Journal: Cancer cell

Article Title: ATRX in-frame fusion neuroblastoma is sensitive to EZH2 inhibition via modulation of neuronal gene signatures

doi: 10.1016/j.ccell.2019.09.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: G401 , ATCC , CRL-1441.

Techniques: Virus, Microarray, Recombinant, Magnetic Beads, Bicinchoninic Acid Protein Assay, Cell Culture, DNA Library Preparation, Extraction, Expressing, Sequencing, Software

Journal: Immunity

Article Title: Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2

doi: 10.1016/j.immuni.2017.11.024

Figure Lengend Snippet: Relationship of In Vitro Activated Monocyte-Derived Cells (A and B) PCA (21,250 present probes); displayed principal components (PCs): (A) 1 versus 2 and (B) 1 versus 3. (C) Heatmap of 1,000 most variable genes in dataset. Log 2 -expression values, z-transformed, scaled (−2 [blue] to 2 [red]). (D) Heatmap, Pearson correlation values (PCV) calculated pairwise between all cell types (top 1,000 most variable genes). (E) PCA (23,952 present probes). (F) Relative fractions of MO, BDCA1 + DC, infM, and infDC gene signatures in CD14 + MOs, different MO-derived cells, DCs. (G and H) Heatmaps of genes specifically expressed in (G) infDCs compared to infMs, BDCA1 + DCs, MOs (dataset 1), and MOs-GM-CSF IL-4(0-72h) versus MO-derived cells, CD14 + MOs, and DCs (dataset 2), or in (H) infMs compared to infDCs, BDCA1 + DCs, and MOs (dataset 1), and in MOs-GM-CSF and MOs-M-CSF versus MOs-GM-CSF IL-4(0-72h) , CD14 + MOs, and DCs (dataset 2). PCVs between indicated group patterns of dataset 1 versus dataset 2, barplot next to heatmaps (correlation cutoff > 0.4). Genes analyzed in (J) and (K) highlighted in red. Log 2 -expression values, z-transformed, scaled (−2 [blue] to 2 [red]). (I) Histograms (flow cytometry analysis), relative expression CD226, MARCO, VSIG4, and CCR7 (representative data, n = 4). (J) Analysis of cell culture supernatants of MOs-M-CSF, MOs-GM-CSF, and MOs-GM-CSF IL-4(0-72h) for CCL22 and CCL2 using ELISA (n = 3, 2 technical replicates each, mean + SEM, one-way RM [repeated-measures] ANOVA, Tukey’s method for multiple test correction, with ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; n.d., not detected). (K) MMP12 quantification (relative) in CD14 + MOs, MOs-M-CSF, MOs-GM-CSF, and MOs-GM-CSF IL-4(0-72h) by immunoblot (n = 3, mean + SEM, one-way ANOVA and Tukey’s method for multiple test correction, with ∗ p < 0.05). Please also see Figure S1 .

Article Snippet: CD14 Microbeads, human , Miltenyi Biotec , Cat# 130-042-401.

Techniques: In Vitro, Derivative Assay, Expressing, Transformation Assay, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Immunity

Article Title: Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2

doi: 10.1016/j.immuni.2017.11.024

Figure Lengend Snippet: MOs-GM-CSF IL-4 Are Most Distinct from MOs-M-CSF and MOs-GM-CSF (A) Schema describing the questions addressed here and in Figure S2 . (B) PCA (18,318 present probes). (C) Heatmap of 1,000 most variable genes in dataset. Log 2 -expression values, z-transformed, scaled (−2 [blue] to 2 [red]). (D) Heatmap of specifically expressed genes in a single out of the three MO-derived cells versus CD14 + MOs and the other two MO-derived cell types. Log 2 -expression values, z-transformed, scaled (−1.5 [blue] to 1.5 [red]). (E) Co-expression networks (union of 2,086 DEG [fold-change > 2 or < −2 and FDR-adjusted p value < 0.05]) between each of three MO-derived cells types versus CD14 + MOs. Fold-change of respective cell type versus overall mean mapped onto networks and displayed blue (negative fold-change) over white to red (positive fold-change). Based on fold-change patterns, networks were divided into four clusters, each cluster representing one of four cell types. (F) Co-expression network (411 TRs expressed in dataset). For each cell type, fold-change of respective cell type versus overall mean mapped onto network. Cell type-specific clusters of upregulated regulators were generated, indicated by color-coded shadings behind network. TRs highlighted in red were predicted as unique master regulators of corresponding cell type. Prediction performed on all genes highlighted in red (fold-change > 1.5 over overall mean) in corresponding cell type-specific cluster in (E). (G) t -SNE display of CD14 + MOs, MOs-M-CSF, MOs-GM-CSF, and MOs-GM-CSF IL-4(0-144h) analyzed by MC (n = 3). (H) Heatmap and HC of mean surface marker expression analyzed using MC. Normalized intensity values, z-transformed, scaled (−6 [blue] to 6 [red]). Color code depicts cluster assignment according to culture condition. Color code as in (G). Please also see Figure S2 .

Article Snippet: CD14 Microbeads, human , Miltenyi Biotec , Cat# 130-042-401.

Techniques: Expressing, Transformation Assay, Derivative Assay, Generated, Marker

Journal: Immunity

Article Title: Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2

doi: 10.1016/j.immuni.2017.11.024

Figure Lengend Snippet: MOs-GM-CSF IL-4(0-144h) Differ from MOs-GM-CSF IL-4(72-144h) MO-Derived Cells (A) Schema describing questions addressed herein and in Figure S4 . (B) PCA (18,857 present probes). (C) Co-expression network (13,691 present genes) describing relationships between CD14 + MOs and four types of MO-derived cells. (D) Heatmap of 1,000 most variable genes in dataset. Log 2 -expression values, z-transformed, scaled (−2 [blue] to 2 [red]). Highly expressed genes grouped together (black boxes) according to cell type. Corresponding group-related cell types highlighted, left side of heatmap. Important genes of each cluster depicted, right side of heatmap. (E) Flow cytometry analysis of MOs-GM-CSF IL-4(72-144h) and MOs-GM-CSF IL-4(0-144h) after incubation with GFP-expressing yeast (1 hr, n = 4–6, mean + SEM, Student’s t test with ∗ p < 0.05). (F) Migration tracks (3 hr) of MOs-GM-CSF IL-4(72-144h) and MOs-GM-CSF IL-4(0-144h) (representative result, n = 3). Please also see Figure S4 .

