Journal: bioRxiv

Article Title: Cardiac fibroblasts regulate cardiomyocyte hypertrophy through dynamic regulation of type I collagen

doi: 10.1101/2022.05.25.493406

Figure Lengend Snippet: (A) Whole ventricle mRNA microarray analysis of Col1a2 -/- mouse hearts compared to Col1a2 +/- at 2 months of age, n=3 per genotype. (B) Mass spectrometry analysis of ECM protein changes in Col1a2 -/- mouse hearts compared to Col1a2 +/- hearts at 3 months of age, n=4 per genotype. (C) Representative immunofluorescence images and (D) Western blot analysis of periostin from hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Scale bar: 25 µm. (E) Flow cytometric gate strategy and (F) analysis of cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from dissociated hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. (G) Representative immunofluorescence images of platelet-derived growth factor receptor (PDGFR)-α (purple) in Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Wheat germ agglutinin (WGA) staining is green and shows outlines of cardiomyocytes. Scale bar: 100 µm. Relative mRNA expression of Col1a2 (H), Postn (I), Col3a1 (J) and Col5a1 (K) in sorted cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from Col1a2 +/- and Col1a2 -/- mice at 9 months of age. Student t -test for panels (F), (H), (I), (J) and (K).

Article Snippet: Antibodies against the following proteins were used: periostin (Novus Biologicals NBP1-30042; 1:300 dilution for IF, 1:1000 for Western blot); collagen I (Abcam ab21286; 1:100 for IF); PDGFRα from (R&D Systems AF1062; 1:1000 for IF); collagen 1a2 (Santa Cruz sc-393573; 1:500 for Western blot) Anti-CD31 was from BioLegend (102423; 1:100 for flow cytometry); anti-CD45 was from BD Biosciences (563890; 1:100 for flow cytometry); anti-MEFSK4 was from Miltenyi Biotec (130-120-802; used 1:30 for flow cytometry).

Techniques: Microarray, Mass Spectrometry, Immunofluorescence, Western Blot, Derivative Assay, Staining, Expressing

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti‐ eIF3d (WB 1:2,000) , Bethyl , Cat # A301‐758A; RRID: AB_1210970.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Magnetic Beads, Software, Microscopy, Microarray, Imaging, Flow Cytometry, Mutagenesis, Gel Extraction, Sample Prep, Clear Native PAGE, Bicinchoninic Acid Protein Assay

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: Parental HCT116 and the five different conditional eIF3‐mAID cell lines were treated with the vehicle DMSO or with IAA for the periods indicated. Metabolic activity (i.e., NAD(P)H) as a proxy of cell proliferation was determined by MTT assay. Graphs represent means ± SD, n = 5–6. Asterisks denote: * P < 0.005, ** P < 0.00005, *** P < 0.000005 (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). 1 × 10 3 cells of the indicated cell lines were plated and grown in DMSO or IAA for 2 weeks. Colony numbers were counted and are shown in a bar graph relative to the DMSO‐treated control. Note that very low numbers of colonies were obtained for IAA‐treated eIF3a‐mAID, eIF3e‐mAID and eIF3f‐mAID – hence no bars are visible. Bars represent means ± SD, n = 3. Numbers indicate P ‐values (unpaired Student's t ‐test). The indicated cell lines were subjected to serum withdrawal (0% FBS) for 16 h. DMSO or IAA was added and cells were stimulated to re‐enter the cell cycle by refeeding with media containing 10% FBS. Cells were fixed after the indicated time points, and the cellular DNA content was measured by flow cytometry. Data are representative of 2 (HCT116, eIF3a‐mAID, eIF3b‐mAID) or 3 (eIF3e‐mAID, eIF3f‐mAID, eIF3k‐mAID) experiments. 1 × 10 6 eIF3‐mAID cells were injected into nude mice. When tumors reached a diameter of ~ 4 mm, mice were treated with vehicle or 500 mg/kg IAA, and tumor growth was measured for 2 weeks. Graphs represent means ± SD, n = 5–7. Numbers indicate P ‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). Expression of the indicated eIF3 proteins in 97 colon adenocarcinomas versus 100 normal colonic mucosae and 110 clear cell renal cell carcinomas versus 84 normal kidney samples. Log2 spectral count data from CPTAC (Chen et al , ) via UALCAN portal (Chandrashekar et al , ). Data information: The median (50 th percentile), 25% percentile (lower box), 75 th percentile (upper box), 5 th percentile (lower whisker), and 95 th percentile (upper whisker) are indicated. n = biologically independent tumor samples. Source data are available online for this figure.

Article Snippet: Rabbit Polyclonal Anti‐ eIF3f (WB 1:2,000) , Bethyl , Cat # A303‐005A; RRID: AB_10748371.

