Journal: Epigenetics

Article Title: A novel lncRNA, Lnc21q22.11, suppresses gastric cancer growth by inhibiting MEK/ERK pathway.

doi: 10.1080/15592294.2025.2512764

Figure Lengend Snippet: Figure 1. Isolation and identification of Lnc21q22.11 in gastric cancer. (a) RNA-seq data showing the Fold changes in lncRNA expression levels in gastric cancer compared to adjacent tissue samples. (b) Real-time PCR results showing reduced expression of Lnc21q22.11 in cancer tissue compared to adjacent tissue samples (n = 245). ***p < 0.001. (c) RT-PCR results showing the expression of Lnc21q22.11 in gastric cancer cell lines. H2O: negative control; GAPDH: internal control. (d) Northern blotting showing the size of Lnc21q22.11, 1000 nt and 1500 nt were marker sizes. GAPDH served as an RNA expressing control. (e) 5'-RACE and 3'-RACE results obtained after walking-PCR extension. (f) Three potential coding patterns of the full length Lnc21q22.11 sequence were constructed into pcDNA3.1 expression vectors. ZSCAN23-Flag fusion protein served as a positive control. FL: full length sequence. (g) Different patterns of Lnc21q22.11 constructs were transfected into AGS cells and detected with Flag antibody. The size of ZSCAN23-Flag was 47 kDa. β-actin: internal control. (h) Cytoplasmic and nuclear RNA were separated from BGC-823 cells, and Lnc21q22.11 was detected by real-time PCR. U6: control for nucleus; GAPDH: control for cytoplasm. (i) FISH assay showing localization of Lnc21q22.11 in BGC- 823 cells. The scale bar represents 25 μm. (j) Histone modification status of the Lnc21q22.11 promoter region predicted by cistrome DB database. H3K4me3 enrichment locations indicate transcriptional activation sites in AGS, BGC-823, and MGC-803 cells. (k) Histone modification status in Lnc21q22.11 promoter region detected by ChIP-PCR assay with H3K4me3, H3K9me2, H3K27me3 antibodies.

Article Snippet: Northern blotting, 5'- and 3'- rapid amplification of cDNA ends Biotin-labeled Lnc21q22.11 probes were synthesized with T7 RNA polymerase using a biotin RNA labeling mix (NEB, USA, #M0251).

Techniques: Isolation, RNA Sequencing, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control, Control, Northern Blot, Marker, Sequencing, Construct, Positive Control, Transfection, Modification, Activation Assay

Journal: Epigenetics

Article Title: A novel lncRNA, Lnc21q22.11, suppresses gastric cancer growth by inhibiting MEK/ERK pathway.

doi: 10.1080/15592294.2025.2512764

Figure Lengend Snippet: Figure 4. Interaction of Lnc21q22.11 and MYH9, and their co-localization in the nucleus. (a) RNA pull-down assay showing Lnc21q22.11 and protein complexes. The red arrow indicates the differential band. LacZ probes were used for the control group. (b) Predicted interaction between Lnc21q22.11 and MYH9 using Alphafold3. Lnc21q22.11 and MYH9 are represented by ribbon and surface, respectively. Short red bands indicate hydrogen bonds between Lnc21q22.11 and MYH9. (c) Interaction between Lnc21q22.11 and MYH9 verified by RNA pull-down and western blotting using labeled Lnc21q22.11 probes and MYH9 antibodies. (d) Interaction between Lnc21q22.11 and MYH9 validated by RNA immunoprecipitation assay. IgG antibody was used for the control group. (e) Confocal laser microscopic images showing the localization of Lnc21q22.11 (red) and MYH9 (green) in cells, and their co- localization in the nucleus. The scale bar represents 25 μm.

Article Snippet: Northern blotting, 5'- and 3'- rapid amplification of cDNA ends Biotin-labeled Lnc21q22.11 probes were synthesized with T7 RNA polymerase using a biotin RNA labeling mix (NEB, USA, #M0251).

