Journal: PLoS Genetics

Article Title: Pregnancy-Induced Noncoding RNA ( PINC ) Associates with Polycomb Repressive Complex 2 and Regulates Mammary Epithelial Differentiation

doi: 10.1371/journal.pgen.1002840

Figure Lengend Snippet: (A) RT-PCR shows multiple splice forms of mPINC1.0 and mPINC1.6 are expressed during mammary gland development. Primers designed to the extreme ends of mPINC1.0 and mPINC1.6 were used to amplify cDNA from mammary gland developmental stages. (w Vir.: weeks old virgin, d Preg.: days pregnancy, d Lac.: days lactation, d Inv.: days involution). PCR products were sequenced and found to be new splice forms of mPINC , including a new splice variant of mPINC1.6 called DCR2 , for deleted conserved region 2. (B) Schematic diagram of mPINC exonic structure. Black boxes represent exons that are always included, grey boxes are sometimes included and clear boxes are never included. Nucleotide length is indicated above each exon along with black lines that overlap the most conserved regions of the PINC locus, CR1 and CR2. Exon 6 sometimes has an additional 24 nucleotides at the 3′ end in the mPINC1.0 and mPINC1.6 splice forms. This alternative splice site does not correlate with the inclusion/exclusion of any particular exon and its function is unknown. (C) qPCR shows mPINC is highest during late pregnancy and early involution. Mammary glands were harvested from 3 female Balb/c mice for each stage (V: adult virgin, dP: days pregnant, dL: days lactation, dI: days involution). Target genes were normalized to Actb and set relative to levels in the virgin mammary gland. (D) mPINC expression is most abundant in the mammary gland. Tissues were harvested from three 10 week old virgin Balb/c female mice and testis, epididymis, and prostate was harvested from three 12 week old male Balb/c mice. ND indicates tissues in which mPINC was not detected by qPCR. Target genes were normalized to Actb and set relative to levels in the lung.

Article Snippet: For Taqman qPCR, the following probe sets were purchased from Applied Biosystems: mPINC1.0 -Mm03456232_m1, mPINC1.6 -Mm03456230_m1, mPINC -Mm03456228_m1, Gapdh -Mm99999915_g1, Csn2 -Mm00839664_m1, Wap -Mm00839913_m1, Ltf -Mm00434787_m1, Edn1 -Mm00438656_ml, Wnt4 -Mm01194003_m1, Dlk2 -Mm01281511_g1, Ly6a -Mm00726565_s1, Pip -Mm00476800_m1, Chi3l1 -Mm00801477_m1, Itgb3 -Mm00443980_m1, Ednra -Mm01243722_m1.

Techniques: Reverse Transcription Polymerase Chain Reaction, Variant Assay, Expressing

Journal: PLoS Genetics

Article Title: Pregnancy-Induced Noncoding RNA ( PINC ) Associates with Polycomb Repressive Complex 2 and Regulates Mammary Epithelial Differentiation

doi: 10.1371/journal.pgen.1002840

Figure Lengend Snippet: (A) Genomic representation of the PINC locus showing alternate isoforms mPINC1.0 , mPINC1.6 and DCR2 . Regions of evolutionarily conserved RNA structures were predicted by SISSIz (see ). (B–D) Circle plot secondary structure representations of interacting nucleotides in mPINC1.0 (B), mPINC1.6 (C) and DCR2 (D). The probability of the interactions is color-coded according to the legend (right). Secondary structures were computed using the RNAstructure software (see ). (E–F) High-confidence localized hairpin structures that represent possible protein-interaction sites and their most stable 3D representations, as modelled by MC-FOLD/MC-SYM. The corresponding regions in B–D are indicated by S1, for structure 1 (E) and by S2, for structure 2 (F) grey arcs.

Article Snippet: For Taqman qPCR, the following probe sets were purchased from Applied Biosystems: mPINC1.0 -Mm03456232_m1, mPINC1.6 -Mm03456230_m1, mPINC -Mm03456228_m1, Gapdh -Mm99999915_g1, Csn2 -Mm00839664_m1, Wap -Mm00839913_m1, Ltf -Mm00434787_m1, Edn1 -Mm00438656_ml, Wnt4 -Mm01194003_m1, Dlk2 -Mm01281511_g1, Ly6a -Mm00726565_s1, Pip -Mm00476800_m1, Chi3l1 -Mm00801477_m1, Itgb3 -Mm00443980_m1, Ednra -Mm01243722_m1.