Techniques: Activity Assay, MTT Assay, Flow Cytometry, Injection, Expressing, Whisker Assay

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: The indicated conditional eIF3 cell lines were exposed to 500 μM IAA for 12 h to induce the degradation of the respective eIF3 subunits. Cell lysates were subjected to immunoblotting to quantify the expression of the indicated eIF3 subunits. Signals were normalized to the reference signal of actin and plotted relative to the vehicle DMSO. Bars represent means ± SD, n = 3. Numbers indicate P ‐values (unpaired Student's t ‐test). The indicated cell lines were exposed to IAA for 12 h, and cell lysates were separated by sucrose density gradient centrifugation. The monosomal (M) and polysomal (P) peaks were quantified, and P/M ratios relative to the vehicle DMSO were plotted. Bars represent means ± SD, n = 3. Numbers indicate P ‐values (unpaired Student's t ‐test). Scatter plots of changes in polysomal mRNA versus total mRNA upon depletion of the indicated eIF3 subunits. Enrichment of Gene Ontology terms in the sets of polysome‐associated mRNAs decreased in the polysomal fractions of eIF3f‐ or eIF3e‐depleted cells or increased in eIF3k‐depleted cells. Distribution histograms of translational efficiencies (TEs) of the mRNA sets shown in (C). The total number of mRNAs and the median TE of each dataset are indicated. The number of up and down changes with FDR ≤ 0.2 as well as the median of all changed items are indicated in red, blue, and black bold print. Distribution histograms of the protein synthesis ratios determined in eIF3 subunit depleted versus replete cells. The total number of measured proteins and the median synthesis change of each dataset are indicated. The number of up and down changes with FDR ≤ 0.1 as well as the median of all changed items are indicated in red and blueprint. Data information: n = number of biological replicates. Source data are available online for this figure.

Article Snippet: Rabbit Polyclonal Anti‐ eIF3f (WB 1:2,000) , Bethyl , Cat # A303‐005A; RRID: AB_10748371.

Techniques: Western Blot, Expressing, Gradient Centrifugation

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: Primary data for the graph shown in Fig . The indicated conditional eIF3 cell lines were exposed to 500 μM IAA for 12 h to induce the degradation of the respective eIF3 subunit. Cell lysates were subjected to immunoblotting to quantify the expression of the indicated eIF3 subunits. Actin signal was used as a reference. Representative primary data for the graph in Fig . The indicated cell lines were exposed to IAA for 12 h, and cell lysates were separated by sucrose density gradient centrifugation. The monosomal (40S, 60S, 80S) and polysomal peaks were quantified, and P/M ratios relative to the vehicle DMSO are plotted in Fig . The region of the gradient used for extraction of polysomal RNA for RNA‐seq is indicated. Strategy of analyzing transcriptomic and proteomic datasets. Total (blue panel) and polysomal (yellow) RNA samples were collected from triplicate cultures of eIF3‐mAID cells exposed to DMSO or IAA for 12 h. Raw RNA‐seq datasets were normalized for sequencing depth by calculating counts per million mapped reads (CPM). For polysomal RNA samples, an additional normalization step was performed to account for the different levels in total polysomes in each sample. This was particularly necessary for those cell lines that showed a strong decrease in polysomes, including eIF3a‐mAID, eIF3e‐mAID, and eIF3f‐mAID; but the normalization was performed for all six cell lines. In the next step, edgeR (Robinson et al , ) was used to identify changed mRNAs. mRNAs changed in polysomes (FDR ≤ 0.05) were analyzed for enriched Gene Ontology functional terms (Fig ). mRNAs changed (FDR ≤ 0.05) in the total and polysomal populations were then graphed in a scatter plot (Fig ). Translational efficiencies (TEs) were calculated for all data points included in the scatter plot with cpm ≥ 10 as Log2 [(polysomal mRNA IAA / total mRNA IAA) / (polysomal mRNA DMSO / total mRNA DMSO)], and TEs were plotted in histograms (Fig ). Triplicate LC–MS/MS datasets were subjected to statistical testing (Benjamini–Hochberg) to identify proteins whose synthesis (Log2 Heavy IAA / Heavy DMSO) was changed upon depletion of eIF3 subunits at a FDR ≤ 0.1. Log2 Heavy IAA / Heavy DMSO values were graphed in a histogram (Fig ). Changes in TE and protein synthesis were compared in a scatter plot of Log2 TE versus Log2 Heavy IAA/Heavy DMSO (Appendix Fig ). Venn diagrams summarizing the changes in the total and polysomal mRNAs (FDR ≤ 0.05) in the indicated cell lines. Source data are available online for this figure.

Article Snippet: Rabbit Polyclonal Anti‐ eIF3f (WB 1:2,000) , Bethyl , Cat # A303‐005A; RRID: AB_10748371.

Techniques: Western Blot, Expressing, Gradient Centrifugation, RNA Sequencing Assay, Sequencing, Functional Assay, Liquid Chromatography with Mass Spectroscopy

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti‐ eIF3f (WB 1:2,000) , Bethyl , Cat # A303‐005A; RRID: AB_10748371.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Magnetic Beads, Software, Microscopy, Microarray, Imaging, Flow Cytometry, Mutagenesis, Gel Extraction, Sample Prep, Clear Native PAGE, Bicinchoninic Acid Protein Assay

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti‐ RPS19 (WB 1:2,000) , Bethyl , Cat # A304‐002A; RRID: AB_2620351.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Magnetic Beads, Software, Microscopy, Microarray, Imaging, Flow Cytometry, Mutagenesis, Gel Extraction, Sample Prep, Clear Native PAGE, Bicinchoninic Acid Protein Assay

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Identification of the minimal human P2P. ( A ) Schematic representation of P2P 5′-truncations and their cloning strategy as used in this study. The translational start site is designated ‘+1’. ( B ) Regulatory DNA regions of the human PHD2 gene were cloned into luciferase reporter vectors that were transiently transfected into human U2OS osteosarcoma cells. One day after transfection, cells were incubated for 24 h at 20 or 0.2% O 2 . Hypoxic IF (mean values ± SD) of relative luciferase activities were calculated from three independent experiments performed in triplicates. Mutation of a single HBS (black rectangles in A) completely abrogated hypoxic inducibility of all constructs. ( C ) HeLa and U2OS cells were incubated at 20 or 0.2% O 2 for 4–24 h and protein levels of HIF-1α, PHD2 and β-actin were analyzed by immunoblotting. Total RNA was isolated from cultures treated as in (B) and mRNA levels of PHD2 and CA9 were determined by RT-qPCR. Transcript levels of CA9 served as positive control to confirm continuous hypoxic responses. Gene expression levels were expressed in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments (±SD).

