid injection. Cyclin D1 expression (A) and apoptosis (B) were analyzed as described in Section 2. (a) s.c. tumors treated with TE only; (b) s.c. tumors treated with plasmid pUT-ccnd1.

Article Snippet: For cyclin D1 detection, the sections were hydrated, and probed with an anti-cyclin D1 monoclonal antibody as mentioned above, and then a biotinized anti-mouse antibody and SABC complex (streptavidin–biotin-peroxidase, Boster Biotech, Wuhan, China) in sequence.

Techniques: Immunohistochemical staining, Expressing, Plasmid Preparation, Injection

Journal: FEBS letters

Article Title: Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

doi: 10.1016/j.febslet.2004.09.040

Figure Lengend Snippet: Fig. 5. Effect of plasmid administration on tumor growth. Four days after inoculation of H22 cells (Day 5), pUT-ccnd1 plasmid was injected into the s.c. tumor xenografts at multiple sites until day 11 or day 15. Tumor volumes were determined by bidimensional caliper measure- ments and were presented as the mean tumor volume (n ¼ 8). The bars stand for standard deviation. (*) Significant (P < 0:05) volume differ- ence between plasmid treated tumors and TE treated control tumors. ðmÞ Significant (P < 0:05) difference between short-term plasmid treated tumors (to day 11) and long-term treated tumors (to day 15). Fig. 4. Cell proliferation assay by MTT method. H22 cells were stained by MTT. Proliferation of the cells was significantly (P < 0:05) reduced by the antisense RNA. The bars represent the standard deviations calculated from three repeated experiments.

Article Snippet: For cyclin D1 detection, the sections were hydrated, and probed with an anti-cyclin D1 monoclonal antibody as mentioned above, and then a biotinized anti-mouse antibody and SABC complex (streptavidin–biotin-peroxidase, Boster Biotech, Wuhan, China) in sequence.

Techniques: Plasmid Preparation, Injection, Standard Deviation, Control, Proliferation Assay, Staining