Article Snippet: CD14 Microbeads, human , Miltenyi Biotec , Cat# 130-042-401.

Techniques: Derivative Assay, Expressing, Transformation Assay, Flow Cytometry, Incubation, Migration

Journal: Immunity

Article Title: Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2

doi: 10.1016/j.immuni.2017.11.024

Figure Lengend Snippet: Timing of IL-4 Determines Transcriptional Regulation in Differentiated MOs (A) Schema describing the IL-4 time kinetic experiment. (B and C) Histograms, relative expression of CD14 and CD209 analyzed by flow cytometry. (D) PCA (12,794 present genes). (E) Heatmap of 1,000 most variable genes across dataset. Log 2 -expression values, z-transformed, scaled (−2 [blue] to 2 [red]). Below, SOM-clustering (12,794 present genes across cell types). (F) Co-expression networks (union of 2,775 DEGs, fold-change > 1.5 or < −1.5 and FDR-corrected p value < 0.05) between MOs-GM-CSF IL-4 and MOs-GM-CSF. For each cell type, fold-change of respective cell type versus overall mean mapped onto networks, displayed in blue (fold-change ≤ 1.5) or red (fold-change ≥ 1.5). (G) Example of genes located in condition-related clusters depicted in (F) and in first column (fold-change ≥ 1.5). First column: condition-specific genes; following columns: genes shared between clusters of two consecutive time points. Please also see Figure S5 .

Article Snippet: CD14 Microbeads, human , Miltenyi Biotec , Cat# 130-042-401.

Techniques: Expressing, Flow Cytometry, Transformation Assay

Journal: Immunity

Article Title: Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2

doi: 10.1016/j.immuni.2017.11.024

Figure Lengend Snippet: NCOR2 Is a Transcriptional Regulator of MOs-GM-CSF IL-4(0-72/144h) (A and B) Co-expression networks (267 TRs) for (A) MOs-GM-CSF, (B) MOs-GM-CSF IL-4(0-72h) , fold-change versus CD14 + MOs mapped onto network. MOs-GM-CSF IL-4(0-72h) -specific cluster of elevated regulators (dark blue). (C) TR Heatmap, specifically upregulated in MOs-GM-CSF IL-4(0-72h) , MOs-GM-CSF IL-4(0-144h) versus CD14 + MOs, MOs-GM-CSF. Log 2 -expression values, z-transformed, scaled (−1.15 [blue] to 1.15 [red]). (D) Immunoblot of NCOR2, Lamin A/C, and β-tubulin in cytoplasm and nucleus of MOs-GM-CSF and MOs-GM-CSF IL-4(0-144h) (representative result, n = 4). (E) Quantification of relative enrichment of NCOR2 in nuclear fractions isolated from MOs-GM-CSF and MOs-GM-CSF IL-4(0-144h) (n = 4, ∗ p < 0.05, mean ± SEM). (F) Representative confocal microscopy images of medial nuclear region (MNR) of MOs-GM-CSF and MOs-GM-CSF IL-4(0-144h) (n = 3, green: NCOR2; blue: DAPI; red: tubulin). (G) Quantification of mean fluorescence intensity of NCOR2 in MNR identified by confocal microscopy of GM-CSF and MOs-GM-CSF IL-4(0-144h) (n = 3, green: NCOR2; blue: DAPI; red: tubulin, ∗ p < 0.05, mean ± SEM). (H) Scatterplot (1,834 variable genes in dataset) containing cells treated with αNCOR2 (y axis), scrambled siRNA (x axis, log 2 -mean expression values). Highlighted genes determined (see Figure S6 F) induced (red) or repressed (blue) by IL-4. (I and J) GSEA of genes upregulated (I), downregulated (J) in IL-4 signature in MOs-GM-CSF IL-4(0-72h) treated with scrambled or α NCOR2 siRNA (nominal p value, empirical phenotype-based permutation test [p < 0.05, FDR < 0.25], 1,000 samples permutations). Please also see Figure S6 .

Article Snippet: CD14 Microbeads, human , Miltenyi Biotec , Cat# 130-042-401.

Techniques: Expressing, Transformation Assay, Western Blot, Isolation, Confocal Microscopy, Fluorescence

Journal: Immunity

Article Title: Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2

doi: 10.1016/j.immuni.2017.11.024

Figure Lengend Snippet: Mass Cytometry Analysis Identifies Unappreciated Phenotypic Heterogeneity in Clinically Relevant Mo-GM-CSF IL-4(0-144h) Cultures (A) Phenograph of CD14 + MOs, MOs-M-CSF, MOs-GM-CSF, and MOs-GM-CSF IL-4(0-144h) based on MC expression data (n = 3, 36 myeloid-related surface markers). Affiliation of cells to the 11 identified clusters indicated by color coding and visualized in t -SNE plot. (B) Heatmap and HC of mean surface marker expression of 11 individual clusters. Right side: Differentiating conditions according to (A) and Figure 2 G. (C) Phenograph of MOs-GM-CSF IL4(0-144h) and visualized in t -SNE plot (representative donor; 3,500 cells). (D) Heatmap and HC of mean surface marker expression of 11 individual clusters. (E) Expression feature plot of the depicted surface markers in MOs-GM-CSF IL-4(0-144h) .

Article Snippet: CD14 Microbeads, human , Miltenyi Biotec , Cat# 130-042-401.

Techniques: Mass Cytometry, Expressing, Marker

Journal: Immunity

Article Title: Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2

doi: 10.1016/j.immuni.2017.11.024

Figure Lengend Snippet:

Article Snippet: CD14 Microbeads, human , Miltenyi Biotec , Cat# 130-042-401.