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Cloning, Clone Assay, Luciferase, Transfection, Incubation, Mutagenesis, Construct, Western Blot, Isolation, Quantitative RT-PCR, Positive Control, Gene Expression

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Hypoxic transactivation of the P2P by ETV4 requires HIF-1α activity. ( A ) Standard dual luciferase reporter gene assays of seven reevaluated hits from the transcription factor overexpression array. Wild-type (left panel) or HBS mutant (right panel) P2P regions controlling firefly luciferase reporter plasmids were cotransfected into U2OS cells together with expression constructs of the aforementioned factors. Transfection of an empty expression vector (empty) served as negative control and differences in transfection efficiency were controlled by cotransfecting SV40 promoter driven renilla luciferase. Cells were cultured at 20% or 0.2% oxygen for 24 h before dual luciferase activities were determined. ( B ) Transient RNAi mediated knock down of HIF-1α fully abrogated hypoxic activation of the P2P by ETV4. U2OS cells were transiently transfected with siRNA oligonucleotides targeting HIF-1α (siHIF1α, right panel) or a control sequence having no human target (siControl, left panel). Reporter gene experiments using the P2P reporter construct with only wild-type HBS were performed as described in (A). The inset shows an immunoblot confirming the robust knock down of HIF-1α in U2OS cells. ( C ) ETV4 and HIF-1 synergism in hypoxic gene activation is not restricted to the P2P. A heterologous hypoxia responsive reporter gene containing two functional HBS from the human Transferrin hypoxia response element (pGL-TfHRE wt) was tested in luciferase reporter assays as described in (A). Mutation of both HBS (pGL-TfHRE mut) caused an abrogation of the signal as seen in (A). ( D and E ) Forced expression of ETV4 in U2OS cells upregulates endogenous PHD2 protein and transcript levels. ( D ) Whole cell lysates were prepared from cells exposed for 16 h to 20 or 0.2% oxygen and analyzed for HIF-1α, ETV4, PHD2 and β-actin levels by immunoblotting. ( E ) Total RNA was extracted of similarly treated cells and mRNA levels of PHD1, PHD2 and L28 were quantified by RT-qPCR. Data are shown in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments (** P < 0.01, paired Student's t -test).

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Activity Assay, Luciferase, Over Expression, Mutagenesis, Expressing, Construct, Transfection, Plasmid Preparation, Negative Control, Cell Culture, Knockdown, Activation Assay, Control, Sequencing, Western Blot, Functional Assay, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Transcriptional cooperation between ETV4 and HIF-1 is disrupted by CITED2. ( A ) Schematic representation of HIF-1α and ETV4 domain structure and fusion constructs used in mammalian two-hybrid assays. PAS, PER-ARNT-SIM; bHLH, basic helix–loop–helix domain; ODD, oxygen-dependent degradation domain; NRR, negative regulatory region; NAD and CAD, amino-carboxy-terminal activation domain and CADs, respectively. A GAL4-DBD was fused to regions encompassing the HIF-1α NAD and CAD. Full-length ETV4 bearing two activation domains (AD, acidic domain; Ct, carboxy-terminal tail) flanking a central ETS domain was fused to a VP16 activation domain (VP16-AD). Numbers indicate the amino acids present in the respective constructs. ( B ) U2OS cells were cotransfected with a Gal4-responsive reporter plasmid and Gal4-HIF-1α (GH1α) constructs alone or in combination with VP16-ETV4. The GH1α fusion constructs are specified by the aminoterminal starting amino acid of the truncated HIF-1α regions (530, 740 and 786, respectively). Following transfection, cells were evenly split and incubated at 20 or 0.2% O 2 before luciferase activities were determined 24 h later. Noninteracting Gal4 DBD-p53 and VP16-AD-CP1 served as negative control (neg. ctrl.), while the interactions between Gal4 DBD-PHD2 and VP16-AD-HIF-2α(ODD) or VP16-AD-FKBP38 were used as positive controls (pos. ctrl. 1 and pos. ctrl. 2, respectively). ( C ) Scheme of the potential interactions between HIF-1, p300/CBP and ETV4 as assessed by mammalian two-hybrid assays. Both CITED2 and FIH can block the interaction between HIF-1α and p300/CBP. ( D ) Cotransfection of the indicated amounts of a CITED2 expression construct together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). ( E ) Cotransfection of siRNA directed against p300 together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The p300 knock down efficiency of different siP300 oligonucleotides was analyzed by immunoblotting (upper panel) and siP300#1 was chosen for further experiments. ( F ) Cotransfection of siRNA directed against FIH together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The efficiency of the siFIH mediated FIH knock down was confirmed by immunoblotting as shown in the inset. ( G ) ChIP of normoxic or hypoxic PC3 cells using antibodies directed against HIF-1α or ETV4, or control serum. The amount of coprecipitated chromatin derived from the human P2P region (encoded by EGLN1 ) containing the HBS was determined by PCR followed by agarose gel electrophoresis.