Techniques: Pull Down Assay, Control, Western Blot, Labeling, RNA Immunoprecipitation

Journal: The American journal of pathology

Article Title: Increased expression of versican in the inflammatory response to UVB- and reactive oxygen species-induced skin tumorigenesis.

doi: 10.1016/j.ajpath.2011.08.042

Figure Lengend Snippet: Figure 3. Expression of continuously up-regulated genes after UVB expo- sure. A: Four genes were significantly up-regulated at both 3 and 24 hours after UVB exposure. Relative expression levels were determined by real-time quantitative PCR. Each of the two groups of RNAs isolated from wild-type and Ogg1 knockout mice was assayed in duplicate. Reactions were normal- ized to GAPDH expression levels. *P 0.05; **P 0.01; and ***P 0.001. B: Immunohistochemical study of versican expression after UVB irradiation. Versican is barely expressed in the wild-type mouse epidermis and dermis in the absence of UVB irradiation. At 24 hours after UVB exposure, versican was expressed in the wild-type epidermis. In Ogg1 knockout mice at 24 hours, versican was strongly expressed in the epidermis (arrowheads), as well as in dermal fibroblasts (arrows). Positive signals are seen as reddish-brown deposits produced on reaction with the 3-amino-9-ethylcarbazole substrate. Scale bar 30 m. C: Versican expression after UVB exposure, as deter- mined by Western blotting. Versican was more strongly up-regulated in Ogg1 knockout than in wild-type mice at 24 and 48 hours after UVB exposure. This up-regulation was time-dependent. The band at approximately 75 kDa indi- cates that the antibody for versican can detect the V1 isoform. /-Tubulin was used as the loading control. Data are representative of three separate determinations.

Article Snippet: Sections were incubated for 16 hours at 4°C with the following primary antibodies: rabbit polyclonal anti-mouse IL-1 (1:1000 dilution; Abcam, Cambridge, MA), rabbit polyclonal anti-mouse versican (1: 100 dilution; LifeSpan Biosciences, Seattle, WA), rabbit polyclonal anti-human versican (1:125 dilution; Atlas Antibodies, Stockholm, Sweden), or rabbit polyclonal antimouse p53 (CM5) (1:500 dilution; Leica Biosystems Newcastle, Newcastle upon Tyne, UK).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Knock-Out, Immunohistochemical staining, Irradiation, Produced, Western Blot, Control

Journal: The American journal of pathology

Article Title: Increased expression of versican in the inflammatory response to UVB- and reactive oxygen species-induced skin tumorigenesis.

doi: 10.1016/j.ajpath.2011.08.042

Figure Lengend Snippet: Figure 4. Versican expression in developing skin tumors of chronically UVB-exposed mice. Representative histological sections of SCC tumors from wild-type and Ogg1 knockout mice red-colored staining positively for versi- can are shown, with a summary of versican-positive wild-type and Ogg1 knockout mouse tumors overall. *P 0.05 for the ratio of malignant tumor to total tumors analyzed for each genotype.

Article Snippet: Sections were incubated for 16 hours at 4°C with the following primary antibodies: rabbit polyclonal anti-mouse IL-1 (1:1000 dilution; Abcam, Cambridge, MA), rabbit polyclonal anti-mouse versican (1: 100 dilution; LifeSpan Biosciences, Seattle, WA), rabbit polyclonal anti-human versican (1:125 dilution; Atlas Antibodies, Stockholm, Sweden), or rabbit polyclonal antimouse p53 (CM5) (1:500 dilution; Leica Biosystems Newcastle, Newcastle upon Tyne, UK).

Techniques: Expressing, Knock-Out, Staining

Journal: The American journal of pathology

Article Title: Increased expression of versican in the inflammatory response to UVB- and reactive oxygen species-induced skin tumorigenesis.

doi: 10.1016/j.ajpath.2011.08.042

Figure Lengend Snippet: Figure 5. Versican expression in human skin tumors. A: Immunohistochemical study of versican expression in malignant (lentigo maligna melanoma, basal cell carcinoma, and squamous cell carcinoma) and benign (seborrheic keratosis and lentigo senilis) tumors. Arrowheads indicate the borders between the seborrheic keratosis tumor and the normal skin. Scale bar 100 m. B: Classification of versican expression in skin tumors, grouped as dermal and stromal versus tumoral.

