Journal: Oncology Letters

Article Title: Oncogenic function of angiopoietin-2 in vitro and its modulation of tumor progression in colorectal carcinoma

doi: 10.3892/ol.2017.6203

Figure Lengend Snippet: Ang-2 expression was investigated by reverse-transcription polymerase chain reaction and western blot analysis in colorectal cancer cell lines, HUVEC and normal cell line (CCD-841 CoN). (A) Among 5 colorectal cancer cell lines, 3 cell lines expressed Ang-2 mRNA, as did the HUVEC cell line. (B) LoVo cells expressed a markedly higher level of Ang-2 protein compared with other colorectal cancer cell lines, as detected by western blotting. (C) The LoVo cell line transfected with Ang-2 target shRNA did not express Ang-2 mRNA. (D) The LoVo cell transfected with Ang-2 target shRNA exhibited markedly decreased expression levels of Ang-2 protein. Ang-2, Angiopoietin-2.

Article Snippet: LoVo cells (1×10 6 ) were grown overnight in RPMI 1640 medium in six-well culture plates to 60–70% confluence prior to transfection with Ang-2 shRNA lentiviral particles (TRCN000005923, TRCN000005924, TRCN000005925, TRCN000005926, and TRCN000005927), which were purchased from Sigma-Aldrich (Merck Millipore) along with a non-target negative control shRNA (SHC002V).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Transfection, shRNA

Journal: Oncology Letters

Article Title: Oncogenic function of angiopoietin-2 in vitro and its modulation of tumor progression in colorectal carcinoma

doi: 10.3892/ol.2017.6203

Figure Lengend Snippet: (A) The proliferation of LoVo cells following Ang-2-targeted shRNA transfection was significantly decreased compared with control LoVo cells and LoVo cell transfected with non-targeted shRNA. (B) The invasion ability of Ang-2 target shRNA transfected LoVo cells was decreased markedly compared with the control and non-target shRNA-transfected LoVo cells. ***P<0.05. Ang-2, Angiopoietin-2; shRNA, short hairpin RNA.

Article Snippet: LoVo cells (1×10 6 ) were grown overnight in RPMI 1640 medium in six-well culture plates to 60–70% confluence prior to transfection with Ang-2 shRNA lentiviral particles (TRCN000005923, TRCN000005924, TRCN000005925, TRCN000005926, and TRCN000005927), which were purchased from Sigma-Aldrich (Merck Millipore) along with a non-target negative control shRNA (SHC002V).

Techniques: shRNA, Transfection, Control

Journal: Oncology Letters

Article Title: Oncogenic function of angiopoietin-2 in vitro and its modulation of tumor progression in colorectal carcinoma

doi: 10.3892/ol.2017.6203

Figure Lengend Snippet: The migration of Ang-2 target shRNA transfected LoVo cells was decreased compared with the control and non-target shRNA transfected LoVo cells. ***P<0.01. Ang-2, Angiopoietin-2; shRNA, short hairpin RNA.

Article Snippet: LoVo cells (1×10 6 ) were grown overnight in RPMI 1640 medium in six-well culture plates to 60–70% confluence prior to transfection with Ang-2 shRNA lentiviral particles (TRCN000005923, TRCN000005924, TRCN000005925, TRCN000005926, and TRCN000005927), which were purchased from Sigma-Aldrich (Merck Millipore) along with a non-target negative control shRNA (SHC002V).