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Construct, Activation Assay, Plasmid Preparation, Transfection, Incubation, Luciferase, Negative Control, Blocking Assay, Cotransfection, Expressing, Activity Assay, Knockdown, Western Blot, Control, Derivative Assay, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Genome-wide microarray expression analysis reveals a broad role for ETV4 in HIF mediated hypoxic gene regulation. ( A ) Efficient knock down of ETV4 in human PC3 prostate cancer cells. PC3 cells were stably transduced with lentiviral shRNA expression vectors encoding either a nontarget control (shNTC) or shETV4. Following 24 h of exposure to 20% O 2 or 0.2% O 2 , ETV4, HIF-1α, PHD2 and β-actin protein levels were analyzed by immunoblotting. ( B ) Total RNA was isolated from cultures treated as in (A) and mRNA levels of ETV4 and its target gene COX2 were determined by RT-qPCR. Gene expression levels were expressed in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments. ( C ) Venn diagram showing the number of transcripts regulated by either an at least twofold induction by hypoxia alone (red), an at least twofold reduction in normoxic cells by the knock down of ETV4 (green), or an at least twofold reduction in hypoxic cells by the knock down of ETV4 (blue), respectively. ( D ) Heatmap of the individual expression levels of the 47 transcripts that required ETV4 for efficient hypoxic induction. ( E ) Expression levels of four randomly chosen transcripts shown in (D) were confirmed by RT-qPCR as described for (B).

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Genome Wide, Microarray, Expressing, Knockdown, Stable Transfection, Transduction, shRNA, Control, Western Blot, Isolation, Quantitative RT-PCR, Gene Expression

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Role of ETV4 in the regulation of established HIF target genes in vitro and in vivo . ( A ) Dot plots showing the correlation between transcripts in normoxic versus hypoxic control cells (left panel) or in hypoxic control versus hypoxic ETV4 knock down cells (right panel) as derived from the gene array data (grey dots). Red dots refer to internal controls and the blue dot shows ETV4 which is downregulated in shETV4 cells. Green dots indicate the positions of a predefined set of 61 well-established HIF target genes. ( B ) Heat map of the 61 HIF target genes ranked by the magnitude of ETV4 requirement for hypoxic induction according to differences in hypoxic expression levels with Δhyp = log 2 (shNTC_hypoxia) − log 2 (shETV4_hypoxia) and mean hypoxic expression levels centered to the mean of normoxic control cells. ( C ) Exemplary mRNA levels of HIF target genes which either require ETV4 for efficient hypoxic induction (PHD3 and CA9) or which remain unaffected by the ETV4 knock down (GLUT1 and PAI1). mRNA was quantified as described for B. ( D and E ) Correlation between ETV4 and established markers for tissue hypoxia in human breast cancer. ( D ) Independent specimens (spec.) of immunohistochemical evaluation of ETV4 expression in primary mammary carcinoma with high (upper panel) or low (lower panel) ETV4 expression levels. ( E ) Rank-order correlations (Spearman's rho) for ETV4 and PHD2 as well as known markers reflecting tissue hypoxia (HIF-1α, HIF-2α, PAI1, GLUT1 and CA9) are summarized in a cross table. The number of cases where both of the correlated markers could be assessed is displayed in parentheses. Asterisks indicate statistical significance with * P < 0.05 and ** P < 0.01.

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: In Vitro, In Vivo, Control, Knockdown, Derivative Assay, Expressing, Immunohistochemical staining

Journal: Cell Reports Methods

Article Title: CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

doi: 10.1016/j.crmeth.2022.100318

Figure Lengend Snippet: SARS-CoV-2 epitope mapping by CasPlay (A) A peptide library tiling the SARS-CoV-2 proteome (excluding ORF1ab) from a previous study ( Barber et al., 2021 ) was presented by CasPlay. (B) CasPlay experiments were performed using serum samples from 8 samples collected prior to December 2020 (“pre-COVID”), 8 patients following COVID-19 infection (“convalescent”), 8 individuals prior to receiving a vaccine, and the same 8 individuals between 2 weeks and 3 months after receiving the second dose of an mRNA vaccine. gRNA barcode enrichment analysis by CasPlay revealed epitope regions in the spike and/or nucleocapsid proteins recognized by patient antibodies in convalescent and vaccinated patient samples. Enrichment is calculated as normalized output gRNA sequencing reads divided by pre-enriched normalized sequencing reads, with data averaged across 4 gRNA barcode replicates and two independent experimental replicates (see for further details).

Article Snippet: Then, approximately 100 ng of purified recombinant SARS-CoV-2 spike protein (Sino Biological 40589-V08H4 was added to the microarray and incubated at 23°C for 1 h. After washing twice with 40 μL PBST, 1:100 anti-SARS-CoV-2 spike CR3022 human IgG antibody (Cell Signaling 37475) was added to the microarray and incubated at 23°C for 1 h. The microarray was then washed twice with PBST and then incubated with 1:40 Alexa 647-conjugated anti-human IgG Fc antibody (Biolegend 410714) at 23°C for 1 h. The microarray was then visualized using a Genepix 4300A microarray scanner, and fluorescence intensities were extracted and analyzed as previously described ( ; ; ; ).