Article Snippet: Sections were incubated for 16 hours at 4°C with the following primary antibodies: rabbit polyclonal anti-mouse IL-1 (1:1000 dilution; Abcam, Cambridge, MA), rabbit polyclonal anti-mouse versican (1: 100 dilution; LifeSpan Biosciences, Seattle, WA), rabbit polyclonal anti-human versican (1:125 dilution; Atlas Antibodies, Stockholm, Sweden), or rabbit polyclonal antimouse p53 (CM5) (1:500 dilution; Leica Biosystems Newcastle, Newcastle upon Tyne, UK).

Techniques: Expressing, Immunohistochemical staining

Journal: The American journal of pathology

Article Title: Increased expression of versican in the inflammatory response to UVB- and reactive oxygen species-induced skin tumorigenesis.

doi: 10.1016/j.ajpath.2011.08.042

Figure Lengend Snippet: Figure 7. Proposed relationships of versican in the inflammatory response leading to the development of skin tumors in terms of UVB-induced 8-oxoG accumulation. The accumulation of UVB/ROS-induced 8-oxoG in the skin leads to inflammatory reactions. High versican expression is induced by a highly inflammatory microenvironment with high numbers of infiltrated neu- trophils; conversely, neutrophil infiltration induces versican overexpression. More ROS will be generated at the inflammatory sites by neutrophils.

Article Snippet: Sections were incubated for 16 hours at 4°C with the following primary antibodies: rabbit polyclonal anti-mouse IL-1 (1:1000 dilution; Abcam, Cambridge, MA), rabbit polyclonal anti-mouse versican (1: 100 dilution; LifeSpan Biosciences, Seattle, WA), rabbit polyclonal anti-human versican (1:125 dilution; Atlas Antibodies, Stockholm, Sweden), or rabbit polyclonal antimouse p53 (CM5) (1:500 dilution; Leica Biosystems Newcastle, Newcastle upon Tyne, UK).

Techniques: Expressing, Over Expression, Generated

Journal: The American journal of pathology

Article Title: Increased expression of versican in the inflammatory response to UVB- and reactive oxygen species-induced skin tumorigenesis.

doi: 10.1016/j.ajpath.2011.08.042

Figure Lengend Snippet: Figure 6. Versican expression and neutrophil localization in human and murine skin tumors. A: Versican expression and inflammatory cells in human squamous cell carcinoma. Versican is strongly expressed in the dermal components (arrows) and inflammatory cells (arrowheads), seen at low magnification (left) and high magnification (right). Two focused areas are taken from different part of sections. Inflammatory cells, especially seg- mented leukocytes (neutrophils) (arrowheads), were strongly positive for versican. Scale bars 30 m. B: Versican expression and neutrophils in mouse skin. In the merged iamge, neutrophils with versican expression (arrows) appear yellow. Scale bar 30 m. C: Neutrophil infiltration after UVB irradiation in wild-type and Ogg1 knockout mice. Arrows indicate Gr-1-positive cells (green) in the dermis at 24 and 48 hours after UVB exposure in the wild-type and Ogg1 knockout mice. Scale bar 30 m. The accompanying graphs show the average number of neutrophils per 800 m2

Article Snippet: Sections were incubated for 16 hours at 4°C with the following primary antibodies: rabbit polyclonal anti-mouse IL-1 (1:1000 dilution; Abcam, Cambridge, MA), rabbit polyclonal anti-mouse versican (1: 100 dilution; LifeSpan Biosciences, Seattle, WA), rabbit polyclonal anti-human versican (1:125 dilution; Atlas Antibodies, Stockholm, Sweden), or rabbit polyclonal antimouse p53 (CM5) (1:500 dilution; Leica Biosystems Newcastle, Newcastle upon Tyne, UK).