Techniques: Migration, shRNA, Transfection, Control

Journal: Journal of Cellular Physiology

Article Title: Overexpression of miR‐181a‐5p inhibits retinal neovascularization through endocan and the ERK1/2 signaling pathway

doi: 10.1002/jcp.29733

Figure Lengend Snippet: miR‐181a‐5p suppresses VEGF‐mediated activation of ERK1/2 pathways through endocan in HRECs. (a) Western blot analysis of phosphorylated ERK1/2 and total ERK. HRECs were incubated with rhVEGFA (20 ng/ml) for 5, 15, 30, 60, and 120 min in a time‐dependent manner. Phosphorylation of ERK1/2 was detected by western blot analyses. (b) Relative protein levels were normalized to total ERK or GAPDH and expressed as the fold change relative to the untreated group. (c) HRECs were transfected with miR‐181a‐5p mimic or nontargeting mimic control (miR‐NC), siRNA_endocan or negative control (siRNA_Ctrl), or miR‐181a‐5p mimic plus endocan expression plasmid (pIRES2_endocan), then exposed to rhVEGFA (20 ng/ml, 30 min). Phosphorylation of ERK1/2 was detected by western blot. (d) Relative protein levels were normalized to total ERK or GAPDH and expressed as the fold change relative to the untreated group. Data in graphs represent means ± SEM . One‐way ANOVA followed by Dunnett's post hoc test was performed for all analyses. ** p < .01, *** p < .001 versus the untreated group. ANOVA, analysis of variance; ERK1/2, extracellular signal‐regulated protein kinases 1 and 2; HREC, human retinal endothelial cell; SEM , standard error of the mean; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor

Article Snippet: AgomiR, agomiR control, miRNA mimic, inhibitor, and nontargeting control oligonucleotides (RiboBio, Guangzhou, China) were transfected into HRECs using riboFECT TM CP Transfection Reagent (RiboBio).

Techniques: Activation Assay, Western Blot, Incubation, Transfection, Negative Control, Expressing, Plasmid Preparation, Small Interfering RNA

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cis -element within the ARF locus mediates repression of p16 INK4A expression via long-range chromatin interactions

doi: 10.1073/pnas.1909720116

Figure Lengend Snippet: The p16INK4A-P2A-mCherry reporter allele recapitulates endogenous transcription of p16INK4A. (A) Schematic diagram of the transcriptional repression of p16INK4A by the CRISPR interference system. The dCas9-KRAB fusion protein was guided to the p16INK4A promoter with 3 individual sgRNAs. (B) qRT-PCR was performed on the p16mCherry/+;dCas9-KRAB cells with 3 different p16INK4A-sgRNAs using primers targeting coding sequences of mCherry and p16INK4A. (C) Flow cytometry analysis was performed on the p16mCherry/+;dCas9-KRAB cells with 2 p16INK4A-sgRNAs. The mean value of the mCherry density in each group was calculated and obtained by 3 replicates. The P value was calculated by a 2-tailed t test. (D) The correlation of transcription reduction in mCherry and p16INK4A in response to dCas9-KRAB–mediated transcription repression (from B) was calculated by Pearson’s correlation test (n = 6). (E) qRT-PCR was performed on the p16mCherry/+ cells with or without the treatment of doxorubicin for 24 h by using specific primers targeting the mRNA sequence of p16INK4A. Three biological replicates were performed. The P value was calculated by a 2-tailed t test. (F) qRT-PCR was performed on the p16mCherry/+ cells with or without the treatment of doxorubicin for 24 h by using specific primers targeting the mRNA sequence of mCherry. Three biological replicates were performed. The P value was calculated by a 2-tailed t test.

Article Snippet: A set of 2,029 sgRNA oligos that target H3K27ac and ATAC-seq positive peaks defined in the TAD containing INK4 / ARF in IMR90, HCT116, and SEM cells as well as an additional 20 nontargeting control sgRNAs with no detectable match to the human genome were designed for array-based oligonucleotide synthesis (CustomArray).

