Journal: Nanoscale

Article Title: Enhanced penetrative siRNA delivery by a nanodiamond drug delivery platform against hepatocellular carcinoma 3D models.

doi: 10.1039/d1nr03502a

Figure Lengend Snippet: Fig. 6 Knockdown efficiency of SALL4 siRNA packaged in lipo or ND in SNU398 spheroids, with NTC (non-targeting control) siRNA as the negative control. (A) SALL4 mRNA level after siRNA treatment in SNU398 spheroids. (B) Western blot of SALL4 and downstream genes, AKT, phosphorylated AKT (pAKT) and Cyclin D2 expression level after knockdown. The blots were cropped and full length blots are presented in the ESI (Fig. S8†). (C) Quantification of western blot results. *p < 0.05, **p < 0.01; n = 3.

Article Snippet: SALL4 (Santa Cruz #sc-101147), phosphor-AKT (Ser473) (Cell Signaling, #4060), AKT (Cell Signaling, #9272), Cyclin D2 (Cell Signaling, #3741), and β-actin (Cell Signaling, #4967).

Techniques: Knockdown, Control, Negative Control, Western Blot, Expressing

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 1. miR-29c regulates the expression of cyclin E at the posttranscriptional level by targeting 3# UTR of cyclin E mRNA in ESCC cells. (A) The public miRNA database (microRNA Targets Version 5) predicted that cyclin E might be a target for miR-29c, and the 3# UTR of human cyclin E mRNA contains a highly conserved binding site from Position 470 to 492 for miR-29c. (B) The full-length 3# UTR of cyclin E complementary DNA containing miR-29c-binding site was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Wt plasmid (Wt). The full-length 3# UTR of cyclin E complementary DNA (cDNA) deleted 22 nt miR-29c-binding sequence was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Mut plasmid (Mut). (C) KYSE150 cells and EC9706 cells were transfected with 800 ng Wt or Mut reporter plasmid and the increasing doses of Pre-miR-29c or Pre-Scramble (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). After transfected for 24 h, luciferase activity was measured by a dual-luciferase reporter assay. The result was expressed as relative luciferase activity (firefly LUC/renilla LUC). Columns, mean for three experiments; bars, SE. (D) EC9706 cells and KYSE150 cells were transfected with either 30 nmol/l Pre-miR-29c or Pre-Scramble for 48 h. Cyclin E protein levels were measured by western blotting and cyclin E mRNA levels were measured by reverse transcription–PCR.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Reporter Assay, Western Blot, Reverse Transcription

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 2. Inverse correlation between miR-29c expression and cyclin E protein in ESCC cell lines. (A) miR-29c expression in KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells was analyzed by quantitative real-time PCR. The results were presented as relative miR-29c expression, RNU6B served as internal control. The relative value of miR-29c expression of KYSE450 is set at 1. (B and C) The cell lysates of KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells were prepared and analyzed by western blotting. The density of each protein band was quantified using LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. The relative value of cyclin E expression in KYSE450 is set at 1. (D) KYSE150 cells were transfected with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). Forty-eight hours after transfection, miR-29c level was detected by using quantitative real-time PCR. (E and F) The expression of cyclin E was measured by western blotting, after transfecting KYSE150 cells with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l) for 48 h. The density of each protein band was quantified by LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Software, Transfection

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 3. miR-29c induced G1/S cell cycle arrest by suppression of cyclin E expression. (A) EC9706 and KYSE150 cells were transfected with 30 nmol/ l Pre-miR-29c, Pre-Scramble or only Lipofectmine 2000 (Mock). Forty-eight hours after transfection was treated with 100 ng/ml nocodazole for 20 h, cells were collected for cell cycle analysis by propidium iodide staining and flow cytometer analysis. The percentage value of G1 fraction between Pre-miR-29c transfected cells and Pre-Scramble or Mock transfected cells was analyzed. P , 0.01. (B) EC9706 cells were transfected with 30 nmol/l Pre-miR-29c along with the expression plasmid pEF-cyclin E, which contains cyclin E open reading frame without 3# UTR. Forty-eight hours after transfection, cells were treated with 100 ng/ml nocodazole for 20 h. The percentage of cells in G1/G0 was determined by flow cytometer. (C) EC9706 cells and KYSE150 cells were transfected with 30 nmol/l Pre-miR-29c, Pre-Scramble or Mock for 48 h. The cells were collected for western blotting using antibody against cyclin D1, cyclin D2, CDK2 and CDK6. b-Actin was used as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Transfection, Cell Cycle Assay, Staining, Cytometry, Plasmid Preparation, Western Blot, Control

Journal: Cell reports

Article Title: Piwil1 Regulates Glioma Stem Cell Maintenance and Glioblastoma Progression

doi: 10.1016/j.celrep.2020.108522

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-CCND2 , Novus Biologicals , Cat# NBP2-14460.