Techniques: Purification, Recombinant, Blocking Assay, Electron Microscopy, cDNA Synthesis, CyQUANT Assay, Proliferation Assay, Labeling, Sample Prep, Library Quantification, Enzyme-linked Immunosorbent Assay, Conjugation Assay, Microarray, Cell Culture, Sequencing, Control, Software

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Expression of CDK11 and CK2 protein complex members in untransformed and malignant breast cells. (A) Immunoblot analysis of cultured breast cell lines, as indicated above the blots. Proteins detected are indicated on the right side of the blots. Actin signal was used as the loading control. (B) Indirect immunofluorescent detection of CDK11, CK2α, and CK2α′ (red color) in breast cell lines. Cell lines are indicated above each set of images and proteins detected are indicated on the left side of the images. Blue, 4′,6-diamidino-2-phenylindole-stained nuclei. Scale bar: 100 μm. (C) Immunohistochemical detection of CDK11 proteins in human normal and malignant breast tissue. Type of breast tissue indicated on the left side of the images. Magnification indicated above the images; dotted ellipse, portion of the 100× image that is shown at 400×. Scale bars: 400 μm for 100× and 100 μm for 400× images. (D) Human microarray tissues stained for CDK11 were scored by two independent observers. The average value was taken and the results plotted for normal ( n = 16) versus triple-negative breast cancer (TNBC; n = 44) tissues. Box, first to third (Q1 to Q3) quartiles; diamond, mean; line inside box, median; whiskers, minimum and maximums of data range. CDK, cyclin-dependent kinase; CK2, casein kinase 2.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Cell Culture, Control, Staining, Immunohistochemical staining, Microarray

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: RNA expression levels in normal breast and breast cancer subtypes. Normalized RNAseq read count data for PAM50 breast cancer subtypes and normal breast from The Cancer Genome Atlas were analyzed for CDK11 and CK2 protein complex genes as shown above each plot. Box, first to third (Q1 to Q3) quartiles; line inside box, median; whiskers, 1.5 maximum interquartile range. Normal, n = 95; basal, n = 141; Her2, n = 67; LumA, n = 421; LumB, n = 192. CDK, cyclin-dependent kinase; CK2, casein kinase 2; Her2, human epidermal growth factor receptor 2; LumA, luminal A; LumB, luminal B.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: RNA Expression

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: mRNA expression levels in normal and breast cancer subtypes

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Comparison

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Immunoblot analyses following small interfering RNA-mediated downregulation of CDK11 and CK2 in breast cancer cells. Immunoblot analysis of MDA-MB-231 and SUM-149 cell lysates following small interfering RNA (siRNA) transfection. Transfected siRNAs are indicated above the blots, proteins detected are indicated on the right side of the blots. CDK11 p110 , cyclin L1α, cyclin L2α, and CK2αα′β lysates are 72 hours post transfection; caspase 3, Bcl-xL, and survivin lysates are 96 hours post transfection. Actin signal was used as the loading control. CDK, cyclin-dependent kinase; CK2, casein kinase 2.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Small Interfering RNA, Transfection, Control

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Protein expression levels following small interfering RNA transfection

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Small Interfering RNA

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Small interfering RNA-mediated downregulation of CDK11 and CK2 in breast cancer cells decreases cell viability and inhibits clonal survival. (A) Breast cancer cells were transfected with 30 nM single small interfering RNA (siRNA) or 15 nM each of the two siRNAs combined as indicated. After 96 hours, cell viability was determined relative to the untreated cells. Means ± standard errors (SEs) are presented. * P <0.05. ** P <0.01, *** P <0.001 relative to untreated. ^ P = 0.055, # P <0.05, ## P <0.01, ### P <0.001 relative to siCtrl. (B) Triple-negative breast cancer (TNBC) cells were transfected twice with 30 nM single siRNAs or 15 nM each of the two siRNAs combined as indicated and as described in . Seven days after the second transfection, cell colonies were stained and counted. Means ± SE are presented. $ P <0.0001 relative to siCtrl and untreated. (C) Representative crystal violet stained colonies on 35 mm plates 7 days after the second siRNA transfection as described in (B). Cell lines are indicated above the plate images and siRNA transfections are indicated to the left of the plate images. CDK, cyclin-dependent kinase; CK2, casein kinase 2; si, small interfering.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Small Interfering RNA, Transfection, Staining

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Nanocapsule morphology and uptake efficiency in cultured cells and in xenograft tumors. (A) Transmission electron micrographs of TBG-siCDK11, TBG-siCK2, and TBG-siCON1 nanocapsules used for in vivo studies. Scale bar: 100 nm. (B) MDA-MB-231 fluorescence-activated cell sorting (FACS) analysis for dysprosium (Dy) in untreated cells (black outline) and in TBG-Dy treated cells (gray). The number of TBG-Dy treatments is indicated above the graphs. (C) FACS analysis of xenograft tumor cells from untreated mouse (left panels) and from intravenous TBG-Dy-treated mouse (right panels). The identity of the tumor type is indicated above each panel. The position of the gate set to define Dy-positive cells is shown as a black line with Dy-positive events to the right of the line. CDK, cyclin-dependent kinase; CK2, casein kinase 2; si, small interfering; SSC, side scatter; TBG, tenfibgen.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Transmission Assay, In Vivo, Fluorescence, FACS

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Therapeutic effects of TBG-siCDK11 and TBG-siCK2 treatment in MDA-MB-231 xenograft tumors. (A) Primary tumor volumes are shown following intravenous treatments at 0.01 mg/kg TBG-siCDK11, TBG-siCK2, or TBG-siCON1 on days 1, 4 and 7 (indicated by arrows). Means ± standard errors (SEs) are presented (TBG-siCDK11 and TBG-siCK2, n = 6; TBG-siCON1, n =7). * P <0.05, ** P <0.01. (B) Primary tumor masses are shown following treatments as described in (A). Thick line, mean; thin bar, SE (TBG-siCDK11 and TBG-siCK2, n = 6; TBG-siCON1, n = 7). # P <0.05. (C) Masses of the mice throughout the study for each treatment group. Means are presented and error bars represent the SE. (D) Percentage of Ki-67-positive cells was analyzed as described in and is shown graphically. Least-squares means are presented and error bars represent SE. ** P <0.01. CDK, cyclin-dependent kinase; CK2, casein kinase 2; si, small interfering; TBG, tenfibgen.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Analysis for RNA-induced silencing complex cleavage products in treated tumors and immunoblot analysis for target protein complexes and death signals in primary tumors. (A) Total RNA was isolated from tumor tissue and used for 5′ ligation-mediated RACE to determine whether RNA-induced silencing complex (RISC)-mediated cleavage of the transcript occurred. The predicted RACE products are indicated to the right, the size (base pairs (bp)) of the DNA standards shown on the left, and the treatment administered indicated above the lanes. (B) Immunoblot analysis of MDA-MB-231 day 10 tumor lysates following intravenous treatments of 0.01 mg/kg TBG-siCDK11, TBG-siCK2, or TBG-siCON1 as indicated above the blots. The signals for three mice per group are shown and the proteins detected are indicated on the right side of the blots. Actin signal was used as the loading control. (C) The protein signals from all mice in each treatment group were quantitated by densitometry using ImageJ software (National Institutes of Health Bethesda, MD, USA). Data presented as mean ± standard error. * P <0.05, ** P <0.01. CDK, cyclin-dependent kinase; CK2, casein kinase 2; si, small interfering; TBG, tenfibgen.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Isolation, Ligation, Control, Software