Techniques: Expressing, Irradiation, Knock-Out

Journal: Molecular Systems Biology

Article Title: A ubiquitous GC content signature underlies multimodal mRNA regulation by DDX3X

doi: 10.1038/s44320-024-00013-0

Figure Lengend Snippet: Reagents and tools.

Article Snippet: For the RNA-seq, RNA was extracted from 25 μl intact lysate (non-digested) using the Direct-zol kit (Zymo Research) and stranded total RNA libraries were prepared using the TruSeq Stranded Total RNA Human/Mouse/Rat kit (Illumina), following manufacturer’s instructions.

Techniques: Sequencing, Software, Flow Cytometry, Staining, Real-time Polymerase Chain Reaction

Journal: STAR Protocols

Article Title: An optimized chromatin immunoprecipitation protocol using Staph-seq for analyzing genome-wide protein-DNA interactions

doi: 10.1016/j.xpro.2022.101918

Figure Lengend Snippet:

Article Snippet: V6.5 mouse embryonic stem cells , Novus Biologicals , Cat#NBP1-41162.

Techniques: Recombinant, SYBR Green Assay, Next-Generation Sequencing, Agarose Gel Electrophoresis, Purification, Software, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: Amino acids-Rab1A-mTORC1 signaling controls whole-body glucose homeostasis

doi: 10.1016/j.celrep.2021.108830

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Proinsulin (253627) , Novus biologicals , Cat# MAB13361; RRID: AB_2126534.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Calcium Assay, esiRNA, Real-time Polymerase Chain Reaction, shRNA, Software, Hybridization, Modification

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: Identification of KAT2A as a host factor promoting cccDNA transcription. (A) The reported succinyltransferases and desuccinylases were summarised in the table. (B) After 4 days of siRNA treatment, total RNA was extracted by TRNzol Universal reagent. The silencing efficiency of related genes in Huh-7 cells was analysed by real-time PCR with specific primers. β-actin was used as the internal control. (C) The indicated siRNA (50 pmol) and monomeric linearised HBV DNA (1 μg) were co-transfected into huh-7 cells by using Lipofectamine™ 3000 Transfection Reagent. The supernatant was collected and HBeAg and HBsAg levels were detected by ELISA. * P < 0.05.

Article Snippet: Mouse anti-HBcAg monoclonal antibody (sc-23945), mouse anti-GAPDH monoclonal antibody (sc-47724) and rabbit anti-β-actin monoclonal antibody (sc-47778) were obtained from Santa Cruz Biotechnology (United States).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: KAT2A knockdown suppresses cccDNA transcription in the HBV infection cell model. HepG2-NTCP cells were infected HBV particles at 1-day post shRNA transduction. At 4 days after infection. (A) The expression of KAT2A was detected by western blot. (B) Northern blotting and real-time PCR were used to analyse the effect of KAT2A knockdown on HBV RNAs. (C) HBV cccDNA was extracted by a modified Hirt DNA extraction protocol and then Southern blotting and Taq-man probe qPCR were used to detect the HBV cccDNA level. The mitochondrial gene Cox1 was hybridised as the loading control for HBV cccDNA. (D) The level of HBV 3.5-kb RNA, total HBV RNAs, and cccDNA were used to calculate the ratio of HBV 3.5-kb RNA to cccDNA and total HBV RNAs to cccDNA. (E) HBV core DNA was quantified by Southern blotting and absolute quantification PCR. The level of β-actin was used as a loading control for HBV core DNA. (F) HBV core protein (HBc) was produced by HBV infected HepG2-NTCP cells. Intracellular HBc was visualised by immunofluorescence staining. The scale bar is 100 μm. (G) Western blot was used to detect the level of HBsAg in cells (left). Cell culture supernatant was collected for HBsAg and HBeAg analysis via ELISA (right). Cox1, cyclooxygenase 1; rRNA, ribosomal RNA. * P < 0.05.

Article Snippet: Mouse anti-HBcAg monoclonal antibody (sc-23945), mouse anti-GAPDH monoclonal antibody (sc-47724) and rabbit anti-β-actin monoclonal antibody (sc-47778) were obtained from Santa Cruz Biotechnology (United States).

