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

Journal: Carcinogenesis

Article Title: Wntless (GPR177) expression correlates with poor prognosis in B-cell precursor acute lymphoblastic leukemia via Wnt signaling.

doi: 10.1093/carcin/bgu166

Figure Lengend Snippet: Fig. 4. Wls expression is critical for Wnt-induced consequent downstream genes’ activation. (A) Ablated Wls protein expression in B-cell leukemia cell lines transfected with Wls shRNAi. (B) Decrease of Wls expression in Reh cells infected with Wls shRNAi was showed to abrogate canonical Wnt signaling induced by Wnt-3a stimulation. (C) Inhibition of c-Myc, cyclin D1 and Skp2 expression induced by Wnt-1 and Wnt-3a in the Reh cells infected with Wntless shRNAi. Arrow head, cyclin D1. (D) Reh cells infected with Wntless shRNAi showed a decrease in pro-inflammatory cytokines (IL-1β, IL-6, IL-8, GM-CSF and cox-2) expression induced by Wnt-3a treatment. Each experiment was repeated at least three. The results are shown as means ± SD. (***p < 0.001).

Article Snippet: The primary antibodies used in this study were β-actin polyclonal antibody (1:2000, Santa Cruz, I-19), cIAP, Bcl-xL polyclonal antibody (1:2000, Santa Cruz), caspase 3, caspase 8, c-Myc (1:1000, Upstate), cyclin D1 (SeroTec.), FITC-conjugated anti-mouse, alkaline phosphatase-conjugated anti-rabbit antibodies (1:500, Jackson ImmunoResearch Lab.) and Wntless antibody (1:2000, Biolegend) (22).

Techniques: Expressing, Activation Assay, Transfection, Infection, Inhibition

Journal: Cancer Cell International

Article Title: LncRNA SNHG12 regulates the radiosensitivity of cervical cancer through the miR-148a/CDK1 pathway

doi: 10.1186/s12935-020-01654-5

Figure Lengend Snippet: Effects of SNHG12 knockdown on radiosensitivity in CC cells. SiHa and Hela cells were transfected with si-SNHG12 or si-con. a , b Clonogenic assay was performed to assess the survival fractions of SiHa and Hela cells. c , d The apoptosis of SiHa and Hela cells treated with different doses of radiation (0 Gy and 2 Gy) was determined by flow cytometry. e , f The caspase-3 activity of SiHa and Hela cells treated with different doses of radiation (0 Gy and 2 Gy) was detected by Caspase-3 Activity Assay Kit. g , h Flow cytometry was used to examine the cell cycle distribution in SiHa and Hela cells treated with different doses of radiation (0 Gy and 2 Gy). i , j WB analysis was performed to measure the protein expression of CCND1 in SiHa and Hela cells treated with different doses of radiation (0 Gy and 2 Gy). * P < 0.05

Article Snippet: After blocking with non-fat milk for 2 h, the membranes were incubated with primary antibodies CDK1 (1:500, BA0027-2, Boster, Wuhan, China), CCND1 (1:2000, PB0403, Boster), γ-H2AX (1:1000, AF5836, Beyotime) and β-actin (1:5000, BA2305, Boster) overnight at 4 °C, followed by incubating with secondary antibody (1:10,000, BA1056, Boster) for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagent (Millipore).

Techniques: Transfection, Clonogenic Assay, Flow Cytometry, Activity Assay, Caspase-3 Activity Assay, Expressing

Journal: Cancer Cell International

Article Title: LncRNA SNHG12 regulates the radiosensitivity of cervical cancer through the miR-148a/CDK1 pathway

doi: 10.1186/s12935-020-01654-5

Figure Lengend Snippet: Effects of miR-148a inhibitor and CDK1 overexpression on radiosensitivity, apoptosis and cell cycle in CC cells. SiHa and Hela cells were transfected with si-SNHG12 and anti-miR-148a or CDK1 overexpression plasmid. a , b The survival fractions of SiHa and Hela cells were assessed by clonogenic assay. c , d Flow cytometry was used to determine the apoptosis of SiHa and Hela cells treated with 2 Gy radiation. e , f Caspase-3 Activity Assay Kit was used to detect the caspase-3 activity of SiHa and Hela cells treated with 2 Gy radiation. g , h The cell cycle distribution in SiHa and Hela cells treated with 2 Gy radiation was determined using flow cytometry. i , j The protein expression of CCND1 in SiHa and Hela cells treated with 2 Gy radiation was tested by WB analysis. * P < 0.05

Article Snippet: After blocking with non-fat milk for 2 h, the membranes were incubated with primary antibodies CDK1 (1:500, BA0027-2, Boster, Wuhan, China), CCND1 (1:2000, PB0403, Boster), γ-H2AX (1:1000, AF5836, Beyotime) and β-actin (1:5000, BA2305, Boster) overnight at 4 °C, followed by incubating with secondary antibody (1:10,000, BA1056, Boster) for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagent (Millipore).

