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: Fig. 4. HILPS binds to PLK1 and enhances PLK1 stability. (A) Volcano plots showing HILPS interacting proteins pulled down from 293T cells exposed to 1% O2 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% O2-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% O2. (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% O2, 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% O2-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: Fig. 5. Inhibition of HILPS-PLK1 axis induces HIF1α degradation and impairs HIF1α signature gene ex- pression. (A to C) Venn diagram of differentially expressed genes (DEGs) resulting from 1% O2-cultured H9 cells expressing shHILPS#1 or shHILPS#2 (A). Heatmap presentation (B) and enrich- ment 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 enrich- ment analysis of HIF1α target gene sets in the expression profiles of H9 cells expressing HILPS shRNA#1 (software. broadinstitute.org/gsea). NES, normal- ized enrichment score; FDR, false dis- covery rate. (E) qPCR analysis of HIF1α target genes in HILPS-depleted H9 cells exposed to 1% O2. (F) qPCR analysis of HIF1α mRNA in HILPS-depleted cells exposed to 1% O2. (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) Immu- noblots of HIF1α and PLK1 in HILPS-de- pleted 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% O2 exposure. (K and L) Time-course analysis of HIF1α degrada- tion in PLK1-depleted (top) or BI6727- treated (bottom) RKO cells subjected to 1% O2 exposure. HIF1α was analyzed by immunoblotting (K) and quantified as shown in (L). (M) Cell death analysis of HILPS-depleted cells cultured in 1% O2 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 bio- logical 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: Inhibition, 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: Fig. 6. PLK1 phosphorylates HIF1α T218 and sustains HIF1α stabilization. (A) Co-IP of endogenous HIF1α and PLK1 using lysates of 1% O2-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 sub- jected 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 ex- pression. 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: Fig. 7. HILPS inhibition impedes human organoid growth under hypoxia. (A) qPCR analysis of HILPS in human normal and tumor organoids exposed to 21 or 1% O2 for 24 hours. (B) Immunoblots of PLK1 and HIF1α in 1% O2-treated human organoids derived from normal colon tissue and colorectal cancer upon HILPS deple- tion. (C to E) Heatmap presentation of all DEG upon HILPS depletion by shRNA#1 in colorectal cancer organo- ids exposed to 1% O2 (C). Pathway enrichment analysis of DEG by Meta- scape (D). Gene set enrichment anal- ysis of HIF1α target gene sets in the expression profiles of HILPS-depleted colorectal cancer organoids (E). (F) Proliferation of colorectal cancer or- ganoids exposed to 1% O2 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% O2 upon HILPS depletion (left). Scale bars, 200 μm. Quantitation of viable organoid colo- nies is shown on the right. (H) Repre- sentative images showing bright-field view and immunofluorescence stain- ing of Ki67 in HILPS-depleted colo- rectal cancer organoids cultured under 1% O2 (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 measure- ment of tumor volumes (n = 5) (right). Data shown are means ± SD from bi- ological 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: Inhibition, Western Blot, Derivative Assay, shRNA, Expressing, CCK-8 Assay, Cell Culture, Quantitation Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A) Schematic representation showing the mouse models used in this study, where Her2 mammary tumors show low levels of CIN and Her2-Plk1 overexpression results in high CIN tumors. B ) Average SCNAs in Her2 and Her2/Plk1 tumors; Unpaired t test, p = 0.012. C ) SCNAs in 10 Her2 and 10 Her2/Plk1 mammary tumors. Shown are whole chromosome gain (WCG) and loss (WCL), partial chromosome gain (PCG) and loss (PCL), focal amplification (AMP), deletion (DEL) and gross chromosomal rearrangement (GCR). D) Total transcriptome of Her2 and Her2-Plk1 tumor tissues was analyzed by bulk RNA-sequencing. Heatmap showing differentially expressed cell cycle and SASP-related genes (Fold change (FC) > 1.5 or < −1.5, p <0.05, n=3 tumors per genotype). E) GSEA using gene sets from KEGG pathways as analyzed by cluster profiler. Pathways overrepresented in Her2-Plk1 tumors are shown in yellow, whereas pathways overrepresented in Her2 tumors are displayed in blue. F) Fluorometric measurement of ß-galactosidase activity in Her2 and Her2-Plk1 tumor lysates (Mann Whitney test p<0.005**, n=10 (Her2) and n=16 (Her2-Plk1)). G) Cytokine array of tumor lysates of Her2 and Her2-Plk1 showing increased CXCL1, IL16 and IL1RN in the high CIN group (p<0.05* p<0.0005***, 2-way ANOVA, Sidak’s multiple comparison test, n=3). H) Characterization of TAM’s and monocytes. Live CD45 + CD11b + cells were divided into two groups based on Ly6C and MHCII. Flow cytometric analysis of inflammatory monocytes (Ly6c high MHCII low ) and M1 TAMs (Ly6c low MHCII high) in endpoint tumors (p<0.05*, n=5 (Her2) and n=8 (Her2-Plk1)).