Techniques: Infection, shRNA, Transduction, Expressing, Western Blot, Northern Blot, Real-time Polymerase Chain Reaction, Modification, DNA Extraction, Southern Blot, Produced, Immunofluorescence, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: KAT2A overexpression promotes HBV transcription in the HBV infection cell model. HepG2-NTCP cells were infected HBV particles at 1-day post lentivirus expressing KAT2A plasmids transduction. At 4 days after infection. (A) The expression of KAT2A was detected by western blot. (B) The effect of KAT2A overexpression on HBV RNAs was analysed by Northern blotting and real-time PCR. (C) Taq-man probe qPCR was used to detect the HBV cccDNA level. (D) The level of HBV 3.5-kb RNA, total HBV RNAs, and cccDNA were used to calculate the ratio of HBV 3.5-kb RNA to cccDNA and total HBV RNAs to cccDNA. (E) HBV core DNA was quantified by Southern blotting and absolute quantification PCR. The level of β-actin was used as a loading control for HBV core DNA. (F) HBc was produced by HBV-infected HepG2-NTCP cells. Intracellular HBc was visualised by immunofluorescence staining. The scale bar is 100 μm. (G) Cell culture supernatant was collected for HBsAg and HBeAg analysis via ELISA. * P < 0.05.

Article Snippet: Mouse anti-HBcAg monoclonal antibody (sc-23945), mouse anti-GAPDH monoclonal antibody (sc-47724) and rabbit anti-β-actin monoclonal antibody (sc-47778) were obtained from Santa Cruz Biotechnology (United States).

Techniques: Over Expression, Infection, Expressing, Transduction, Western Blot, Northern Blot, Real-time Polymerase Chain Reaction, Southern Blot, Produced, Immunofluorescence, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 1. miR-206 targets cyclin D1. (A) Sequence alignment between miR-206 and the 3′UTRs of cyclin D1 from different species. In brackets the 3′UTR size. (B) Diagram of the luciferase reporter construct with the putative miR-206 binding site (WT 3′UTR) and mutations (3′UTR MUT). (C) Relative lucif- erase activity was measured in HeLa cells after transfection of reporter constructs along with pSP65-U1 (CTR) or pSP65–206 (miR-206). Relative Firefly luciferase values were determined by a ratio of Firefly to Renilla luciferase with the control set to 1.00. Values are the means ± SD of 3 separate experi- ments. **A Student t test performed between control and miR-206 transfected cells yielded P values < 0.01.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Sequencing, Luciferase, Construct, Binding Assay, Activity Assay, Transfection, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 2. Expression kinetics of miR-206 and cyclin D1 in differentiating C2C12 cells. C2C12 myoblasts were seeded in GM at 1.5 × 104/cm2. Cells were shifted in DM 24 h after plating and left to differentiate for further 72 h. (A) Northern blot analysis of miR-206 expression in C2C12 cells after 24 h in GM (0) and at different time points upon shift to DM. (B) Western blot analysis of cyclin D1 and MyHC expression in C2C12 cells cultured as in (A). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) MyHC immunofluorescence staining (green) of C2C12 cells after 24 h in GM (DM 0 h) and after 72 h in DM (DM 72 h). Nuclei were counterstained in blue (DAPI) and individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 20 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Western Blot, Cell Culture, Immunofluorescence, Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 3. miR-206 controls cyclin D1 accumulation in C2C12 cells. C2C12 myoblasts were seeded in GM at 2.5 × 103/cm2. Cells were transfected 24 h after plaiting. (A) Northern blot analysis of miR-206 expression (upper) and western blot analysis of cyclin D1 expression (lower) in C2C12 cells 48 h after transfection with a control vector (CTR) or with a miR-206 expression vector (miR-206). Cells were kept in GM throughout the experiment. (B) The effect of miR-206 overexpression on C2C12 cell proliferation and differentiation was evaluated 48 h after transfection by 1 h BrdU incorporation and MyHC staining, respectively. Results are represented relative to the BrdU+ nuclei or nuclei in MyHC+ cells in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. (C) Immunofluorescence staining of cyclin D1 (pink) and MyHC (green) 48 h after transfection. Nuclei were counterstained in blue with DAPI. Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. To obtain cyclin D1 images, before merging, individual pictures were pseudocol- ored using a LEICA Microsystems Imaging software. Bar = 10 μm. (D) C2C12 myoblasts were seeded at low (LD) and high (HD) density in GM. Cells were shifted to DM the day after plating and analyzed after further 3 d. The panels show a northern blot analysis of miR-206 expression (left panel) and a western blot analysis of cyclin D1 and differentiation associ- ated marker expression (right panel) after 24 h in GM and 72 h after shift- ing to DM. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Northern Blot, Expressing, Western Blot, Control, Plasmid Preparation, Over Expression, BrdU Incorporation Assay, Staining, Immunofluorescence, Imaging, Software, Marker