Techniques: Recombinant, Staining, Imaging, Membrane, Cell Culture, SYBR Green Assay, Plasmid Preparation, shRNA, Software

Journal: Experimental and Therapeutic Medicine

Article Title: miR-206 serves an important role in polycystic ovary syndrome through modulating ovarian granulosa cell proliferation and apoptosis

doi: 10.3892/etm.2021.9610

Figure Lengend Snippet: miR-206 directly targets CCND2. (A) Predicted WT miR-206 binding sites in the 3'-UTR of CCND2. (B) 293 cells were co-transfected with the CCND2-WT/MUT 3'-UTR luciferase reporter plasmid and the miR-206 mimic or mimic control. Following 48 h of transfection, the relative luciferase activity was analyzed using a Dual-Luciferase reporter assay. ** P<0.01 vs. mimic control group. (C) mRNA expression levels of CCND2 in IOSE80 and KGN cells were analyzed using reverse transcription-quantitative PCR. (D) Western blotting was performed to analyze the relative protein expression levels of CCND2 in IOSE80 and KGN cells. (E) Semi-quantification of CCND2 expression levels from part (D). ## P<0.01 vs. IOSE80. miR, microRNA; CCND2, cyclin D2; WT, wild-type; UTR, untranslated region; MUT, mutant.

Article Snippet: Following the overnight culture, KGN cells (5x10 4 cells per well) were transfected with the 100 nM mimic control (5'-UUUGUACUACACAAAAGUACUG-3'; Shanghai GenePharma Co., Ltd.), 100 nM miR-206 mimic (5'-TGGAATGTAAGGAAGTGTGTGG-3'; Shanghai GenePharma Co., Ltd.), 1 μg cyclin D2 CRISPR Activation Plasmid (CCND2-plasmid; cat. no. sc-401236-ACT; Santa Cruz Biotechnology, Inc.), 1 μg Control CRISPR Activation Plasmid (control-plasmid; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.), 100 nM miR-206 mimic + 1 μg control-plasmid or 100 nM miR-206 mimic + 1 μg CCND2-plasmid using Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Binding Assay, Transfection, Luciferase, Plasmid Preparation, Control, Activity Assay, Reporter Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: miR-206 serves an important role in polycystic ovary syndrome through modulating ovarian granulosa cell proliferation and apoptosis

doi: 10.3892/etm.2021.9610

Figure Lengend Snippet: Transfection efficiency of the miR-206 mimic and CCND2-plasmid in KGN cells. (A) mRNA expression levels of miR-206 in KGN cells following the transfection with the miR-206 mimic or mimic control were analyzed using RT-qPCR. ** P<0.01 vs. mimic control group. (B) mRNA expression levels of CCND2 in KGN cells following the transfection with the control-plasmid or CCDN2-plasmid were analyzed using RT-qPCR. ## P<0.01 vs. control-plasmid group. (C) mRNA expression levels of CCND2 in KGN cells following the different transfections were analyzed using RT-qPCR. ** P<0.01 vs. mimic control group; && P<0.01 vs. miR-206 mimic + control-plasmid group. (D) Protein expression levels of CCND2 in KGN cells following the different transfections were analyzed using western blotting. miR, microRNA; CCND2, cyclin D2; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: Following the overnight culture, KGN cells (5x10 4 cells per well) were transfected with the 100 nM mimic control (5'-UUUGUACUACACAAAAGUACUG-3'; Shanghai GenePharma Co., Ltd.), 100 nM miR-206 mimic (5'-TGGAATGTAAGGAAGTGTGTGG-3'; Shanghai GenePharma Co., Ltd.), 1 μg cyclin D2 CRISPR Activation Plasmid (CCND2-plasmid; cat. no. sc-401236-ACT; Santa Cruz Biotechnology, Inc.), 1 μg Control CRISPR Activation Plasmid (control-plasmid; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.), 100 nM miR-206 mimic + 1 μg control-plasmid or 100 nM miR-206 mimic + 1 μg CCND2-plasmid using Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: miR-206 serves an important role in polycystic ovary syndrome through modulating ovarian granulosa cell proliferation and apoptosis