Journal: Cell reports

Article Title: A molecular interactome of the glioblastoma perivascular niche reveals integrin binding sialoprotein as a mediator of tumor cell migration

doi: 10.1016/j.celrep.2022.111511

Figure Lengend Snippet:

Article Snippet: Anti-human CD31 antibody, REA730 , Miltenyi Biotec , Cat#: 130-110-670; RRID:AB_2657283.

Techniques: Plasmid Preparation, Virus, Clone Assay, Recombinant, CCK-8 Assay, Viability Assay, Microarray, Knock-Out, Software, Functional Assay

Journal: Redox Biology

Article Title: Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution

doi: 10.1016/j.redox.2019.101234

Figure Lengend Snippet: NOX4 Antibody validation, NOX4 expression pattern in the normal human glomerular compartment, and in renal nephropathies. Representative Western blot analysis of T-Rex cells overexpressing (right lane) or not (left lane) the human NOX4 protein ( A ). Representative images of NOX4 immunohistochemical analysis of T-REX cells overexpressing (tetracycline inducible) ( C ) or not ( B ), the human NOX4 protein. D–F: Representative images of NOX4 immunohistochemical analysis of normal human kidney, diabetic and IgA nephropathies. In normal glomeruli, NOX4 expression is restricted to parietal glomerular epithelial cells ( D , upper and lower panel). NOX4 expression is observed in the glomerular mesangium of diabetic nephropathy ( E, upper and lower panel) and IgA nephropathy ( F, upper and lower panel).

Article Snippet: The following primary antibodies were used: NOX4 (rabbit monoclonal, 1:500) [ , ], BCL-2 (Rabbit, 1:500, sc-492), NRF-2 (Rabbit, 1:500, sc-722), α-SMA (Mouse, 1:2000, kind gift of Dr. Marie-Luce Bochaton-Piallat, Department of Immunology, University of Geneva), Collagen1α (Rabbit, 1:500, Rockland 600-401-103S), Fibronectin (Rabbit, 1:500, ab-2413), HIF1α (Rabbit, 1:500, NB100-479).

Techniques: Biomarker Discovery, Expressing, Western Blot, Immunohistochemical staining

Journal: Redox Biology

Article Title: Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution

doi: 10.1016/j.redox.2019.101234

Figure Lengend Snippet: NOX4 expression in normal renal tubular cells and in tubulo-interstitial injury. A-B: Representative images of NOX4 immunohistochemical analysis of normal human kidney. A marked cytoplasmic NOX4 immunostaining (brown) is observed in proximal tubules, whereas expression is much lower in distal segments of the nephron (lower magnification of the image shown in D) ( A ). NOX4 and Na, K-ATPase (red) double immunostaining. NOX4 (in brown) appears predominant at the basolateral side of proximal tubular cells and colocalizes with basolateral sodium-potassium pump Na, K-ATPase ( B ). C–D: Representative images of NOX4 immunohistochemical analysis in chronic tubulo-interstitial injury. NOX4 expression is decreased and almost absent in atrophic tubules in diabetes nephropathy ( C ). Periodic acid Schiff staining ( D ) showing area of fibrosis. Analysis of NOX4, GSTA1, NOX2 and TGFβ1 gene expression in the Affymetrix microarray expression dataset generated by the European Renal cDNA Kröner-Fresenius Biopsy bank demonstrating a marked decrease in NOX4 mRNA expression correlating with the different stages of CKD ( E ). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following primary antibodies were used: NOX4 (rabbit monoclonal, 1:500) [ , ], BCL-2 (Rabbit, 1:500, sc-492), NRF-2 (Rabbit, 1:500, sc-722), α-SMA (Mouse, 1:2000, kind gift of Dr. Marie-Luce Bochaton-Piallat, Department of Immunology, University of Geneva), Collagen1α (Rabbit, 1:500, Rockland 600-401-103S), Fibronectin (Rabbit, 1:500, ab-2413), HIF1α (Rabbit, 1:500, NB100-479).

Techniques: Expressing, Immunohistochemical staining, Immunostaining, Double Immunostaining, Staining, Gene Expression, Microarray, Generated

Journal: Redox Biology

Article Title: Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution

doi: 10.1016/j.redox.2019.101234

Figure Lengend Snippet: Generation and validation of tubular cell specific Nox4 knock-in mice. NOX4 protein expression in renal cortex harvested from wild-type (Wt) mice and mice subjected to 10 days of Unilateral Ureteral Obstruction ( A ). B–C: Gene expression and Western blot analysis of renal cortex and medulla dissected from Wt and tubular cell specific Nox4 KI kidneys. Nox4 mRNA expression ( B ) and NOX4 protein levels ( C ) are increased in both cortex and medulla of Nox4 KI kidneys. HE-higher exposure of the blot. Quantification of high (70 kDa) and low molecular weight (35 kDa) NOX4 protein bands are shown. D–F: Detection of H 2 O 2 production in the primary epithelial cells and renal organoids isolated from Wt and Nox4 KI kidneys. Fold increase in Amplex red fluorescence in the primary epithelial cells is shown in D . Expression levels of Nox4 mRNA in the cultured primary epithelial cells ( E ). Panel showing the visualization of increased H 2 O 2 generation in renal organoids isolated from Nox4 KI kidney by the production of intense florescent pink color and the fold increase in H 2 O 2 production ( F ). Representative Sirius red images of Wt and Nox4 KI kidney sections demonstrating that the overall kidney structure was not modified ( G ). Transcutaneous glomerular filtration rate measurement by sinistrin-FITC excretion rate on Wt and Nox4 KI mice ( H ). Estimation of glomerular filtration rate by endogenous creatinine clearance ( I ). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following primary antibodies were used: NOX4 (rabbit monoclonal, 1:500) [ , ], BCL-2 (Rabbit, 1:500, sc-492), NRF-2 (Rabbit, 1:500, sc-722), α-SMA (Mouse, 1:2000, kind gift of Dr. Marie-Luce Bochaton-Piallat, Department of Immunology, University of Geneva), Collagen1α (Rabbit, 1:500, Rockland 600-401-103S), Fibronectin (Rabbit, 1:500, ab-2413), HIF1α (Rabbit, 1:500, NB100-479).