Techniques: Infection, Sequencing

Journal: Cell Reports Methods

Article Title: CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

doi: 10.1016/j.crmeth.2022.100318

Figure Lengend Snippet: CasPlay is compatible with full-length protein display and applications (A) Synthetic antibodies were expressed as C-terminal fusions to dCas9 with unique gRNA barcodes. A nanobody recognizing a peptide from β-catenin ( Braun et al., 2016 ; Traenkle et al., 2015 ) and an scFv recognizing the spike protein from SARS-CoV-2 ( Wang et al., 2021 ) were paired with unique gRNAs. (B) Target antigens of the synthetic antibodies were adsorbed to a plate surface and then incubated with the dCas9-antibody fusions. Primers specific to the gRNA sequences were then used to RT-PCR the gRNAs associated with each antibody to detect binding of the antibody to its target antigen. (C) Densitometry of RT-PCR amplicons on an agarose gel reveal specific retention and detection of each synthetic antibody only in the presence of its respective analyte, indicating synthetic antibody functionality. n = 3 independent replicates for each condition. Mean with SD plotted.

Article Snippet: Then, approximately 100 ng of purified recombinant SARS-CoV-2 spike protein (Sino Biological 40589-V08H4 was added to the microarray and incubated at 23°C for 1 h. After washing twice with 40 μL PBST, 1:100 anti-SARS-CoV-2 spike CR3022 human IgG antibody (Cell Signaling 37475) was added to the microarray and incubated at 23°C for 1 h. The microarray was then washed twice with PBST and then incubated with 1:40 Alexa 647-conjugated anti-human IgG Fc antibody (Biolegend 410714) at 23°C for 1 h. The microarray was then visualized using a Genepix 4300A microarray scanner, and fluorescence intensities were extracted and analyzed as previously described ( ; ; ; ).

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Agarose Gel Electrophoresis

Journal: Cell Reports Methods

Article Title: CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

doi: 10.1016/j.crmeth.2022.100318

Figure Lengend Snippet:

Article Snippet: Then, approximately 100 ng of purified recombinant SARS-CoV-2 spike protein (Sino Biological 40589-V08H4 was added to the microarray and incubated at 23°C for 1 h. After washing twice with 40 μL PBST, 1:100 anti-SARS-CoV-2 spike CR3022 human IgG antibody (Cell Signaling 37475) was added to the microarray and incubated at 23°C for 1 h. The microarray was then washed twice with PBST and then incubated with 1:40 Alexa 647-conjugated anti-human IgG Fc antibody (Biolegend 410714) at 23°C for 1 h. The microarray was then visualized using a Genepix 4300A microarray scanner, and fluorescence intensities were extracted and analyzed as previously described ( ; ; ; ).

Techniques: Synthesized, Recombinant, Plasmid Preparation, Expressing, Software, Microarray

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Comparison of the expression of POSTN (in cells ( A ) and in stroma ( B )) and the expression of pro-angiogenic markers (VEGF-A ( C ); CD31 ( D ); CD34 ( E ); CD105 ( F )) between non-malignant lung tissue and cancer cells. The significance of the differences was determined using the Mann–Whitney U test. The images in ( G – K ) show punches from tissue microarrays illustrating an example of the immunohistochemical (IHC) reaction for each individual protein, as described above. Error bars represent the standard deviation (SD). *** p < 0.001.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Comparison, Expressing, MANN-WHITNEY, Immunohistochemical staining, Standard Deviation

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Bar charts showing the expression of POSTN in cells ( A – C ) and in stroma ( D – F ) across all cancer types with regard to the T-stage ( A , D ) and N-stage ( B , E ) of the TNM classification as well as the tumor stage ( C – F ). The significance of the differences was determined using the Kruskal–Wallis ANOVA test and the differences between individual groups were evaluated using the appropriate post hoc test. Error bars represent the standard deviation (SD). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Expressing, Standard Deviation

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Bar charts showing the expression of POSTN in cells ( A – D ) and in stroma ( E – H ), across all cancer types ( A , E ), in adenocarcinoma ( B , F ), and in squamous-cell carcinoma ( C , G ), with regard to the tumor grade. The significance of the differences was determined using the Kruskal–Wallis ANOVA test ( p < 0.001 in bar plots ( A – C , E – G )), and differences between individual groups were evaluated with the appropriate post hoc test. The results obtained for large-cell carcinoma were statistically insignificant ( D , H ). The images in ( I – Q ) show representative punches demonstrating the IHC reaction for POSTN in adenocarcinoma ( I – K ), squamous-cell carcinoma ( L – N ), and large-cell carcinoma ( O – Q ) with respect to the tumor grade. Error bars represent the standard deviation (SD). *** p < 0.001.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Expressing, Standard Deviation

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Mann–Whitney U test results for the differences between males and females. Only POSTN in stroma yielded statistically significant results.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Mann-Whitney U-Test

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Correlation plots comparing POSTN expression in cells to POSTN expression in stroma, across all cancer types ( A ), in adenocarcinoma ( B ), in squamous-cell carcinoma ( C ), and in large-cell carcinoma ( D ). The plots include p -values and R-values (calculated with Spearman’s correlation test) and the number of cases, which can be seen in the lower right corner of each plot.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Expressing