Techniques: CRISPR, Quantitative RT-PCR, Flow Cytometry, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cis -element within the ARF locus mediates repression of p16 INK4A expression via long-range chromatin interactions

doi: 10.1073/pnas.1909720116

Figure Lengend Snippet: Noncoding pooled CRISPR screening identified a distal repressive element of p16INK4A residing within the ARF locus. (A) Schematic diagram of a working model of dCas9-KRAB– and Cas9-mediated noncoding screening. (B) The global correlation of sgRNA distribution in dCas9-KRAB and Cas9 screens in the p16mCherry/+ reporter cell line. (C) The global distribution of all sgRNAs in a selected region of the INK4/ARF locus from 3 screens in the p16mCherry/+ reporter cell line using dCas9-KRAB, Cas9, and no effector control. The red arrow indicates the most enriched sgRNAs in a 42-bp region of the ARF exon1β and intron 1. (D) Two candidate sgRNAs binding to the plus and minus strands of the most enriched 42-bp core DNA sequence near the ARF promoter were designed for validation (ARF-sgRNA-3 and -4). The red arrows indicate the orientation of the sgRNA binding sequences. (E) Western blotting analysis of ARF and p16INK4A in dCas9-KRAB– and Cas9-expressing SEM cells infected with ARF-sgRNA-3, -4, and the nontargeting sgRNA control.

Article Snippet: A set of 2,029 sgRNA oligos that target H3K27ac and ATAC-seq positive peaks defined in the TAD containing INK4 / ARF in IMR90, HCT116, and SEM cells as well as an additional 20 nontargeting control sgRNAs with no detectable match to the human genome were designed for array-based oligonucleotide synthesis (CustomArray).

Techniques: CRISPR, Control, Binding Assay, Sequencing, Biomarker Discovery, Western Blot, Expressing, Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cis -element within the ARF locus mediates repression of p16 INK4A expression via long-range chromatin interactions

doi: 10.1073/pnas.1909720116

Figure Lengend Snippet: Physical interactions between ARF, p16INK4A, and p15INK4B were detected by 3C. (A) Next-generation Capture-C was performed on parental human SEM cells for 2 replicates. Two specific anchor probes (Bait 1 and Bait 2) were designed to hybridize to the H3K27ac peaks that overlap each of the promoters of p16INK4A, ARF, and p15INK4B to capture chromatin interactions with the respective promoters within the INK4/ARF locus. (B) CUT&RUN was performed on p16mCherry/+;dCas9-KRAB cells infected individually with 2 sgRNAs targeting the p16INK4A repressive element adjacent to the ARF promoter and 2 sgRNAs targeting the p16INK4A promoter. A Cas9 antibody that recognizes dCas9 was used for a pull-down assay. ChIP-seq tracks of CTCF and H3K27ac were included to indicate the open chromatin status of the locus.

Article Snippet: A set of 2,029 sgRNA oligos that target H3K27ac and ATAC-seq positive peaks defined in the TAD containing INK4 / ARF in IMR90, HCT116, and SEM cells as well as an additional 20 nontargeting control sgRNAs with no detectable match to the human genome were designed for array-based oligonucleotide synthesis (CustomArray).

Techniques: Capture-C, Infection, Pull Down Assay, ChIP-sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cis -element within the ARF locus mediates repression of p16 INK4A expression via long-range chromatin interactions

doi: 10.1073/pnas.1909720116

Figure Lengend Snippet: The ARF/p16INK4A chromatin interaction and transcriptional regulation is functionally detected in NBL cells. (A) qRT-PCR analysis of p16INK4A in the human NBL cell line SK-N-SH infected with lentiviral dCas9-KRAB and ARF-sgRNAs-3 and -4. NT-sgRNA is a nontargeting sgRNA control. Four biological replicates were performed. The P value was calculated by a 2-tailed t test. (B) qRT-PCR analysis of ARF in the human NBL cell SK-N-SH infected with lentiviral dCas9-KRAB and ARF-sgRNAs-3 and -4. Four biological replicates were performed. The P value was calculated by a 2-tailed t test. (C) Western blotting analysis of ARF and p16INK4A in the human NBL cell line SK-N-SH infected with lentiviral dCas9-KRAB and ARF-sgRNA-3 and -4 compared to NT-infected controls. (D) Representative image of SK-N-SH cells stably expressing dCas9-KRAB and ARF-sgRNAs-3, -4, and NT-sgRNA at day 3. (E) Quantification of cell proliferation by cell number count. Two biological replicates were performed. The P value was calculated by a 2-tailed t test. (F) Next-generation Capture-C was performed on SK-N-SH cells stably expressing dCas9-KRAB and ARF-sgRNAs-3, -4, and NT-sgRNA. Anchor probes that hybridized to the p16INK4A promoter were utilized to capture chromatin interactions. Boxes 2 and 3 highlight ARF and p15INK4B promoters which were interacting with the indicated p16INK4A anchor regions. Box 1 indicates a negative control noncoding region. (G) Quantification of interaction frequency between the anchor regions to Boxes 1, 2, and 3. Signal value from the .bw file of Capture-C was normalized by probe signal for each experiment.