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Over Expression, Amplification, RNA Sequencing, Activity Assay, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A) Expression of P38a and NF-kB-p65 in breast tumors from Her2 and Her2-Plk1 mice. Tumor cell lysates were used for immunodetection by using anti-PLK1, anti-NFkB-p65, anti-RELB, anti-P38, anti-pP38 and anti-vinculin as a loading control. Bar graphs represent the relative intensity of the signal from immunodetection (*, p<0.05, **, p<0.005, Mann-Whitney test, n=11 (Her2), n=14 (Her2-Plk1); numbers represent different tumors). B) Immunohistochemistry of PLK1, PD-L1 and CD206 in Her2 and Her2-Plk1 tumor sections and their corresponding bar graphs representing total sum area (μm 2 ). Positive regions per sample was calculated by dividing DAB positive area in each ROI/total area of the ROI (region of interest) and total sum area (μm 2 ) and adding positive regions in all the individual samples. Scale bar: 100μm. (*, p<0.05, **** p< 0.0001, Mann-Whitney test, n=5 (Her2), n=8 (Her2-Plk1)). C) Quantification of tumor-infiltrating lymphocytes in Her2 and Her2-Plk1 tumors and spleens. Representative two-parameter dot plots of CD45 + gated Tregs (CD25 + CD4 + Foxp3 + ), dendritic cells (CD11c + MHCII high ), NK cells (CD11c − Nkp46/Nk1.1 + )) and T cells (CD3 + CD4 + , CD3 + CD8 + ) in both spleen (n=5 (Her2), n=7 (Her2-Plk1)) and tumors (*, p<0.05, ** p<0.005, ***, p< 0.0005, Mann-Whitney test n=6 (Her2), n=10 (Her2-Plk1)) and bar graphs represented as % of CD45 positive cells. The numbers depicted in the boxes refer to the percentage of each cell type in different samples (Her2 and Her2-Plk1).

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Expressing, Immunodetection, Control, MANN-WHITNEY, Immunohistochemistry

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A) Schematic depiction of isolation of hyperplastic mammary glands and FACS sorting of individual cells to obtain CD45 hematopoietic cells at early stage Her2 (16 days on doxycycline) and Her2-Plk1 (22 days on doxycycline) samples. Samples were extracted from three different mice per genotype from two mammary glands each. B) Clustered immune cell populations displayed by UMAP in all samples. The cell types in the clusters were annotated by using SingleR and gene expression information from the ImmGen reference database and displayed in a color-coded fashion. C) Volcano graph showing differentially expressed genes in the CD11b + CD11c − macrophage subtype with log2-fold change in gene expression versus -log10 of adjusted p-values. Genes that were found to be downregulated in Her2-Plk1 with respect to Her2 are shown in blue, while genes that were upregulated are shown in yellow (by a fold change of at least ±0.6 and an adjusted p-value of ≤0.05). D) Violin graphs showing the normalized expression levels of CD11b + CD24 − macrophages for genes related to antigen presentation, EMT and anti-inflammatory wound-healing phenotypes. Her2 samples are in blue and Her2-Plk1 in yellow. p-values adjusted for multiple testing are shown above each graph. Each dot represents the gene expression level of an individual cell.