Techniques: Over Expression, Transfection, Plasmid Preparation, Clonogenic Assay, Flow Cytometry, Caspase-3 Activity Assay, Activity Assay, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Magnolol alleviates hypoxia-induced pulmonary vascular remodeling through inhibition of phenotypic transformation in pulmonary arterial smooth muscle cells.

doi: 10.1016/j.biopha.2022.113060

Figure Lengend Snippet: Fig. 3. Effects of magnolol on the phenotypic transformation of pulmonary arteries in hypoxia-induced PH rats. (A) Representative images of immunohistochemistry staining for Ki67 (cell proliferation markers) in each group; (B-F) The protein expression of SM-22α, CyclinD1, OPN, and PCNA in the pulmonary artery of rats; Data are expressed as mean ± SD. +Mag(L): hypoxia+ magnolol (10 mg/kg/d); +Mag(H): hypoxia+ magnolol (20 mg/kg/d). **P < 0.01 vs Control; #P < 0.05, ##P < 0.01 vs Hypoxia.

Article Snippet: The PVDF membranes were blocked with 5% BSA and then incubated with the following primary antibodies: collagen I (No. ab34710, 1:1000, Abcam), collagen III (No. ab7778, 1:1000, Abcam), SM-22α (No. ab14106, 1:1000, Abcam), α-SMA (No. 19245, 1:1000, Cell Signaling Technology), OPN (No. ab91655, 1:1000, Abcam), PCNA (No. AF0261, 1:1000, Beyotime), calponin (No. AF1393, 1:1000, Beyotime), CyclinD1 (No. BM4272, 1:2000, BOSTER), p-JAK2 (No. ab32101, 1:1000, Abcam), JAK2 (No. AF1489, 1:2000, Beyotime), p-STAT3 (No. 13332, 1:1000, Signalway Antibody), STAT3 (No. 41465, 1:1000, Signalway Antibody), α-tubulin (No. sc-5286, 1:200, Santa Cruz) and β-actin (No. AF5001, 1:1000, Beyotime).

Techniques: Transformation Assay, Immunohistochemistry, Staining, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Magnolol alleviates hypoxia-induced pulmonary vascular remodeling through inhibition of phenotypic transformation in pulmonary arterial smooth muscle cells.

doi: 10.1016/j.biopha.2022.113060

Figure Lengend Snippet: Fig. 5. Effects of magnolol on the proliferation, migration, and phenotypic transformation of PASMCs induced by hypoxia. (A) The cell viability was detected by the CCK-8 array. (B-C) Representative pictures of EdU staining. (D-E) The cell migration ability was detected by Transwell and scratch arrays. (F-G) The protein expression of SM-22α, α-SMA, Calponin, OPN, CyclinD1, PCNA, and collagen I in PASMCs. Data are expressed as mean ± SD. +Mag(L): hypoxia+ magnolol (10 mg/ kg/d); +Mag(H): hypoxia+ magnolol (20 mg/kg/d). **P < 0.01 vs Control; #P < 0.05, ##P < 0.01 vs Hypoxia.

Article Snippet: The PVDF membranes were blocked with 5% BSA and then incubated with the following primary antibodies: collagen I (No. ab34710, 1:1000, Abcam), collagen III (No. ab7778, 1:1000, Abcam), SM-22α (No. ab14106, 1:1000, Abcam), α-SMA (No. 19245, 1:1000, Cell Signaling Technology), OPN (No. ab91655, 1:1000, Abcam), PCNA (No. AF0261, 1:1000, Beyotime), calponin (No. AF1393, 1:1000, Beyotime), CyclinD1 (No. BM4272, 1:2000, BOSTER), p-JAK2 (No. ab32101, 1:1000, Abcam), JAK2 (No. AF1489, 1:2000, Beyotime), p-STAT3 (No. 13332, 1:1000, Signalway Antibody), STAT3 (No. 41465, 1:1000, Signalway Antibody), α-tubulin (No. sc-5286, 1:200, Santa Cruz) and β-actin (No. AF5001, 1:1000, Beyotime).

Techniques: Migration, Transformation Assay, CCK-8 Assay, Staining, Expressing, Control