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 4. Inhibition of miR-206 rescues cyclin D1 in myotubes (A) Experimental scheme. C2C12 myoblasts were induced to differ- entiate in DM in the presence of AraC. After 3 d, AraC was washed out and cells left to recover in DM for further 24 h. Finally, pure myotubes were transfected with LNA against miR-206 and analyzed 48 h later. (B) Northern blot analysis of miR-206 and miR-1 expression (left panel) and western blot analysis of cyclin D1 expression (right panel) in pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Cyclin D1 expression in proliferating myoblasts is also shown (GM). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) Double immunofluorescence staining of MyHC and cyclin D1 of pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 10 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Inhibition, Transfection, Northern Blot, Expressing, Western Blot, Control, Double Immunofluorescence Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 5. miR-206 inhibits cell proliferation in Ras-transformed fibro- blasts. (A) Expression levels of cyclin D1 in NIH3T3(Ras) cells as compared with NIH3T3(BN) cells. (B) Real-time PCR analysis of miR-206 expres- sion in NIH3T3(Ras) cells. Results are shown relative to untransformed NIH3T3(BN) cells set to value 1.00. Each sample was analyzed in tripli- cate, and values are the means ± SD of 3 independent experiments. **A Student t test performed between untransformed and transformed cells yielded P values < 0.01. (C) NIH3T3(Ras) cells were transfected with a control vector (CTR) or with a miR-206 expression vector (miR-206) and analyzed 24 h later. Upper, northern blot analysis of miR-206 expression; lower, western blot analysis of cyclin D1 expression. (D) Effect of miR-206 forced expression on cell proliferation as determined by 1 h BrdU incor- poration. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. Equal RNA and protein loading was confirmed by detecting, snRNA U2, and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 6. Relationship between miR-206 downregulation and cyclin D1 expression in NSCLCs. (A) Northern blot analysis of miR-206 in different murine tissues. snRNA U2 levels were used as a loading control. (B) Real-time PCR analysis of miR-206 expression in human NSCLC tissues. Results are shown relative to the matched normal lung tissues set to value 1.00. Each sample was analyzed in triplicate, and values are the means ± SD of three independent experiments. **A Student t test performed between normal and tumor tissues yielded P values < 0.01. (C) Western blot analysis of cyclin D1 expression in normal and neoplastic lung tissues. Equal protein loading was confirmed by detecting actin. n, normal tissue; t = tumor tissue

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

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

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 7. miR-206 inhibits cancer cell proliferation through repression of cyclin D1. (A) A549 and HeLa cells were transfected with a control vec- tor (CTR) or with a miR-206 expression vector (miR-206) and analyzed 72 h later. Top panel: northern blot analysis of miR-206 expression; lower panel, western blot analysis of cyclin D1 expression. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (B) Effect of miR-206 forced expression on cell prolifera- tion as determined by 1 h BrdU incorporation and immunofluorescence staining. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values <0.05.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Northern Blot, Western Blot, BrdU Incorporation Assay, Immunofluorescence, Staining