Techniques: Biomarker Discovery, Knock-In, Expressing, Gene Expression, Western Blot, Molecular Weight, Isolation, Fluorescence, Cell Culture, Modification, Filtration

Journal: Redox Biology

Article Title: Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution

doi: 10.1016/j.redox.2019.101234

Figure Lengend Snippet: Pro-survival, antioxidant and angiogenesis pathways are activated in Nox4 KI mice at baseline. Gene expression analysis of pro-survival ( Nrf2 , Bcl2 ) and antioxidant pathway ( Gstα, Nqo1 ) genes at baseline. The gene expression level for each gene is normalized to the housekeeping gene Rplp0 ( A ). Representative Western blot images of proteins involved in the cell survival, angiogenesis and fibrosis processes. Quantitation of Western blot experiments are shown in ( B ).

Article Snippet: The following primary antibodies were used: NOX4 (rabbit monoclonal, 1:500) [ , ], BCL-2 (Rabbit, 1:500, sc-492), NRF-2 (Rabbit, 1:500, sc-722), α-SMA (Mouse, 1:2000, kind gift of Dr. Marie-Luce Bochaton-Piallat, Department of Immunology, University of Geneva), Collagen1α (Rabbit, 1:500, Rockland 600-401-103S), Fibronectin (Rabbit, 1:500, ab-2413), HIF1α (Rabbit, 1:500, NB100-479).

Techniques: Gene Expression, Western Blot, Quantitation Assay

Journal: Redox Biology

Article Title: Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution

doi: 10.1016/j.redox.2019.101234

Figure Lengend Snippet: Gene expression changes in Wt and Nox4 KI animals subjected to 3 days and 10 days of UUO. A-B: Gene expression analysis on Wt and Nox4 KI kidneys harvested 3 days ( A ) and 10 days ( B ) after subjecting the animals to UUO. The non-obstructed kidneys of the same animals were used as controls. Gene expression levels of Nox4, Nox2 , pro-survival pathway genes ( Bcl2, Nrf2) and antioxidant genes ( Gstα, Nqo1 ) are shown. The gene expression level for each gene is normalized to the housekeeping gene Rplp0 .

Article Snippet: The following primary antibodies were used: NOX4 (rabbit monoclonal, 1:500) [ , ], BCL-2 (Rabbit, 1:500, sc-492), NRF-2 (Rabbit, 1:500, sc-722), α-SMA (Mouse, 1:2000, kind gift of Dr. Marie-Luce Bochaton-Piallat, Department of Immunology, University of Geneva), Collagen1α (Rabbit, 1:500, Rockland 600-401-103S), Fibronectin (Rabbit, 1:500, ab-2413), HIF1α (Rabbit, 1:500, NB100-479).

Techniques: Gene Expression

Journal: Redox Biology

Article Title: Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution

doi: 10.1016/j.redox.2019.101234

Figure Lengend Snippet: Renal NOX4 protein expression is decreased in a severe fibrotic renal disease mouse model but not in NOX4 KI mice. A–B: Western blot analysis on Wt and Nox4 KI kidneys harvested 3 days ( A ) and 10 days ( B ) after subjecting to UUO. The non-obstructed kidneys were used as controls. Protein expression levels of NOX4, and proteins implicated in cell survival pathways (NRF2, BCL2) and fibrosis (Collagen 1α, Fibronectin and α-SMA) and the quantification are shown.

Article Snippet: The following primary antibodies were used: NOX4 (rabbit monoclonal, 1:500) [ , ], BCL-2 (Rabbit, 1:500, sc-492), NRF-2 (Rabbit, 1:500, sc-722), α-SMA (Mouse, 1:2000, kind gift of Dr. Marie-Luce Bochaton-Piallat, Department of Immunology, University of Geneva), Collagen1α (Rabbit, 1:500, Rockland 600-401-103S), Fibronectin (Rabbit, 1:500, ab-2413), HIF1α (Rabbit, 1:500, NB100-479).

Techniques: Expressing, Western Blot

Journal: Redox Biology

Article Title: Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution

doi: 10.1016/j.redox.2019.101234

Figure Lengend Snippet: Constitutive Nox4 expression in renal tubular cells does not prevent fibrotic disease progression. A, B, E: Representative images of Sirius red staining and quantification on the sections of Wt and Nox4 KI kidneys harvested 3 days ( A, E ) and 10 days ( B, E ) after subjecting to UUO. Endomucin immunostaining and quantification on the sections of Wt and Nox4 KI kidneys harvested 3 days ( C, F ) and 10 days ( D, F ) after subjecting to UUO are presented. The non-obstructed kidneys were used as controls. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following primary antibodies were used: NOX4 (rabbit monoclonal, 1:500) [ , ], BCL-2 (Rabbit, 1:500, sc-492), NRF-2 (Rabbit, 1:500, sc-722), α-SMA (Mouse, 1:2000, kind gift of Dr. Marie-Luce Bochaton-Piallat, Department of Immunology, University of Geneva), Collagen1α (Rabbit, 1:500, Rockland 600-401-103S), Fibronectin (Rabbit, 1:500, ab-2413), HIF1α (Rabbit, 1:500, NB100-479).