Techniques: Software

Journal: International Journal of Molecular Sciences

Article Title: ceRNA Network of lncRNA/miRNA as Circulating Prognostic Biomarkers in Non-Hodgkin Lymphomas: Bioinformatic Analysis and Assessment of Their Prognostic Value in an NHL Cohort

doi: 10.3390/ijms23010201

Figure Lengend Snippet: In silico analysis of the lncRNAs targeting hsa-miR-150-5p and hsa-miR-335-5p. ( a ) LncRNAs that target hsa-miR-150-5p according to StarBase database analysis; ( b ) details about the binding site of hsa-miR-150-5p on MALAT1, predicted by StarBase database; ( c ) lncRNAs that target hsa-miR-335-5p according to StarBase database analysis; ( d ) details about the binding site of hsa-miR-335-5p on NEAT1, predicted by StarBase database.

Article Snippet: Quantification of the miRNA and lncRNA expression levels were performed by qPCR using a StepOneTM qPCR Real-Time PCR machine, using 1× TaqMan ® Gene Expression Master mix (Thermo Fisher Scientific ® ) and 1× probes TaqMan ® MicroRNA Assays (hsa-miR-150-5p: 000473 and hsa-miR-335-5p: 002185) and using TaqMan ® Noncoding RNA assays (MALAT1: Hs00273907 and NEAT1: Hs01008264_s1) (Thermo Fisher Scientific ® ).

Techniques: In Silico, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: ceRNA Network of lncRNA/miRNA as Circulating Prognostic Biomarkers in Non-Hodgkin Lymphomas: Bioinformatic Analysis and Assessment of Their Prognostic Value in an NHL Cohort

doi: 10.3390/ijms23010201

Figure Lengend Snippet: Expression levels of MALAT1 and NEAT1 in plasma samples of NHL patients’ groups according to lymphoma grade. MALAT1 and NEAT1 presented higher plasma levels in patients diagnosed with high grade lymphoma compared to low grade lymphoma ( p = 0.035 and p = 0.018, respectively). * p < 0.05.

Article Snippet: Quantification of the miRNA and lncRNA expression levels were performed by qPCR using a StepOneTM qPCR Real-Time PCR machine, using 1× TaqMan ® Gene Expression Master mix (Thermo Fisher Scientific ® ) and 1× probes TaqMan ® MicroRNA Assays (hsa-miR-150-5p: 000473 and hsa-miR-335-5p: 002185) and using TaqMan ® Noncoding RNA assays (MALAT1: Hs00273907 and NEAT1: Hs01008264_s1) (Thermo Fisher Scientific ® ).

Techniques: Expressing, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: ceRNA Network of lncRNA/miRNA as Circulating Prognostic Biomarkers in Non-Hodgkin Lymphomas: Bioinformatic Analysis and Assessment of Their Prognostic Value in an NHL Cohort

doi: 10.3390/ijms23010201

Figure Lengend Snippet: OS ( a ) and PFS ( b ) of NHL patients according to plasma levels of hsa-miR-150-5p, hsa-miR-335-5p, MALAT1 and NEAT1.

Article Snippet: Quantification of the miRNA and lncRNA expression levels were performed by qPCR using a StepOneTM qPCR Real-Time PCR machine, using 1× TaqMan ® Gene Expression Master mix (Thermo Fisher Scientific ® ) and 1× probes TaqMan ® MicroRNA Assays (hsa-miR-150-5p: 000473 and hsa-miR-335-5p: 002185) and using TaqMan ® Noncoding RNA assays (MALAT1: Hs00273907 and NEAT1: Hs01008264_s1) (Thermo Fisher Scientific ® ).

