Journal: BMC Genomics

Article Title: A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains

doi: 10.1186/1471-2164-8-39

Figure Lengend Snippet: Subcellular localization of human and mouse NEAT1 and NEAT2/MALAT-1 . (A) Tig1 cells: Human NEAT1 RNA (green) distributes in discrete foci through the nucleus and is not detected in the cytoplasm. (B) Tig1 cells: NEAT1 RNA (red) is transcribed from 2 loci early in G1 (left) and occasionally shows a highly polarized pattern (right). (C) Tig1 cells: NEAT2/MALAT-1 RNA (red) is not detected in the cytoplasm at interphase and concentrates in domains in the nucleus with weaker signal apparent throughout the nucleoplasm. (D) NIH-3T3s: Mouse Neat2/Malat-1 RNA (green) localizes as bright nucleoplasmic signal in some cells and concentrates in domains in others. (E) NEAT1 RNA (red) does not localize to its parent chromosome 11 (green). (F) NEAT2/MALAT-1 RNA (red) rapidly disperses from its site of transcription (Chromosome 11 (green)) and distributes throughout the nucleus. (G) Tig1 cells: NEAT1 RNA (green) and NEAT2/MALAT-1 RNA are associated in the interphase nucleus. (H) MEFs: Mouse Neat1 RNA (red) is exclusively nuclear, and is found in fewer, larger foci than in human cells, yet shows the same peripheral association with Neat2/Malat-1 RNA (green), and neither RNA associates significantly with DAPI dense heterochromatin (blue).

Article Snippet: Normal human female diploid fibroblast cells TIG1 (Coriell); mouse fibroblasts NIH-3T3 (ATCC); primary mouse embryo fibroblasts (MEFs) (Cell Essentials) were cultured under normal conditions.

Techniques:

Journal: BMC Genomics

Article Title: A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains

doi: 10.1186/1471-2164-8-39

Figure Lengend Snippet: Human and mouse NEAT1 and NEAT2/MALAT-1 are intimately associated with SC35 domains . (A) Tig1 cells: NEAT2 /MALAT-1 RNA (red) is found in every SC35 domain (green); (B) NIH-3T3s: Neat2/Malat-1 RNA (green) concentrates in domains in about 50% of the population, when it does, it overlaps with SRM-300 speckles (red); (C,D,E) NEAT2/MALAT-1 RNA (green (C)) and a probe to poly(A) (red (D)) overlap (yellow (E)) in domains in the nucleus; (F) Neat1 RNA (red) preferentially localizes to the periphery of SRM-300 domains (green) in mouse cells; (G) NEAT1 RNA (red) preferentially associates at the periphery of SC35 domains (green) in human cells; (H) Two transcription sites of NEAT2/MALAT-1 RNA (red) localize with SC35 domains (green) in early G1 daughter cells; (I) Poly(A) domains (red) are apparently formed prior6=- to the spread of NEAT2/MALAT-1 RNA (green) in early G1.

Article Snippet: Normal human female diploid fibroblast cells TIG1 (Coriell); mouse fibroblasts NIH-3T3 (ATCC); primary mouse embryo fibroblasts (MEFs) (Cell Essentials) were cultured under normal conditions.

Techniques:

Journal: Nucleic Acids Research

Article Title: Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability

doi: 10.1093/nar/gkv222

Figure Lengend Snippet: Outline and validation of the experimental approach. ( A ) Workflow scheme for the SILAC analysis. siRNA-mediated knockdown of XRCC1 or control was performed with Tig-1 cells grown in light (R0K0) or medium (R6K4) containing DMEM for 72 hours. Cells were harvested, mixed at a ratio 1:1 and fractionated into cytoplasmic and nuclear fractions. Precipitated proteins were digested with trypsin and analyzed via liquid chromatography MS/MS to quantify differences in peptide abundance. Results were further analyzed using the Ingenuity Pathway Analysis (IPA). ( B ) Western blot analysis of the XRCC1 levels in WCE made from cells used in ( A ). α-tubulin was used as loading control. ( C ) Representative Comets for Alkaline Comet assay of siXRCC1 and control cells. Scale bar = 50 μm. ( D ) Quantification of three independent Alkaline Comet assay experiments. Values are mean ± SD. The P -value was calculated using Student's two-tailed t -test. (E and F) Venn diagrams showing all detected proteins (E) or proteins up- or downregulated ≥2-fold (F) upon XRCC1 KD, as detected by IPA.

Article Snippet: Human primary fibroblasts (TIG-1) were purchased from Coriell and cultured under standard conditions in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, 21885-108) supplemented with 15% Fetal Calf Serum (FCS).

Techniques: Liquid Chromatography, Tandem Mass Spectroscopy, Western Blot, Alkaline Single Cell Gel Electrophoresis, Two Tailed Test

Journal: BMC Genomics

Article Title: A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains

doi: 10.1186/1471-2164-8-39

Figure Lengend Snippet: Subcellular localization of human and mouse NEAT1 and NEAT2/MALAT-1 . (A) Tig1 cells: Human NEAT1 RNA (green) distributes in discrete foci through the nucleus and is not detected in the cytoplasm. (B) Tig1 cells: NEAT1 RNA (red) is transcribed from 2 loci early in G1 (left) and occasionally shows a highly polarized pattern (right). (C) Tig1 cells: NEAT2/MALAT-1 RNA (red) is not detected in the cytoplasm at interphase and concentrates in domains in the nucleus with weaker signal apparent throughout the nucleoplasm. (D) NIH-3T3s: Mouse Neat2/Malat-1 RNA (green) localizes as bright nucleoplasmic signal in some cells and concentrates in domains in others. (E) NEAT1 RNA (red) does not localize to its parent chromosome 11 (green). (F) NEAT2/MALAT-1 RNA (red) rapidly disperses from its site of transcription (Chromosome 11 (green)) and distributes throughout the nucleus. (G) Tig1 cells: NEAT1 RNA (green) and NEAT2/MALAT-1 RNA are associated in the interphase nucleus. (H) MEFs: Mouse Neat1 RNA (red) is exclusively nuclear, and is found in fewer, larger foci than in human cells, yet shows the same peripheral association with Neat2/Malat-1 RNA (green), and neither RNA associates significantly with DAPI dense heterochromatin (blue).