Techniques: Expressing, Biomarker Discovery, Staining, Immunostaining

Journal: Cell death & disease

Article Title: Epigenetic priming of immune/inflammatory pathways activation and abnormal activity of cell cycle pathway in a perinatal model of white matter injury.

doi: 10.1038/s41419-022-05483-4

Figure Lengend Snippet: Fig. 1 Experimental strategy and major transcriptomic impact of neuroinflammation on the immune/inflammatory pathway in O4+ cells. A We used a mouse model of encephalopathy of prematurity that we previously validated and in which we mimic the systemic and neuroinflammatory insults as undergone by human infants from approximately (23–32)-week gestational age equivalent. Neuroinflammation is induced via i.p. IL1B from postnatal days 1–5, and this leads to OPC maturation blockade, defective myelination, and behavioral anomalies as seen clinically [8]. O4+ cell populations (green circles) were respectively isolated from P5 and P10 pup cortices by MACS (see “Materials and methods”). B Quality-control validation of OPC maturation arrest in the model of perinatal neuroinflammation. RT-qPCR analysis of the expression of myelination and progenitor markers in O4+ cells showing that neuroinflammation, induced by IL1B peritoneal injections in pups, results in defects in the myelination gene expression program and inappropriate elevation of progenitor markers at P5 and P10. Myelin markers: Mbp, Myelin binding protein; Mog, Myelin oligodendrocyte glycoprotein; Mag Myelin-associated glycoprotein. Plp1 proteolipid protein 1, a transmembrane, predominant component of myelin; Cnp, 2′,3′-Cyclic Nucleotide 3′ Phosphodiesterase, abundant protein in myelin in the central nervous system. Progenitor (OPC) markers: Id2, Inhibitor of differentiation 2; Pdgfra, Platelet Derived Growth Factor Receptor Alpha. Number of independent experiments at P5: n = 7 for Cnp, Mag, and Pdgfra; and n = 15 for Mbp, Mog, and Id2; at P10: n = 14 for Mag and Mog; and n = 19 for Cnp, Pdgfra; and Mbp, and Id2. ns not statistically significant; *p < 0.05; ***p < 0.001. C (Left) Graphical representation of the total number of probes dysregulated upon neuroinflammation induced by IL1B injections in O4+ cells at P5 and P10 (white; 2572 probes at P5 and 2038 probes at P10) including upregulated (yellow; 1873 probes at P5 and 1385 probes at P10) and downregulated (blue; 699 probes at P5 and 653 probes at P10) probes; gray bars: number of probes dysregulated at P5 and P10 (common), and dysregulated at least at one stage (P5 and/or P10). (Right) Number of upregulated (UP) and downregulated genes (DOWN), at P5 or P10.

Article Snippet: Five μL volume of phosphate-buffered saline (PBS) containing 10 μG/kG/injection of recombinant mouse IL1B (R&D Systems, Minneapolis, MN) or of PBS alone (control) was injected intraperitoneally (i.p.) twice a day on days P1 to P4 and once a day, on day P5 (see Fig. 1).

Techniques: Isolation, Control, Biomarker Discovery, Quantitative RT-PCR, Expressing, Gene Expression, Binding Assay, Derivative Assay

Journal: Cell death & disease

Article Title: Epigenetic priming of immune/inflammatory pathways activation and abnormal activity of cell cycle pathway in a perinatal model of white matter injury.

doi: 10.1038/s41419-022-05483-4

Figure Lengend Snippet: Fig. 6 TFBS motifs identified in significant ATAC-seq peaks associated with cluster C1 genes and expression of the corresponding TF genes in O4+ cell populations and the Oli-neu cell line. A TFBS motif analysis reveals the presence of TF binding motifs of the immune/ inflammatory pathways in significant ATAC-seq peaks that are adjacent to the upregulated genes (UP) correspond to TF of gene cluster C1. TFBS were identified using HOMER known motifs. (See also Fig. S5). B Heat map illustrating the expression of TFs of the IRF and NFκB families in O4+ cell populations. Microarray data from microglia [9]; n = 3 PBS samples and n = 3 IL1B samples isolated from the same cortices and at the same stage is given for comparison. M: Microglia. O_P5: O4+ cells at P5. O_P10: O4+ cells at P10. C The oligodendroglial Oli-neu cell line recapitulates the expression of TF genes of the immune/inflammatory pathways in physiological and inflammatory conditions. As in Fig. 4C. Cyclin-dependent kinase 6 (Cdk6) mRNA levels were analyzed in parallel.

Article Snippet: Five μL volume of phosphate-buffered saline (PBS) containing 10 μG/kG/injection of recombinant mouse IL1B (R&D Systems, Minneapolis, MN) or of PBS alone (control) was injected intraperitoneally (i.p.) twice a day on days P1 to P4 and once a day, on day P5 (see Fig. 1).

Techniques: Expressing, Binding Assay, Microarray, Isolation, Comparison

Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet:

Article Snippet: Anti dsRED, dsRFP Purified (Rabbit Polyclonal IgG) , Rockland , #600-401-379.

Techniques: Purification, Avidin-Biotin Assay, Virus, Recombinant, High Molecular Weight, Cell Isolation, Microarray, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF1 is a transcriptional regulator of ZBP1 promoting NLRP3 inflammasome activation and cell death during influenza virus infection

doi: 10.4049/jimmunol.1701538

Figure Lengend Snippet: (A) Microarray analysis of IRF family transcription factors differentially regulated in Ifnar1−/− BMDMs infected with IAV compared with WT BMDMs. (B) Transcript levels of Irf1 in WT and Ifnar1−/− BMDMs infected with IAV for 9 h. (C) Immunoblot analysis of IRF1, IAV-NS1 and GAPDH in WT, Irf1−/−, Ifnar1−/− and Irf9−/− BMDMs at various time points after IAV infection. (D) Immunoblot analysis of caspase-1 activation in uninfected or IAV-infected WT, Irf1−/− and Nlrp3−/− BMDMs 16 h after infection. (E) Quantification of caspase-1 p20 in IAV-infected WT, Irf1−/− and Nlrp3−/− BMDMs. (F - G) Levels of IL-1β and IL-18 in cell culture supernatants from WT and Irf1−/− BMDMs infected with IAV for 16 h. (H) Transcript levels of Nlrp3, Il1b and Il6 in WT and Irf1−/− BMDMs infected with IAV for 9 h. (I) Immunoblot analysis of NLRP3, ASC and GAPDH in WT and Irf1−/− BMDMs infected with IAV at various time points. (J) Levels of IL-6, TNF and KC in cell culture supernatants from WT and Irf1−/− BMDMs infected with IAV for 16h. (K) Transcript levels of Nlrp3 in WT and Irf1−/− lung fibroblasts infected with IAV for 9h. (L) Immunoblot analysis of NLRP3, ASC and β-actin in lung fibroblasts infected with IAV. (M) Immunoblot analysis of pro-IL-1β and IL-1 β (p17) in lung fibroblasts infected with IAV for 16 h. Data are representative (B-D, G-H, (K-M)) or pooled (E-F, I) from 3–4 independent experiments. ** denotes P<0.01, *** denotes P<0.001.