doi: 10.3892/etm.2021.9610

Figure Lengend Snippet: Effects of the miR-206 mimic and CCND2-plasmid on cell viability and apoptosis in KGN cells. (A) Cell viability of transfected KGN cells was analyzed using an MTT assay. (B) Flow cytometric analysis of apoptosis in transfected KGN cells. (C) Semi-quantification of the levels of cell apoptosis from part (B). ** P<0.01 vs. mimic control group; ## P<0.01 vs. miR-206 mimic + control-plasmid group. miR, microRNA; CCND2, cyclin D2; PI, propidium iodide.

Article Snippet: Following the overnight culture, KGN cells (5x10 4 cells per well) were transfected with the 100 nM mimic control (5'-UUUGUACUACACAAAAGUACUG-3'; Shanghai GenePharma Co., Ltd.), 100 nM miR-206 mimic (5'-TGGAATGTAAGGAAGTGTGTGG-3'; Shanghai GenePharma Co., Ltd.), 1 μg cyclin D2 CRISPR Activation Plasmid (CCND2-plasmid; cat. no. sc-401236-ACT; Santa Cruz Biotechnology, Inc.), 1 μg Control CRISPR Activation Plasmid (control-plasmid; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.), 100 nM miR-206 mimic + 1 μg control-plasmid or 100 nM miR-206 mimic + 1 μg CCND2-plasmid using Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Plasmid Preparation, Transfection, MTT Assay, Control

Journal: Experimental and Therapeutic Medicine

Article Title: miR-206 serves an important role in polycystic ovary syndrome through modulating ovarian granulosa cell proliferation and apoptosis

doi: 10.3892/etm.2021.9610

Figure Lengend Snippet: Effects of the miR-206 mimic and CCND2-plasmid on the activity and expression levels of caspase-3 in KGN cells. (A) Caspase-3 activity in KGN cells following the different transfections was detected using a caspase-3 activity assay kit. (B) Protein expression levels of cleaved-caspase-3 and pro-caspase-3 in transfected KGN cells were analyzed using western blot analysis. (C) Semi-quantification of the ratio of cleaved-caspase-3/pro-caspase-3 expression levels in transfected KGN cells from part (B). ** P<0.01 vs. mimic control group; ## P<0.01 vs. miR-206 mimic + control-plasmid group. miR, microRNA; CCND2, cyclin D2.

Article Snippet: Following the overnight culture, KGN cells (5x10 4 cells per well) were transfected with the 100 nM mimic control (5'-UUUGUACUACACAAAAGUACUG-3'; Shanghai GenePharma Co., Ltd.), 100 nM miR-206 mimic (5'-TGGAATGTAAGGAAGTGTGTGG-3'; Shanghai GenePharma Co., Ltd.), 1 μg cyclin D2 CRISPR Activation Plasmid (CCND2-plasmid; cat. no. sc-401236-ACT; Santa Cruz Biotechnology, Inc.), 1 μg Control CRISPR Activation Plasmid (control-plasmid; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.), 100 nM miR-206 mimic + 1 μg control-plasmid or 100 nM miR-206 mimic + 1 μg CCND2-plasmid using Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Plasmid Preparation, Activity Assay, Expressing, Transfection, Caspase-3 Activity Assay, Western Blot, Control

Journal: Nature genetics

Article Title: De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome.

doi: 10.1038/ng.2948

Figure Lengend Snippet: Figure 1 Images of children with MPPH and CCND2 mutations. Photos and brain MRI scans of four affected individuals are shown. (a–d) LR07-041. (e–h) LR02-064. (i–l) JT-144. (m–p) JT-210. Photographs demonstrate macrocephaly with a prominent forehead (a,e,i,m,n) and postaxial polydactyly of the hands (b,f,j). T1- or T2-weighted midsagittal images demonstrate large brain size in relation to facial structures (c,g,k,o), and axial images show polymicrogyria that appears to be most severe in the perisylvian regions but also involves other regions (d,h,l,p). The parents of all subjects gave informed consent for publication of photographs.

