cdna sequence encoding plk1  (Cusabio)

Journal: Science Advances

Article Title: HILPS , a long noncoding RNA essential for global oxygen sensing in humans

doi: 10.1126/sciadv.adi1867

Figure Lengend Snippet: ( A ) Volcano plots showing HILPS interacting proteins pulled down from 293T cells exposed to 1% O 2 using biotin-labeled HILPS . Proteins significantly enriched (fold change > 2 and P < 0.01) from three independent experiments are displayed as red dots. ( B ) Biotin-labeled HILPS pull-down assay using 293T cell lysates with respective hemagglutinin (HA)–tagged protein expression. IB, immunoblot. ( C ) Biotin-labeled HILPS pull-down assay to detect endogenous PLK1 in 1% O 2 -treated cells. ( D ) In vitro RNA pull-down assay using biotin-labeled HILPS and recombinant GST-PLK1. ( E ) Immunoblots of PLK1 in HILPS -depleted NCM460 and RKO cells exposed to 1% O 2 . ( F ) Immunoblots of PLK1 in HILPS -depleted NCM460 and RKO cells treated with MG132 (10 μM) for 6 hours before harvest. ( G ) Time-course analysis of PLK1 degradation. HILPS -depleted NCM460 and RKO cells were cultured in 1% O 2 , treated with cycloheximide (CHX; 100 μg/ml) and harvested at the indicated time points, followed by immunoblotting of PLK1. ( H ) Analysis of PLK1 polyubiquitination in the presence or absence of HILPS in 293T cells. ( I ) Immunoblots of PLK1 and CDH1 in 1% O 2 -cultured NCM460 and RKO cells expressing shRNA targeting HILPS and/or CDH1 . Asterisk denotes a nonspecific band detected by CDH1 antibody. ( J ) Immunoblots of indicated proteins in 293T cells transfected with HA-PLK1, Flag-CDH1, and/or HILPS . ( K ) Co-IP to detect protein interaction between PLK1 and CDH1 in the absence or presence of HILPS . 293T cells were treated with MG132 (10 μM) for 6 hours before harvest. ( L ) In vitro protein binding assay to detect PLK1-CDH1 interaction in the absence or presence of in vitro transcribed HILPS .

Article Snippet: His-CDH1 protein was purchased from CUSABIO. cDNA sequence encoding PLK1 was cloned into the pGEX-4T vector, in frame with GST.

Techniques: Labeling, Pull Down Assay, Expressing, Western Blot, In Vitro, Recombinant, Cell Culture, shRNA, Transfection, Co-Immunoprecipitation Assay, Protein Binding

Journal: Science Advances

Article Title: HILPS , a long noncoding RNA essential for global oxygen sensing in humans

doi: 10.1126/sciadv.adi1867

Figure Lengend Snippet: ( A to C ) Venn diagram of differentially expressed genes (DEGs) resulting from 1% O 2 -cultured H9 cells expressing sh HILPS #1 or sh HILPS #2 (A). Heatmap presentation (B) and enrichment analysis (C) of 4453 merged DEG in (A). Pathway enrichment was analyzed by Metascape ( www.metascape.org ). TNFα, tumor necrosis factor–α; NFκB, nuclear factor κB. ( D ) Gene set enrichment analysis of HIF1α target gene sets in the expression profiles of H9 cells expressing HILPS shRNA#1 ( software.broadinstitute.org/gsea ). NES, normalized enrichment score; FDR, false discovery rate. ( E ) qPCR analysis of HIF1α target genes in HILPS -depleted H9 cells exposed to 1% O 2 . ( F ) qPCR analysis of HIF1 α mRNA in HILPS -depleted cells exposed to 1% O 2 . ( G ) Immunoblots of HIF1α in HILPS -depleted cells treated with MG132 (10 μM) for 6 hours before harvest. ( H ) Biotin-labeled HILPS pull-down assay using lysates from 293T cells expressing tagged proteins. ( I ) Immunoblots of HIF1α and PLK1 in HILPS -depleted cells with or without Flag-PLK1 overexpression. ( J ) Immunoblots of HIF1α and PLK1 in PLK1 -depleted (top) or BI6727-treated (bottom) RKO cells subjected to 1% O 2 exposure. ( K and L ) Time-course analysis of HIF1α degradation in PLK1 -depleted (top) or BI6727-treated (bottom) RKO cells subjected to 1% O 2 exposure. HIF1α was analyzed by immunoblotting (K) and quantified as shown in (L). ( M ) Cell death analysis of HILPS -depleted cells cultured in 1% O 2 with or without ectopic HIF1α. ( N and O ) Tumor growth of HILPS -depleted HCT116 xenografts with or without ectopic HIF1α ( n = 5) (N). Tumors were dissected 2 weeks after engraftment (O). Data shown are means ± SD from biological triplicates. ** P < 0.01 and *** P < 0.001; unpaired two-tailed Student’s t test [(E) and (F)] and one-way ANOVA [(M) and (N)].