Techniques: Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: ceRNA Network of lncRNA/miRNA as Circulating Prognostic Biomarkers in Non-Hodgkin Lymphomas: Bioinformatic Analysis and Assessment of Their Prognostic Value in an NHL Cohort

doi: 10.3390/ijms23010201

Figure Lengend Snippet: Definition of high-, intermediate- and low-risk groups considering the combination of the plasma levels of each ceRNA pair, hsa-miR-150hsa-miR-150-5p/MALAT1 plasma levels and hsa-miR-335hsa-miR-335-5p/NEAT1 plasma levels.

Article Snippet: Quantification of the miRNA and lncRNA expression levels were performed by qPCR using a StepOneTM qPCR Real-Time PCR machine, using 1× TaqMan ® Gene Expression Master mix (Thermo Fisher Scientific ® ) and 1× probes TaqMan ® MicroRNA Assays (hsa-miR-150-5p: 000473 and hsa-miR-335-5p: 002185) and using TaqMan ® Noncoding RNA assays (MALAT1: Hs00273907 and NEAT1: Hs01008264_s1) (Thermo Fisher Scientific ® ).

Techniques: Clinical Proteomics

Journal: Cell metabolism

Article Title: The MYC Oncogene Cooperates with Sterol Regulated Element-Binding Protein To Regulate Lipogenesis Essential for Neoplastic Growth

doi: 10.1016/j.cmet.2019.07.012

Figure Lengend Snippet: Upregulation of lipid metabolic genes by MYC. (A) Diagram of MYC’s regulation of the fatty acid synthesis pathway. (B) qPCR shows fatty acid synthesis mRNA expression in high MYC (MYC ON, N=3 per cell line) compared to 24 hours of MYC inactivation (MYC OFF, N=3 per cell line) in BCL (P493–6) and RCC (E28). Expression of lipogenesis proteins for BCL (P493–6) and RCC (E28) cells with MYC ON vs. MYC OFF, by Western blot analysis. Statistical significance by t-test, ***P<0.001. (C) RNA-seq analysis of lipid metabolic pathways in fold-change of RPKM comparing MYC ON/MYC OFF in BCL (P493–6).

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-SREBP1 (clone 2A4) [1:1000] home-made antibody N/A Rabbit monoclonal anti-MYC (Y69) [1:1000] Abcam CAS# ab32072 IgG from mouse serum [1:1000] Sigma CAS# I5006 IgG from rabbit serum [1:1000] Sigma CAS# I5381 MYC [1:1000] Abcam Ab32072 SREBP1 [1:1000] Novus Biologicals NB600–582 SCD1 [1:1000] Cell Signaling Tech CST 2794 FASN [1:500] Sigma-Aldrich F9554 ACLY [1:1000] Cell Signaling Tech CST 4332S ACACA [1:1000] Cell Signaling Tech CST 3661 Chemicals, Peptides, and Recombinant Proteins Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) Sigma-Aldrich CAS# C2920 Rotenone Sigma-Aldrich CAS# R8875 Seahorse XFe96 FluxPak Agilent Technologies CAS# 102416–100 XF Base Medium Seahorse Bioscience CAS# 102353–100 D-Glucose-1- 13 C Sigma 297046 L-Glutamine- 13 C 5 Sigma 605166 Critical Commercial Assays ChIP-IT® Express Chromatin Immunoprecipitation Kits Active Motif 53008 Qiagen RNA Extraction kit (RNEasy Plus Kit) Qiagen 74134 Deposited Data MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels.

Techniques: Expressing, Western Blot, RNA Sequencing Assay

Journal: Cell metabolism

Article Title: The MYC Oncogene Cooperates with Sterol Regulated Element-Binding Protein To Regulate Lipogenesis Essential for Neoplastic Growth