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Isolation, Gene Expression, Expressing, Immunopeptidomics

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A ) Survival analysis of Her2 (left) and Her2-Plk1 (right) tumors of mice treated with NK1.1 blocking antibody. Survival probability over time (upper graphs) and number and percentage of mice alive over time (lower table). The curves in light blue and light yellow depict untreated mice, whereas the curves in dark blue and dark yellow show mice treated with NK block, respectively. B) Normalized gene expression of CD27 versus Itgam ( CD11b ) in NK cells at the single cell level (individual dots) (Her2: blue, Her2-Plk1: yellow). The Spearman rank correlation coefficient across samples as well as the fold change and p-values adjusted for multiple testing for differential expression between Her2 and Her2-Plk1 samples are provided. C) Violin graphs showing the log-normalized gene expression values of NK cells in Her2 (blue) and Her2-Plk1 (yellow) tumors exemplarily shown for genes involved in cytotoxic response and surface receptors of NK cells. D) Heatmap showing normalized gene expression levels of differentially expressed effector genes, chemokines and their receptors, and activating receptor genes in NK cells. Each line represents a single cell. E) Gene set enrichment analysis for identification of pathways with enrichment of differentially expressed genes for two macrophage subsets and NK cells. Y-axis: hallmark signatures and X-axis: normalized enrichment scores with adjusted p-values, color-coded by the direction of the effect (yellow: upregulation in Her2-Plk1, blue: upregulation in Her2).

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Blocking Assay, Gene Expression, Quantitative Proteomics

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A) Volcano graph with reduced axes to focus on selected differentially expressed genes (labeled) in the B cell subset of Her2 and Her2-Plk1 tumors displayed as log2 fold change versus - log10 of the adjusted p-value. Genes that were found to be downregulated in Her2-Plk1 with respect to Her2 are shown in blue, while genes that were overexpressed are shown in yellow (by a fold change of at least ±0.4 and an adjusted p-value of ≤0.05). B) UMAP graph of T cells clustered into different T cell subtypes. Seven clusters with different cell types were identified after cell type annotation. C ) Relative distribution of the number of cells in each of the T cell subtypes with Her2 in blue and Her2-Plk1 in yellow. D) Normalized gene expression of Sell ( Cd62L ) versus Ccr7 in regulatory T cells at the single cell level (individual dots) (Her2: blue, Her2-Plk1: yellow). The Spearman rank correlation coefficient across samples as well as the fold change and p-values adjusted for multiple testing for differential expression between Her2 and Her2-Plk1 samples are provided. E) Volcano graph with reduced axes to focus on selected differentially expressed genes in the cluster of regulatory T cells displayed as log2 fold change versus -log10 of the adjusted p-value. Genes downregulated in Her2-Plk1 compared to Her2 are shown in blue, genes upregulated in Her2-Plk1 are displayed in yellow (by a fold change of at least ±0.4 and an adjusted p-value of ≤0.05).