Article Snippet: A C-terminal Myc-tagged cDNA clone of CCND2 transcript NM_001759 was purchased from Origene (Origene, RC210316).

Techniques:

Journal: Nature genetics

Article Title: De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome.

doi: 10.1038/ng.2948

Figure Lengend Snippet: Figure 2 CCND2 mutations and cyclin D2 protein conservation. Schematic showing the intron-exon structure of human CCND2. The cDNA sequence from position 805 and the corresponding peptide sequence from Ser269 are shown, with positions altered in subjects with MPPH marked in red (Supplementary Fig. 1).

Article Snippet: A C-terminal Myc-tagged cDNA clone of CCND2 transcript NM_001759 was purchased from Origene (Origene, RC210316).

Techniques: Sequencing

Journal: Nature genetics

Article Title: De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome.

doi: 10.1038/ng.2948

Figure Lengend Snippet: Figure 4 Comparative ability of mutant and wild-type CCND2 to enhance neural progenitor proliferation in the embryonic mouse brain. Two patient mutations (encoding p.Thr280Ala and p.Pro281Arg) are compared to wild-type and phosphomimetic (p.Thr280Asp) CCND2. Wild-type and mutant cDNAs were cloned into an IRES-eGFP vector, affording independent translation of both CCND2 and, from an IRES, eGFP. Electroporated (GFP+) cells expressed either GFP alone or GFP together with human wild-type or mutant CCND2. Constructs (1 µg each) were delivered to the cortex by IUEP at E13.5, and embryos were harvested at E15.5. (a–e) Cortical sections were immunostained with an antibody to GFP. (f–j) The same sections were double immunolabeled (yellow) with antibodies to GFP (green) and Ki67 (red). Ki67 is a proliferation marker. (a,f) For the most part, cells receiving vector without CCND2 are no longer dividing, and many have migrated up to the cortical plate. (b,g) More cells continue to divide (yellow cells, mostly in the SVZ and intermediate zone (IZ)) when overexpressing wild-type CCND2. (c,h,e,j) Progenitors expressing either the phosphodeficient p.Thr280Ala CCND2 or putative phosphodeficient p.Pro281Arg CCND2 found in patients with MPPH show strikingly more proliferation and fewer GFP+ cells residing in the cortical plate. (d,i) A phosphomimetic (p.Thr280Asp) form of CCND2 is significantly less effective in promoting proliferation than the forms carrying MPPH-associated mutations. (k) Quantification of the proportions of transfected (GFP+) cells that are proliferating (GFP+Ki67+ cells/total GFP+ cells). Error bars, s.e.m. *P < 0.05, **P < 0.0001 with respect to cells expressing GFP only (control); §P < 0.0001 with respect to cells expressing wild- type or p.Thr280Asp CCND2, as indicated. A full statistical summary is shown in Supplementary Table 2, and the P values shown in the figure were calculated by one-way analysis of variance (ANOVA) with post-hoc tests (LSD). n = 7 embryos (a), n = 5 embryos each (b–e), with each value an average of five sections per embryo. MZ, marginal zone; CP, cortical plate. Scale bar, 20 µm.

Article Snippet: A C-terminal Myc-tagged cDNA clone of CCND2 transcript NM_001759 was purchased from Origene (Origene, RC210316).

Techniques: Mutagenesis, Clone Assay, Plasmid Preparation, Construct, Immunolabeling, Marker, Expressing, Transfection, Control

Journal: Nature genetics

Article Title: De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome.