doi: 10.1016/j.cmet.2019.07.012

Figure Lengend Snippet: MYC regulates SREBP and together they regulate lipogenesis. (A) mRNA expression of fatty acid synthesis genes: Acly, Acaca, Fasn, and Scd1 upon siRNA knockdown of SREBP1 or scramble RNA in HCC(EC4) under MYC ON and OFF conditions (N=3 per condition). Statistical significance by two-way ANOVA using Tukey’s multiple comparisons post tests, ***P<0.001. Overall, ***P<0.001 for SREBP1 effect, ***P<0.001 for MYC effect and ***P<0.001 for interaction between the two. (B) SREBP1 ChIP-qPCR on BCL P493–6 line shows decreased binding of SREBP1 upon MYC inactivation (top panel). MYC ChIP-qPCR on HCC EC4 line shows no change of MYC binding upon SREBP1 knockdown (bottom panel). (C) Glucose and glutamine incorporation into lipids by NMR in MYC ON (upper panel) and OFF (bottom panel) in BCL(P493–6). For close-up look see Figure S3B. (D) Incorporation of glucose and glutamine carbons to de novo synthesized fatty acids by mass spectrometry based on the fold change of 2C13 peak in BCL(P493–6) as measured upon 24 hours of MYC inactivation (N=5). Fold change in the de novo fatty acid synthesis when MYC is activated or inactivated (left panel); and glucose and glutamine incorporation into fatty acids when MYC is activated (middle panel), or inactivated (right panel). Statistical significance by t-test, **P<0.01, ***P<0.001.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-SREBP1 (clone 2A4) [1:1000] home-made antibody N/A Rabbit monoclonal anti-MYC (Y69) [1:1000] Abcam CAS# ab32072 IgG from mouse serum [1:1000] Sigma CAS# I5006 IgG from rabbit serum [1:1000] Sigma CAS# I5381 MYC [1:1000] Abcam Ab32072 SREBP1 [1:1000] Novus Biologicals NB600–582 SCD1 [1:1000] Cell Signaling Tech CST 2794 FASN [1:500] Sigma-Aldrich F9554 ACLY [1:1000] Cell Signaling Tech CST 4332S ACACA [1:1000] Cell Signaling Tech CST 3661 Chemicals, Peptides, and Recombinant Proteins Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) Sigma-Aldrich CAS# C2920 Rotenone Sigma-Aldrich CAS# R8875 Seahorse XFe96 FluxPak Agilent Technologies CAS# 102416–100 XF Base Medium Seahorse Bioscience CAS# 102353–100 D-Glucose-1- 13 C Sigma 297046 L-Glutamine- 13 C 5 Sigma 605166 Critical Commercial Assays ChIP-IT® Express Chromatin Immunoprecipitation Kits Active Motif 53008 Qiagen RNA Extraction kit (RNEasy Plus Kit) Qiagen 74134 Deposited Data MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels.

Techniques: Expressing, Binding Assay, Synthesized, Mass Spectrometry

Journal: Cell metabolism

Article Title: The MYC Oncogene Cooperates with Sterol Regulated Element-Binding Protein To Regulate Lipogenesis Essential for Neoplastic Growth

doi: 10.1016/j.cmet.2019.07.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-SREBP1 (clone 2A4) [1:1000] home-made antibody N/A Rabbit monoclonal anti-MYC (Y69) [1:1000] Abcam CAS# ab32072 IgG from mouse serum [1:1000] Sigma CAS# I5006 IgG from rabbit serum [1:1000] Sigma CAS# I5381 MYC [1:1000] Abcam Ab32072 SREBP1 [1:1000] Novus Biologicals NB600–582 SCD1 [1:1000] Cell Signaling Tech CST 2794 FASN [1:500] Sigma-Aldrich F9554 ACLY [1:1000] Cell Signaling Tech CST 4332S ACACA [1:1000] Cell Signaling Tech CST 3661 Chemicals, Peptides, and Recombinant Proteins Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) Sigma-Aldrich CAS# C2920 Rotenone Sigma-Aldrich CAS# R8875 Seahorse XFe96 FluxPak Agilent Technologies CAS# 102416–100 XF Base Medium Seahorse Bioscience CAS# 102353–100 D-Glucose-1- 13 C Sigma 297046 L-Glutamine- 13 C 5 Sigma 605166 Critical Commercial Assays ChIP-IT® Express Chromatin Immunoprecipitation Kits Active Motif 53008 Qiagen RNA Extraction kit (RNEasy Plus Kit) Qiagen 74134 Deposited Data MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels.

Techniques: Recombinant, Chromatin Immunoprecipitation, RNA Extraction, Transgenic Assay, Software