Article Snippet: The primary antibodies used are: caspase-1 (AG-20B-0042, Adipogen), ZBP-1 (AG-20B-0010-C100, Adipogen), Influenza A virus NS1 (sc-130568, Santa Cruz Biotechnology), NLRP3 (AG-20B-0014, Adipogen), ASC (04–147, Millipore), caspase-8 (#4927, CST), cleaved caspase-8 (#8592, (CST), cleaved caspase-3 (#9664, CST), Caspase-3 (bs-0081R, Bioss), pIRF3 (clone 4D4G, CST), IRF3 (D83B9, CST), and pSTAT1 (D4A7, CST); IL-1β (AF-401-NA; R&D systems) and GAPDH (#5174, CST) followed by secondary anti-rabbit or anti-mouse HRP antibodies (Jackson Immuno Research Laboratories).

Techniques: Microarray, Infection, Western Blot, Activation Assay, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Casz1 expression pattern during mouse embryogenesis. A, cartoon of the Casz1 gene trap that inserted with a βgeo reporter after Casz1 exon 9 resulting in a truncated Casz1. B, genotyping of Casz1-trapped mice by RT-PCR. C, whole mount X-gal staining (blue) of E9.5 Casz1+/βgeo embryos shows that Casz1 was expressed in the hindbrain, neural tube, and heart, which are further demonstrated by dissected embryo sagittal sections 1, 2, and 3 (100× magnification). Sagittal section 3b (200× magnification) shows that Casz1 was expressed in the cardiomyocytes. D and E, whole mount X-gal staining of E12.5 Casz1+/+, Casz1+/βgeo, and Casz1βgeo/βgeo embryos showed that Casz1 is expressed in the eye, dorsomedial telencephalon, cranial ganglia, nasal placode, somite, neural tube, and heart. F, real time PCR to detect wild type Casz1 allele and Gata4 mRNA levels in E12.5 hearts. G, the protein levels of Casz1a/Casz1b and GAPDH were visualized by immunoblotting E14.5 whole heart lysate with anti-Casz1 antibody and anti-GAPDH antibody. H, whole mount anti-β-galactosidase staining (red) of E14.5 Casz1+/+, Casz1+/βgeo, and Casz1βgeo/βgeo hearts.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Hypoplasia in the myocardium of Casz1βgeo/βgeo heart. A, phenotype of Casz1+/+, Casz1+/βgeo and Casz1βgeo/βgeo E 15.5 embryos. The edema was seen at the neck region on the back of Casz1βgeo/βgeo embryos but not other embryos (green arrow). B–D, column 1, H&E staining of transverse sections of E15.5 Casz1+/+ (B1), Casz1+/βgeo (C1), and Casz1βgeo/βgeo (D1) embryos. Column 2, right ventricle (RV) magnification of the respective green squares from column 1. Column 3, septum (SP) magnification of respective yellow squares from column 1. Column 4, left ventricle (LV) magnification of respective blue squares from column 1. E, representative photomicrographs indicate accumulation of blood cells in the liver of the Casz1βgeo/βgeo embryo (right panel) but not in the Casz1+/+ (left panel) or Casz1+/βgeo (middle panel) embryo.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Decreased proliferation in Casz1βgeo/βgeo heart. A, the E14.5 Casz1βgeo/βgeo hearts were morphologically different compared with the Casz1+/βgeo or wild type heart (compare the green arrow targeted region). B, lower magnification of H&E staining of transverse sections from E14.5 embryos (upper panels). The lower panels represent a magnification of the regions that are depicted in the blue squares in the upper panel. The green arrow denotes the septal defect apparent in the serial sectioning of the Casz1βgeo/βgeo heart compared with the Casz1+/+ heart. C, paraffin sections from E14.5 hearts from Casz1+/+ and Casz1βgeo/βgeo hearts were immunostained with antibodies for MF20 (red) and the mitosis marker phosphohistone H3 (pH3, green). The nuclei were stained with DAPI (blue; left top panels, original overview; left bottom panels, high magnification). In the high magnification images, arrows indicate pH3+ cells. The graph (right panel) represents the relative percentage of pH3 positive cells compared with the total number of DAPI positive Casz1βgeo/βgeo or Casz1+/+ cardiomyocytes from at least five sections from each embryo. The Casz1βgeo/βgeo cardiomyocytes proliferate significantly slower than Casz1+/+ cardiomyocytes (p < 0.00001). D, tunnel staining (green) was performed using E14.5 heart paraffin sections (upper panels), and nuclei were stained with DAPI (blue). The lower panels represent magnified areas delineated in the red squares in the upper panels. There was no significant difference in the number of apoptotic cells observed in Casz1βgeo/βgeo cardiomyocytes compared with Casz1+/+ cardiomyocytes.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Staining, Marker