Article Snippet: His-CDH1 protein was purchased from CUSABIO. cDNA sequence encoding PLK1 was cloned into the pGEX-4T vector, in frame with GST.

Techniques: Cell Culture, Expressing, shRNA, Software, Western Blot, Labeling, Pull Down Assay, Over Expression, Two Tailed Test

Journal: Science Advances

Article Title: HILPS , a long noncoding RNA essential for global oxygen sensing in humans

doi: 10.1126/sciadv.adi1867

Figure Lengend Snippet: ( A ) Co-IP of endogenous HIF1α and PLK1 using lysates of 1% O 2 -cultured NCM460 and RKO cells. ( B ) Immunoblots of indicated proteins in 293T cells expressing Flag-HIF1α, HA-VHL, and/or PLK1 (T210D and K82R). ( C ) Time-course analysis of Flag-HIF1α in 293T cells coexpressing PLK1 T210D or K82R. Flag-HIF1α protein levels were analyzed by immunoblotting. ( D ) In vitro kinase assay of recombinant GST-HIF1α catalyzed by Flag-tagged PLK1 T210D or K82R purified from 293T cells. Phosphorylated proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography. Loading controls were shown as Coomassie blue staining in the bottom panels. ( E ) In vitro kinase analysis of recombinant GST-HIF1α. Active human WT PLK1 proteins were incubated with GST-HIF1α (WT, T218A, or S463A) for kinase reaction. HIF1α phosphorylation signals were detected as in (D). ( F ) Peptide sequence alignment of HIF1α (amino acid 215 to 221) in multiple species. The threonine-218 residue is highlighted in red. ( G and H ) Time-course analysis of Flag-HIF1α WT or mutants (T218D or T218A) degradation in 293T cells by immunoblot (G) with quantification shown in (H). ( I ) Analysis of Flag-HIF1α WT or mutants (T218D or T218A) polyubiquitylation. Myc-tagged ubiquitin was cotransfected with Flag-HIF1α (WT or mutants) into 293T cells, and the resulting cell lysates were subjected to co-IP, followed by polyubiquitylation detection. ( J ) Co-IP of Flag-HIF1α (WT or mutants) and HA-VHL using 293T cell lysate with epitope-tagged proteins expression. MG132 (10 μM) was added to prevent HIF1α degradation. Data shown are means ± SD from biological triplicates.

Article Snippet: His-CDH1 protein was purchased from CUSABIO. cDNA sequence encoding PLK1 was cloned into the pGEX-4T vector, in frame with GST.

Techniques: Co-Immunoprecipitation Assay, Cell Culture, Western Blot, Expressing, In Vitro, Kinase Assay, Recombinant, Purification, Polyacrylamide Gel Electrophoresis, Autoradiography, Staining, Incubation, Phospho-proteomics, Sequencing, Residue, Ubiquitin Proteomics

Journal: Science Advances

Article Title: HILPS , a long noncoding RNA essential for global oxygen sensing in humans

doi: 10.1126/sciadv.adi1867

Figure Lengend Snippet: ( A ) qPCR analysis of HILPS in human normal and tumor organoids exposed to 21 or 1% O 2 for 24 hours. ( B ) Immunoblots of PLK1 and HIF1α in 1% O 2 -treated human organoids derived from normal colon tissue and colorectal cancer upon HILPS depletion. ( C to E ) Heatmap presentation of all DEG upon HILPS depletion by shRNA#1 in colorectal cancer organoids exposed to 1% O 2 (C). Pathway enrichment analysis of DEG by Metascape (D). Gene set enrichment analysis of HIF1α target gene sets in the expression profiles of HILPS -depleted colorectal cancer organoids (E). ( F ) Proliferation of colorectal cancer organoids exposed to 1% O 2 upon HILPS depletion. Organoid proliferation was assessed by CCK-8. OD, optical density. ( G ) Representative bright field images of colorectal cancer organoids cultured under 1% O 2 upon HILPS depletion (left). Scale bars, 200 μm. Quantitation of viable organoid colonies is shown on the right. ( H ) Representative images showing bright-field view and immunofluorescence staining of Ki67 in HILPS -depleted colorectal cancer organoids cultured under 1% O 2 (left). DAPI was stained to visualize the organoids in dark field. Scale bar, 200 μm. Quantitation of Ki67 positive staining cells per budding are presented (right). ( I ) Schematic depicting patient-derived organoid–based xenograft model. ( J ) Representative images of colorectal cancer organoid–based tumors in the subrenal capsule with or without HILPS depletion (left) and measurement of tumor volumes ( n = 5) (right). Data shown are means ± SD from biological triplicates. ** P < 0.01 and *** P < 0.001; unpaired two-tailed Student’s t test [(A), (F), (G), and (H)] and one-way ANOVA (J).

Article Snippet: His-CDH1 protein was purchased from CUSABIO. cDNA sequence encoding PLK1 was cloned into the pGEX-4T vector, in frame with GST.

Techniques: Western Blot, Derivative Assay, shRNA, Expressing, CCK-8 Assay, Cell Culture, Quantitation Assay, Immunofluorescence, Staining, Two Tailed Test