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Correlation plots comparing the expression of POSTN in stroma with the expression of pro-angiogenic markers, as quantified via the Weidner ( A – C ) or Chalkley ( D – F ) method. The pro-angiogenic factors are CD31 ( A , D ), CD34 ( B , E ), and CD105 ( C , F ). The plots include p -values and R-values (calculated with Spearman’s correlation test) and the number of cases, which can be seen in the lower right corner of each plot.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Expressing

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Correlation plots comparing the expression of POSTN in cells ( A – C ) and in stroma ( D – F ) with the expression of pro-angiogenic markers, as quantified via the Chalkley method, assessed in adenocarcinoma. The pro-angiogenic factors are CD31 ( A , D ), CD34 ( B , E ), and CD105 ( C , F ). The plots include p -values and R-values (calculated with Spearman’s correlation test) and the number of cases, which can be seen in the lower right corner of each plot. The images in ( G – I ) show punches from tissue microarrays demonstrating an example of the immunohistochemical (IHC) reaction for each individual protein, as previously described. The images in ( J – L ) show representative magnified regions of the microarray punches.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Expressing, Immunohistochemical staining, Microarray

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Correlation plots comparing the expression of POSTN in cells ( A – C ) and in stroma ( D – F ) with the expression of pro-angiogenic markers, as quantified via the Chalkley method, assessed in squamous-cell carcinoma. The pro-angiogenic factors are CD31 ( A , D ), CD34 ( B , E ), and CD105 ( C , F ). The plots include p -values and R-values (calculated with Spearman’s correlation test) and the number of cases, which can be seen in the lower right corner of each plot. The images in ( G – I ) show punches from tissue microarrays demonstrating an example of the immunohistochemical (IHC) staining for each individual protein, as previously described. The images in ( J – L ) show representative magnified regions of the microarray punches.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Microarray

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Correlation plots comparing the expression of POSTN in stroma ( A – D ) with the expression of VEGF-A, as assessed with the semi-quantitative IRS method, across all cancer types ( A ), in adenocarcinoma ( B ), in squamous-cell carcinoma ( C ), and in large-cell carcinoma ( D ). The images in ( E – G ) show punches from tissue microarrays demonstrating an example of the immunohistochemical (IHC) staining for each individual protein, as previously described. The images in ( H – J ) show representative magnified regions of the microarray punches.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Microarray

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Kaplan–Meier plots of survival for patients expressing POSTN in their cells ( A ), POSTN in their stroma ( B ), and VEGF-A ( C ) at levels below or above the median. The graphs in ( D , E ) show the plots of survival for patients with regard to the quartile classifications of the levels of POSTN expression in their cells ( D ) or stroma ( E ). Quartiles are shown in ascending order, with I representing the first 25% of results, II representing 26–50%, III representing 51–75%, and IV representing 76% and above. The significance of the differences was determined with the log-rank test. The p -value and the number of cases are given in the lower right corner of each plot.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Expressing

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Kaplan–Meier plots of survival for patients with POSTN in their cells ( A , B ) or stroma ( C , D ) with regard to the tumor type (adenocarcinoma ( A , C ) or squamous-cell carcinoma ( B , D )). The significance of the differences was determined using the log-rank test. The p -value and the number of cases are given in the lower right corner of each plot.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques:

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Linear regression model constructed for the purpose of predicting survival days. It was determined that other predictors were not valid. The model yielded an R-value of 0.473 when the variables presented below were used.

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Construct

Journal: Cells

Article Title: Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma

doi: 10.3390/cells13171406

Figure Lengend Snippet: Logistic regression model constructed for the prediction of 5-year survival. It was determined that other predictors were not significant. Measures of model effectiveness are shown on the ROC curve ( Figure 13 ).

Article Snippet: The following specific primary antibodies were used in the IHC experiments: anti-POSTN rabbit polyclonal antibody (1:200 dilution, NBP1-82472, Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-VEGF-A antibody (1:50 dilution + linker, VG1 clone, M7273, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD31 antibody (RTU, JC70A clone, IR610, Agilent Technologies, Santa Clara, CA, USA), monoclonal mouse anti-CD34 antibody (RTU, QBEnd/10 clone, IR632, Agilent Technologies, Santa Clara, CA, USA), and rabbit polyclonal anti-CD105 antibody (1:1000 dilution, 10862-1-AP, Proteintech, Manchester, UK).

Techniques: Construct

Journal: Chinese Journal of Cancer

Article Title: Follistatin-like protein 1 plays a tumor suppressor role in clear-cell renal cell carcinoma

doi: 10.1186/s40880-018-0267-2

Figure Lengend Snippet: Major signaling pathways enriched by up-regulated genes in FSTL1 -knockdown ccRCC cells by microarray assay. a Heat map of 105 differentially expressed genes (48 down-regulated and 57 up-regulated). b Reproducibility of the 105 differentially expressed genes in NRCC-shFSTL1-1 and NRCC-shFSTL1-2 cells (Pearson correlation coefficient r = 0.92; scope of linear model k = 0.86, P < 0.001). c Ranked by normalized enrichment score (NES), the top 11 gene sets (blue squares) enriched by up-regulated genes in response to FSTL1 knockdown are selected to plot the network. Intersection genes (green dots) with fold change > 2 were plotted to connect the gene sets. The gene sets fell into nuclear factor-кB (NF-κB)- and hypoxia-inducible factor (HIF)-related signaling subnetworks, respectively, which is connected by a histone deacetylase 1 (HDAC1)-related gene sets. d Enrichment plot of the representative gene set with the highest NES score in the NF-κB-related signaling subnetwork. e Enrichment plot of the representative gene set with the highest NES score in the HIF-related signaling subnetwork. The vertical dashed line in each plot denoted the point at which NES reached its maximum

Article Snippet: Rabbit polyclonal antibodies to human FSTL1 (C-term) (1:50 dilution; Abgent No. AP10534b), anti-HIF-1α (1:30 dilution; Novus Biologicals No. NB100-105 Littleton, CO, USA), and anti-HIF-2α antibodies (1:300 dilution; Novus Biologicals No. NB100-132) were applied according to the manufacturers’ protocols.