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Disorganized fiber orientation in Casz1βgeo/βgeo heart. To visualize thin filaments, Casz1+/+ and Casz1βgeo/βgeo E14.5 hearts were stained with phalloidin-488 (green) to detect F-Actin and co-stained with TOPRO3 to show cell nuclei. The left panels show the co-staining results of the left ventricles of the hearts. The right panels represent magnified areas delineated in the red squares in the left panels. Enlarged images showed complete disruption of the fiber orientation/cell alignment in the left ventricle (LV) of Casz1βgeo/βgeo heart.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Abnormal gene expression in Casz1βgeo/βgeo heart. A, microarray analysis of RNA from three E12.5 Casz1+/+ hearts and three E12.5 Casz1βgeo/βgeo hearts. The heat map was generated using Partek software. Two-thirds of these genes are aberrantly up-regulated, and one-third are aberrantly down regulated in Casz1βgeo/βgeo hearts. B, Partek gene ontology oncology analysis showed that the top two categories of enriched genes are “signaling” and the process of biological adhesion. Biological adhesion genes in Casz1βgeo/βgeo hearts that are up-regulated (red) or down-regulated (blue) are listed below. C, IPA assays showed gene enrichment in the categories of physiological system development and function (panel 1) with a key subcategory being skeletal and muscular system development and function genes that are up-regulated (red) or down-regulated (blue) in Casz1βgeo/βgeo hearts listed below and molecular and cellular function (panel 2) with a key subcategory of genes in molecular transport and the ion transport genes that are up-regulated (red) or down-regulated (blue) listed below. D, verification of microarray results. The mRNA levels of representative genes encoding cell adhesion molecules, muscle contraction, and muscular development proteins and ion channels were evaluated by real time PCR and normalized to GAPDH gene. The mRNA levels of these genes in Casz1βgeo/βgeo hearts were significantly different compared with their levels in Casz1+/+ hearts. The bar graph represents means ± S.D. (all p < 0.005).

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Expressing, Microarray, Generated, Software, Cell Function Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Abnormal expression of genes involved in cell cycle and regulation of cell proliferation in Casz1βgeo/βgeo heart. A, IPA assays showed gene enrichment in the categories of cell cycle and cellular growth and proliferation. The genes that are up-regulated (red) or down-regulated (blue) in Casz1βgeo/βgeo hearts were listed below. B, verification of microarray results. The mRNA levels of representative genes were evaluated by real time PCR and normalized to GAPDH gene. The mRNA levels of these genes in Casz1βgeo/βgeo hearts were significantly different compared with their levels in Casz1+/+ hearts. The bar graph represents means ± S.D. (all p < 0.05).

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Validation of Casz1 target genes in cellular models. A, human cardiac fibroblasts were transfected with empty vector (EMV) or CASZ1b vector and cultured for 5 days. Panel a, relative CASZ1b mRNA level was evaluated by real time PCR using human CASZ1 primer. The bar graph represents means ± S.E.(#, p < 0.05). Panel b, Western blot to show the overexpression of Casz1b in CASZ1b overexpressed cells. Panel c, after overexpression of CASZ1b, relative mRNA levels of representative genes were evaluated by real time PCR. The bar graph represents means ± S.E. (#, p < 0.05). B, knockdown or overexpression of Casz1 in HL-1 cells. Panel a, HL-1 cells were transfected with nontargeting control (con) siRNA or Casz1 siRNA, and after a 2-day culture, the relative mRNA levels of representative genes were evaluated by real time. The bar graph represents means ± S.D. (*, p < 0.005). Panel b, HL-1 cells were transfected with EMV or CASZ1b vector, and after a 2-day culture, the relative CASZ1b mRNA levels were evaluated by real time PCR using human CASZ1 primers. The graph represents mean ± S.D. (*, p < 0.005). Panel c, after transfection with CASZ1b and a two-dimensional culture, the relative mRNA levels of representative genes were evaluated by real time PCR. The bar graph represents means ± S.D. (#, p < 0.05; ns represents no statistic significant difference).

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Sarcomeric organization in Casz1βgeo/βgeo heart. A, GSEA assay indicated the negative enrichment of genes encoding contractile fiber proteins (panels a and b), as well as genes encoding contractile fiber part proteins (panel c). NES, normalized enrichment score; Nom, nominal; FDR, false discovery rate. B, sections containing the trabecular regions of E14.5 hearts were labeled for desmin (red), and nuclei were stained with DAPI (blue). There is a striated pattern for desmin labeling in both wild type and Casz1-deficient hearts, but the striated patterns are not as uniform or apparent in the Casz1-deficient heart.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Labeling, Staining

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Abnormal Z line organization in E14.5 Casz1βgeo/βgeo heart. A, cardiomyocytes isolated from Casz1+/+ and Casz1βgeo/βgeo E14.5 embryo hearts were cultured in vitro for 2 days and immunostained for α-actinin (red) and F-actin (green), whereas the nuclei were stained with DAPI (blue). Representative images show that clear Z lines were presented in the wild type cardiomyocytes, but not in the Casz1βgeo/βgeo cardiomyocytes. B, the CHITEST histogram shows the percentage of Casz1+/+ or Casz1βgeo/βgeo cardiomyocytes with clear Z lines (Z line+) or abnormal Z lines (Z line−) (p < 0.00001). The data are from two independent experiments (>100 Casz1+/+ or Casz1βgeo/βgeo cardiomyocytes were counted in each experiment). C, more representative images show that clear Z lines are present in the Casz1+/+ cardiomyocytes. There are no clear Z lines in these Casz1βgeo/βgeo cardiomyocytes, and the α-actinin staining pattern is distinct compared with the Casz1+/+ cardiomyocytes.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Isolation, Cell Culture, In Vitro, Staining

Journal: Cancer cell

Article Title: Integrative Analysis Identifies Four Molecular and Clinical Subsets in Uveal Melanoma

doi: 10.1016/j.ccell.2017.07.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Biological Samples Primary tumour samples Multiple tissue source sites, processed through the Biospecimen Core Resource See Methods: Experimental Model and Subject Details Critical Commercial Assays Genome-Wide Human SNP Array 6.0 ThermoFisher Scientific Cat: 901153 Infinium HumanMethylation450 BeadChip Kit Illumina Cat: WG-314-1002 EZ-96 DNA Methylation Kit Zymo Research Cat: D5004 Illumina Barcoded Paired-End Library Preparation Kit Illumina https://www.illumina.com/techniques/sequencing/ngs-library-prep.html TruSeq RNA Library Prep Kit Illumina Cat: RS-122-2001 TruSeq PE Cluster Generation Kit Illumina Cat: PE-401-3001 Phusion High-Fidelity PCR Master Mix with HF Buffer New England Biolabs Cat: M0531L VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard) Vector Lab Catalog: PK-6100 Deposited Data Raw and processed clinical, array and sequence data.

Techniques: Genome Wide, DNA Methylation Assay, Plasmid Preparation, Sequencing, Software, In Silico, Microarray, Expressing