Article Snippet: A set of 2,029 sgRNA oligos that target H3K27ac and ATAC-seq positive peaks defined in the TAD containing INK4 / ARF in IMR90, HCT116, and SEM cells as well as an additional 20 nontargeting control sgRNAs with no detectable match to the human genome were designed for array-based oligonucleotide synthesis (CustomArray).

Techniques: Quantitative RT-PCR, Infection, Control, Western Blot, Stable Transfection, Expressing, Capture-C, Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cis -element within the ARF locus mediates repression of p16 INK4A expression via long-range chromatin interactions

doi: 10.1073/pnas.1909720116

Figure Lengend Snippet: Loss-of-function CRISPR screening of human transcriptional factors in the p16INK4A reporter cell line. (A) Schematic diagram of a working model of Cas9-mediated CRISPR screening targeting 1,639 human TFs. (B) Gene ranking of looping factors (YY1, first; CTCF, 1,001st) enriched from screening. The enrichment score of 7 sgRNAs against each TF was combined by MAGeCK algorithm. (C, Top) The overall distribution of all sgRNAs from the screening. (C, Bottom) The Log2[Fold Change (top10/bottom10)] ratio for all sgRNAs targeting CTCF, YY1, and the top 10 negative regulators from D and NT sgRNAs were overlaid on a gray gradient depicting the overall distribution. NT: 100 gRNAs; TF: 7 sgRNAs/each. (D) Gene ranking of top 10 negative candidate regulators enriched from screening analysis by MAGeCK algorithm. (E) Immunoblotting of YY1 in p16mCherry/+;Cas9 cells targeted individually with 2 sgRNAs against the coding sequence of human YY1. GAPDH was probed as a loading control. (F) The p16mCherry/+;Cas9 cells were targeted with Cas9 and 2 sgRNAs identified from TF screening targeting the coding region of human YY1. Flow cytometry analysis of mCherry reporter activity was subsequently conducted in YY1-targeted cells compared with NT control. (G) YY1-binding motif prediction by JASPAR algorithm. (H) CUT&RUN of YY1 was conducted in p16mCherry/+;dCas9-KRAB cells infected with ARF-sgRNA-4 and NT-sgRNA for 2 replicates. Tracks were shown at the viewpoint of the INK4/ARF locus. (I) CUT&RUN of ZNF217 was conducted in p16mCherry/+;dCas9-KRAB cells infected with ARF-sgRNA-4 and NT-sgRNA. Tracks were shown at the viewpoint of the INK4/ARF locus. (J) CUT&RUN of ZNF217 was conducted in p16mCherry/+;dCas9-KRAB cells infected with ARF-sgRNA-4 and NT-sgRNA. Tracks of ZNF217 were shown for viewpoint at a randomly chosen locus.

Article Snippet: A set of 2,029 sgRNA oligos that target H3K27ac and ATAC-seq positive peaks defined in the TAD containing INK4 / ARF in IMR90, HCT116, and SEM cells as well as an additional 20 nontargeting control sgRNAs with no detectable match to the human genome were designed for array-based oligonucleotide synthesis (CustomArray).

Techniques: CRISPR, Western Blot, Sequencing, Control, Flow Cytometry, Activity Assay, Binding Assay, Infection