Techniques: Protein-Protein interactions, Knockdown, Microarray, Histone Deacetylase Assay

Journal: Chinese Journal of Cancer

Article Title: Follistatin-like protein 1 plays a tumor suppressor role in clear-cell renal cell carcinoma

doi: 10.1186/s40880-018-0267-2

Figure Lengend Snippet: FSTL1 and HIF-1α/2α levels in ccRCCs and adjacent tissues and their effects on postoperative prognosis. a – c Representative immunostaining of FSTL1, HIF-1α, and HIF-2α in adjacent renal tissues, respectively. The HIF-2α positive parts are indicated by black arrows. d – f Representative immunostaining of FSTL1, HIF-1α, and HIF-2α in ccRCC tissues, respectively. g – i Effect of intratumoral expression of FSTL1, HIF-1α, and HIF-2α on disease-specific survival, respectively

Article Snippet: Rabbit polyclonal antibodies to human FSTL1 (C-term) (1:50 dilution; Abgent No. AP10534b), anti-HIF-1α (1:30 dilution; Novus Biologicals No. NB100-105 Littleton, CO, USA), and anti-HIF-2α antibodies (1:300 dilution; Novus Biologicals No. NB100-132) were applied according to the manufacturers’ protocols.

Techniques: Immunostaining, Expressing

Journal: Chinese Journal of Cancer

Article Title: Follistatin-like protein 1 plays a tumor suppressor role in clear-cell renal cell carcinoma

doi: 10.1186/s40880-018-0267-2

Figure Lengend Snippet: The correlation of FSTL1 level with the expression of HIF-1α/2α in ccRCC tissues and tumor stage

Article Snippet: Rabbit polyclonal antibodies to human FSTL1 (C-term) (1:50 dilution; Abgent No. AP10534b), anti-HIF-1α (1:30 dilution; Novus Biologicals No. NB100-105 Littleton, CO, USA), and anti-HIF-2α antibodies (1:300 dilution; Novus Biologicals No. NB100-132) were applied according to the manufacturers’ protocols.

Techniques: Expressing

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti‐ eIF3g (WB 1:2,000) , Bethyl , Cat # A301‐757A; RRID: AB_1210991.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Magnetic Beads, Software, Microscopy, Microarray, Imaging, Flow Cytometry, Mutagenesis, Gel Extraction, Sample Prep, Clear Native PAGE, Bicinchoninic Acid Protein Assay

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: Parental HCT116 and the five different conditional eIF3‐mAID cell lines were treated with the vehicle DMSO or with IAA for the periods indicated. Metabolic activity (i.e., NAD(P)H) as a proxy of cell proliferation was determined by MTT assay. Graphs represent means ± SD, n = 5–6. Asterisks denote: * P < 0.005, ** P < 0.00005, *** P < 0.000005 (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). 1 × 10 3 cells of the indicated cell lines were plated and grown in DMSO or IAA for 2 weeks. Colony numbers were counted and are shown in a bar graph relative to the DMSO‐treated control. Note that very low numbers of colonies were obtained for IAA‐treated eIF3a‐mAID, eIF3e‐mAID and eIF3f‐mAID – hence no bars are visible. Bars represent means ± SD, n = 3. Numbers indicate P ‐values (unpaired Student's t ‐test). The indicated cell lines were subjected to serum withdrawal (0% FBS) for 16 h. DMSO or IAA was added and cells were stimulated to re‐enter the cell cycle by refeeding with media containing 10% FBS. Cells were fixed after the indicated time points, and the cellular DNA content was measured by flow cytometry. Data are representative of 2 (HCT116, eIF3a‐mAID, eIF3b‐mAID) or 3 (eIF3e‐mAID, eIF3f‐mAID, eIF3k‐mAID) experiments. 1 × 10 6 eIF3‐mAID cells were injected into nude mice. When tumors reached a diameter of ~ 4 mm, mice were treated with vehicle or 500 mg/kg IAA, and tumor growth was measured for 2 weeks. Graphs represent means ± SD, n = 5–7. Numbers indicate P ‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). Expression of the indicated eIF3 proteins in 97 colon adenocarcinomas versus 100 normal colonic mucosae and 110 clear cell renal cell carcinomas versus 84 normal kidney samples. Log2 spectral count data from CPTAC (Chen et al , ) via UALCAN portal (Chandrashekar et al , ). Data information: The median (50 th percentile), 25% percentile (lower box), 75 th percentile (upper box), 5 th percentile (lower whisker), and 95 th percentile (upper whisker) are indicated. n = biologically independent tumor samples. Source data are available online for this figure.

Article Snippet: Rabbit Polyclonal Anti‐ eIF3e (WB 1:2,000) , Bethyl , Cat # A302‐985A; RRID: AB_10749034.