Article Snippet: Normal human female diploid fibroblast cells TIG1 (Coriell); mouse fibroblasts NIH-3T3 (ATCC); primary mouse embryo fibroblasts (MEFs) (Cell Essentials) were cultured under normal conditions.

Techniques:

Journal: BMC Genomics

Article Title: A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains

doi: 10.1186/1471-2164-8-39

Figure Lengend Snippet: Human and mouse NEAT1 and NEAT2/MALAT-1 are intimately associated with SC35 domains . (A) Tig1 cells: NEAT2 /MALAT-1 RNA (red) is found in every SC35 domain (green); (B) NIH-3T3s: Neat2/Malat-1 RNA (green) concentrates in domains in about 50% of the population, when it does, it overlaps with SRM-300 speckles (red); (C,D,E) NEAT2/MALAT-1 RNA (green (C)) and a probe to poly(A) (red (D)) overlap (yellow (E)) in domains in the nucleus; (F) Neat1 RNA (red) preferentially localizes to the periphery of SRM-300 domains (green) in mouse cells; (G) NEAT1 RNA (red) preferentially associates at the periphery of SC35 domains (green) in human cells; (H) Two transcription sites of NEAT2/MALAT-1 RNA (red) localize with SC35 domains (green) in early G1 daughter cells; (I) Poly(A) domains (red) are apparently formed prior6=- to the spread of NEAT2/MALAT-1 RNA (green) in early G1.

Article Snippet: Normal human female diploid fibroblast cells TIG1 (Coriell); mouse fibroblasts NIH-3T3 (ATCC); primary mouse embryo fibroblasts (MEFs) (Cell Essentials) were cultured under normal conditions.

Techniques:

Journal: Nucleic Acids Research

Article Title: MicroRNA-296 is enriched in cancer cells and downregulates p21 WAF1 mRNA expression via interaction with its 3′ untranslated region

doi: 10.1093/nar/gkr492

Figure Lengend Snippet: Exogenous miR-296 expression inactivated the p53-21 WAF1 pathway. ( A ) Immortal JFCF6-derived cells (6B, telomerase-positive and 3C, telomerase-negative) cells transfected with miR-296 showed decreased levels of p53. p21 WAF1 showed decrease in telomerase-positive (JFCF6-6B) cells only. ( B ) Normal human fibroblasts transfected with miR-296 expression vector showing GFP fluorescence. ( C ) Normal human fibroblasts transfected with miR-296 showed a decrease in p21 WAF1 protein. ( D ) Single-cell immunostaining for p53 in control and pCXbG-miR-296 transfected U-2 OS cells. Cells showing GFP fluorescence (GFP-miR-296) either lacked or showed faint nuclear staining for p53 (red).

Article Snippet: Breast carcinoma cells (MCF 7) and normal human fibroblasts (TIG-1 and MRC5) were obtained from the Japanese Collection of Research Bioresources (JCRB, Japan).

Techniques: Expressing, Derivative Assay, Transfection, Plasmid Preparation, Fluorescence, Immunostaining, Control, Staining

Journal: Nucleic Acids Research

Article Title: MicroRNA-296 is enriched in cancer cells and downregulates p21 WAF1 mRNA expression via interaction with its 3′ untranslated region

doi: 10.1093/nar/gkr492

Figure Lengend Snippet: miR-296 regulates p53-p21 WAF1 pathway. ( A ) RT–PCR amplification of miR-296 from cells treated with either precursor PmiR-296 or antagonist AmiR-296. ( B ) p53 and p21 WAF1 protein levels in cells transfected with synthetic precursor miR-296 (PmiR-296) and antagonist (AmiR-296) of miR-296. PmiR-296 transfected cells showed decreased levels of p53 and p21 WAF1 proteins. The decrease was more pronounced in telomerase-positive (MCF 7 and JFCF6-6B) than in the telomerase-negative (U-2 OS and JFCF6-3C) cells. PmiR-296 transfected normal human fibroblasts showed a sharp decrease in p21 WAF1 . Cells transfected with miR-296 antagonist (AmiR-296, Lane 3) showed an increase in p21 WAF1 and co-transfections of AmiR-296 with PmiR-296 (lane 4) partly antagonized the effect of PmiR-296 (lane 2). ( C ) RT–PCR analysis for miR-296 transcript in cells transfected with synthetic precursor miR-296 (PmiR-296) and antagonist (AmiR-296) of miR-296. PmiR-296 caused reduction in p53 and p21 WAF1 transcripts (lane 2). AmiR-296 caused increase in miR-296 transcript (lane 3) and decrease in p53 and p21 WAF1 obtained by PmiR-296 (lane 2) was compensated by co-transfection with Amir-296 (lane 4).

Article Snippet: Breast carcinoma cells (MCF 7) and normal human fibroblasts (TIG-1 and MRC5) were obtained from the Japanese Collection of Research Bioresources (JCRB, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Transfection, Cotransfection