doi: 10.1038/ng.2948

Figure Lengend Snippet: Figure 5 Phosphodeficient CCND2 more effectively promotes proliferation and prevents neural progenitor cells from exiting the cell cycle. (a–d) Tissue sections immunostained with antibodies to GFP (green) and pH3 (purple) after electroporation with constructs encoding wild-type, phosphodeficient (p.Thr280Ala) and phosphomimetic (p.Thr280Asp) CCND2. (a–c) Compared to transfection with wild-type CCND2, introduction of phosphodeficient CCND2 resulted in a significant increase in the number of GFP+ and pH3+ double-labeled cells in the VZ and SVZ (a,b), whereas transfection with phosphomimetic CCND2 was no different from transfection with wild-type CCND2 (c). (d) Quantification shows significantly more GFP+pH3+ cells after introduction of phosphodeficient CCND2 compared to wild-type (***P = 0.000054) or phosphomimetic (***P = 0.00049) sequences. There was no difference between transfection with the phosphomimetic and wild-type CCND2 forms (P = 0.41). (e–k) Comparison of actively proliferating (P fraction) and nonproliferating (exit fraction) cortical progenitors transfected with wild-type or mutant CCND2 constructs. (e–g) Cells were colabeled with antibodies to GFP (green) and BrdU (red) after BrdU injection at E14.5 (24 h after IUEP) and harvested at E15.5. (h–j) The same samples were also colabeled with antibodies to Ki67 (blue). Expression of either the wild-type form of CCND2 (e,h,k) or phosphomimetic (p.Thr280Asp) forms of CCND2 (g,j,k) resulted in a similar increase in the percentage of progenitors in the exit fraction over those in the P fraction (Online Methods), thereby indicating that more cells had exited the cell cycle 24 h after BrdU injection. (f,i,k) Introduction of the phosphodeficient (p.Thr280Ala) form of CCND2 significantly increased the number of progenitors in the P fraction (still proliferating 24 h after BrdU pulse) and correspondingly decreased the number in the exit fraction compared to transfection with either the wild-type or phosphomimetic form of CCND2. Thus, phosphodeficient CCND2 was more effective in promoting mitotic events and keeping cells in cycle. Full statistical analyses are shown in Supplementary Table 3. GFP and pH3 experiment: n = 4 embryos, wild-type CCND2; n = 5 embryos, p.Thr280Ala; n = 5 embryos, p.Thr280Asp. GFP, BrdU and Ki67 experiment: n = 5 embryos, wild-type CCND2; n = 5 embryos, p.Thr280Ala; n = 5 embryos, p.Thr280Asp. Each value shown is an average of five sections per embryo. Data are presented as the mean ± s.e.m. Scale bars, 20 µm. *P < 0.02 compared to wild-type CCND2, §P < 0.01 compared to p.Thr280Ala CCND2. Throughout the figure, unpaired Student’s t-test was used to calculate all P values.

Article Snippet: A C-terminal Myc-tagged cDNA clone of CCND2 transcript NM_001759 was purchased from Origene (Origene, RC210316).

Techniques: Electroporation, Construct, Transfection, Labeling, Comparison, Mutagenesis, Injection, Expressing

Journal: Oncology letters

Article Title: Ubiquitin-specific peptidase 22 promotes proliferation and metastasis in human colon cancer.

doi: 10.3892/ol.2019.10872

Figure Lengend Snippet: Figure 5. USP22 regulates the expression of Bmi‑1 and Cyclin D2. (A) Protein expression levels of USP22, Bmi‑1 and Cyclin D2 were examined following transfection of HCT116 cells with USP22‑siRNA1 or negative control siRNA. β‑Actin was used as a loading control. *P<0.05, **P<0.01. (B) Cell cycle distribution was determined by flow cytometry analysis. The percentage of HCT116 cells in each cell cycle phase is shown. *P<0.05. ***P<0.001. USP22, ubiquitin‑specific peptidase 22; siRNA, small interfering RNA; PI, propidium iodide.

Article Snippet: Membranes were blocked at room temperature with 5% skimmed milk for 45 min and incubated at room temperature for 2 h with the following antibodies: USP22 rabbit polyclonal antibody (LifeSpan BioSciences, Inc.; cat. no. LS‐C99567; 1:800), Bmi‐1 rabbit polyclonal antibody (Abcam; cat. no. ab126783; 1:500) and Cyclin D2 rabbit polyclonal antibody (OriGene Technologies, Inc.; cat. no. TA323121; 1:500), and subsequently with horseradish peroxidase‐conjugated goat anti‐rabbit/mouse secondary antibodies (Abcam; cat. no. ab150077; 1:1,000) at room temperature for 1 h. The blots were incubated by WesternBright chemiluminescence (ECL) (Western blotting detection kit; Advansta, Inc.) for 1 min, and developed using ImageQuantTM LAS4000 (GE Healthcare Life Sciences).

Techniques: Expressing, Transfection, Negative Control, Control, Flow Cytometry, Small Interfering RNA