Techniques: Activity Assay, MTT Assay, Flow Cytometry, Injection, Expressing, Whisker Assay

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: The indicated conditional eIF3 cell lines were exposed to 500 μM IAA for 12 h to induce the degradation of the respective eIF3 subunits. Cell lysates were subjected to immunoblotting to quantify the expression of the indicated eIF3 subunits. Signals were normalized to the reference signal of actin and plotted relative to the vehicle DMSO. Bars represent means ± SD, n = 3. Numbers indicate P ‐values (unpaired Student's t ‐test). The indicated cell lines were exposed to IAA for 12 h, and cell lysates were separated by sucrose density gradient centrifugation. The monosomal (M) and polysomal (P) peaks were quantified, and P/M ratios relative to the vehicle DMSO were plotted. Bars represent means ± SD, n = 3. Numbers indicate P ‐values (unpaired Student's t ‐test). Scatter plots of changes in polysomal mRNA versus total mRNA upon depletion of the indicated eIF3 subunits. Enrichment of Gene Ontology terms in the sets of polysome‐associated mRNAs decreased in the polysomal fractions of eIF3f‐ or eIF3e‐depleted cells or increased in eIF3k‐depleted cells. Distribution histograms of translational efficiencies (TEs) of the mRNA sets shown in (C). The total number of mRNAs and the median TE of each dataset are indicated. The number of up and down changes with FDR ≤ 0.2 as well as the median of all changed items are indicated in red, blue, and black bold print. Distribution histograms of the protein synthesis ratios determined in eIF3 subunit depleted versus replete cells. The total number of measured proteins and the median synthesis change of each dataset are indicated. The number of up and down changes with FDR ≤ 0.1 as well as the median of all changed items are indicated in red and blueprint. Data information: n = number of biological replicates. Source data are available online for this figure.

Article Snippet: Rabbit Polyclonal Anti‐ eIF3e (WB 1:2,000) , Bethyl , Cat # A302‐985A; RRID: AB_10749034.

Techniques: Western Blot, Expressing, Gradient Centrifugation

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: Primary data for the graph shown in Fig . The indicated conditional eIF3 cell lines were exposed to 500 μM IAA for 12 h to induce the degradation of the respective eIF3 subunit. Cell lysates were subjected to immunoblotting to quantify the expression of the indicated eIF3 subunits. Actin signal was used as a reference. Representative primary data for the graph in Fig . The indicated cell lines were exposed to IAA for 12 h, and cell lysates were separated by sucrose density gradient centrifugation. The monosomal (40S, 60S, 80S) and polysomal peaks were quantified, and P/M ratios relative to the vehicle DMSO are plotted in Fig . The region of the gradient used for extraction of polysomal RNA for RNA‐seq is indicated. Strategy of analyzing transcriptomic and proteomic datasets. Total (blue panel) and polysomal (yellow) RNA samples were collected from triplicate cultures of eIF3‐mAID cells exposed to DMSO or IAA for 12 h. Raw RNA‐seq datasets were normalized for sequencing depth by calculating counts per million mapped reads (CPM). For polysomal RNA samples, an additional normalization step was performed to account for the different levels in total polysomes in each sample. This was particularly necessary for those cell lines that showed a strong decrease in polysomes, including eIF3a‐mAID, eIF3e‐mAID, and eIF3f‐mAID; but the normalization was performed for all six cell lines. In the next step, edgeR (Robinson et al , ) was used to identify changed mRNAs. mRNAs changed in polysomes (FDR ≤ 0.05) were analyzed for enriched Gene Ontology functional terms (Fig ). mRNAs changed (FDR ≤ 0.05) in the total and polysomal populations were then graphed in a scatter plot (Fig ). Translational efficiencies (TEs) were calculated for all data points included in the scatter plot with cpm ≥ 10 as Log2 [(polysomal mRNA IAA / total mRNA IAA) / (polysomal mRNA DMSO / total mRNA DMSO)], and TEs were plotted in histograms (Fig ). Triplicate LC–MS/MS datasets were subjected to statistical testing (Benjamini–Hochberg) to identify proteins whose synthesis (Log2 Heavy IAA / Heavy DMSO) was changed upon depletion of eIF3 subunits at a FDR ≤ 0.1. Log2 Heavy IAA / Heavy DMSO values were graphed in a histogram (Fig ). Changes in TE and protein synthesis were compared in a scatter plot of Log2 TE versus Log2 Heavy IAA/Heavy DMSO (Appendix Fig ). Venn diagrams summarizing the changes in the total and polysomal mRNAs (FDR ≤ 0.05) in the indicated cell lines. Source data are available online for this figure.

Article Snippet: Rabbit Polyclonal Anti‐ eIF3e (WB 1:2,000) , Bethyl , Cat # A302‐985A; RRID: AB_10749034.

Techniques: Western Blot, Expressing, Gradient Centrifugation, RNA Sequencing Assay, Sequencing, Functional Assay, Liquid Chromatography with Mass Spectroscopy

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti‐ eIF3e (WB 1:2,000) , Bethyl , Cat # A302‐985A; RRID: AB_10749034.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Magnetic Beads, Software, Microscopy, Microarray, Imaging, Flow Cytometry, Mutagenesis, Gel Extraction, Sample Prep, Clear Native PAGE, Bicinchoninic Acid Protein Assay