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Labeling, Gene Expression, Quantitative Proteomics

Journal: bioRxiv

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

doi: 10.1101/2022.06.16.496429

Figure Lengend Snippet: A) Schematic description of sorting PLK1 high versus PLK1 low tumors from the TCGA-BRCA cohort. A total of 186 (PLK1 high) and 165 (PLK1 low) tumors with a Z-score of +1 and –1, respectively, were identified. B) Unsupervised clustering of PLK1 high versus PLK1 low tumors using principal component analysis. Samples color-coded based on the groups (PLK1 low: blue, PLK1 high: yellow). C) Cellular deconvolution of PLK1 high and PLK1 low tumors using relative CIBERSORT to characterize the heterogeneity in immune cell composition of tumors with different levels of CIN. D) Heatmap showing gene expression data (Log 2 norm_count+1) and clustered into three different signatures associated with senescence, T-cell exhaustion and immune suppression from both PLK1 high (yellow) and PLK1 low (blue) tumor cohorts.

Article Snippet: These cells were then infected with an inducible Tet-ON lentivirus carrying the human PLK1 cDNA (pLenti CMVtight Hygro DEST from Addgene #26433) and selected with hygromycin (350 μg/ml).

Techniques: Gene Expression

Journal: Cell reports

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer.

doi: 10.1016/j.celrep.2023.113266

Figure Lengend Snippet: Figure 1. Plk1 overexpression drives inflammatory monocyte recruitment as a consequence of the SASP (A) Schematic showing the mouse models used in this study, where Her2 mammary tumors show low levels of CIN and Her2-Plk1 overexpression results in high CIN tumors. (B) Average SCNAs in 10 Her2 and 10 Her2-Plk1 tumors; unpaired t test, p = 0.0004. (C) SCNAs in 10 Her2 and 10 Her2-Plk1 mammary tumors. Shown are whole-chromosome gain (WCG) and whole-chromosome loss (WCL), focal amplification (AMP), deletion (DEL) and gross chromosomal rearrangement (GCR). (D) Total transcriptome of Her2 and Her2-Plk1 tumor tissues was analyzed by bulk RNA-seq. Heatmap showing differentially expressed cell cycle and SASP- related genes (fold change [FC] > 1.5 or < 1.5, p < 0.05, n = 3 tumors per genotype).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Gene expression data Breast cancer METABRIC cohort, Pereira et al.50 https://cbioportal-datahub.s3. amazonaws.com/brca_metabric.tar.gz Experimental models: Cell lines MCF-7 Obtained from Guillermo de Cárcer Laboratory N/A Cal51 Obtained from Uri-Ben David Laboratory N/A Experimental models: Organisms/strains Mouse: TetO-Her2/Plk1/MMTV-rtTA, FVB Carcer et al.7 https://doi.org/10.1038/s41467-018-05429-5 Oligonucleotides Mycoplasma antisense primer MGSO (50-TGCACCATCTGTCACTCTGTTA ACCTC-30), (10 mM) Integrated DNA Technologies https://doi.org/10.1038/nprot.2010.43 Mycoplasma sense primer GPO-3 (50-GGGAGCAAACAGGATTAGATA CCCT-30), (10 mM) Integrated DNA Technologies https://doi.org/10.1038/nprot.2010.43 Recombinant DNA pLenti CMVtight Hygro DEST Addgene #26433 Human PLK1 cDNA Carcer et al.7 https://doi.org/10.1038/s41467-018-05429-5 Software and algorithms GraphPad Prism 8 and 9 GraphPad Software https://www.graphpad.com/ scientific-software/prism/ FlowJo ImageJ software FlowJo https://imagej.net/plugins/flowj StrataQuest software TissueGnostics https://tissuegnostics.com/products/ contextual-image-analysis/strataquest R, Version 4.3.0 R Project for Statistical Computing https://www.r-project.org/ Seurat Hao et al.58; https://satijalab.org/seurat/ https://cloud.r-project.org/web/ packages/Seurat/index.html SingleR Aran et al.29 https://bioconductor.org/packages/ release/bioc/html/SingleR.html clusterProfiler Yu et al.59 https://bioconductor.org/packages/ release/bioc/html/clusterProfiler.html Python Python Software Foundation https://www.python.org/ matplotlib Hunter et al.60 https://matplotlib.org/

Techniques: Over Expression, RNA Sequencing