Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in BT-549 cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Transformation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: H3K4me2 XH lncRNAs regulate the transcriptional activity of their protein coding partners. ( A ) Nuclear expression levels of H3K4me2 XH lncRNAs and non-CAR XH lncRNAs. ( B ) Expression of H3K4me2 XH lncRNA-protein coding pairs (pPCGs) in nuclear input and H3K4me2 ChRIP samples. The P -value denotes the significance of difference between nuclear input and H3K4me2. ( C ) Gene ontology based functional enrichment analysis of protein coding genes that are partners (pPCGs) to H3K4me2 lncRNAs: divergent (XH), other antisense (XT, XI, and XO) and sense lncRNAs. The bar graph shows number of genes in corresponding ontology term and the heatmap represents the significance of individual terms ( P -values obtained using GeneSCF). The highlighted box, by the heatmap, is listed with different transcription factors from the enriched transcription related terms. ( D ) Gene expression analysis of three transcription factors ( FOXD3, GATA6 and HOXC13 ) using RT–qPCR analysis following downregulation of their neighboring H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) with siRNA in BT-549 cells. LncRNA expression is in green bars while transcription factors expression is in grey. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( E ) RT–qPCR analysis of FOXD3, GATA6 and HOXC13 gene expression after strand-specific downregulation of FOXD3-AS1, GATA6-AS1 and HOXC13-AS respectively, using LNA. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( F ) Gene expression analysis of three H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) using RT–qPCR analysis following downregulation of their respective transcription factor partners with siRNA or esiRNA in BT-549 cells. LncRNAs expression is depicted as green bars while transcription factors expression is in grey bars. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ns denotes non-significant.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Activity Assay, Expressing, Functional Assay, Quantitative RT-PCR, esiRNA

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: XH lncRNAs are overrepresented in active lncCARs. ( A ) Scatter plot showing enrichment of WDR5 lncRNAs over nuclear input. The X-axis denotes log transformed expression and Y-axis denotes log-fold change of WDR5 lncRNAs over nuclear input. Venn diagram, in the Scatter plot, shows 209 lncRNAs that are commonly enriched in both H3K4me2 and WDR5 ChRIP pulldowns. ( B ) Distribution of different patterns of genomic arrangements as described in Figure for WDR5 lncRNAs and non-CAR lncRNAs. ( C ) Distribution of different patterns of genomic arrangements as described in Figure for active lncRNA (ChRIP pulldown using H3K4me2 and WDR5 antibodies) and inactive lncRNA (ChRIP pulldown using EZH2 and H3K27me3 antibodies). ( D ) Nuclear expression levels of WDR5 XH lncRNAs, active XH lncCAR (WDR5 and H3K4me2) and non-CAR XH lncRNAs. ( E ) Enrichment of H3K4me2, H3K4me3, WDR5 ChIP-seq signals over active XH lncCAR (in green, n = 98) and non-CAR XH lncRNA (in black, n = 335) promoters (±2 kb) in BT-549 cells. The signals presented in the plots represent log 2 ratio between ChIP and input sample. TSS denotes the transcription start site of lncRNAs. ( F ) RT-qPCR analysis of WDR5 UV-RIP. The Y-axis shows fold enrichment relative to IgG. The significance of FOXD3-AS1 and HOXC13-AS enrichment in WDR5 RIP is calculated compared to the enrichment of FOXD3 and HOXC13 mRNAs, respectively. *** P ≤ 0.001. ( G ) Left panel: f-RIP of WDR5 WT and WDR5 F266A using FLAG antibody. The Y-axis shows percentage of Input. The significance of decrease in FOXD3-AS1, HOXC13-AS and positive control HOTTIP enrichment in WDR5 F266A RIP is calculated compared to their respective enrichments in WDR5-WT. GAPDH was used as negative control. Right panel: Expression of FLAG tagged WDR5 WT and WDR5 F266A mutant in HeLa cells by western blot. Data are shown as mean ± SD. ** P ≤ 0.01.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Transformation Assay, Expressing, ChIP-sequencing, Quantitative RT-PCR, Positive Control, Negative Control, Mutagenesis, Western Blot

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: Active XH lncCARs promote transcription of their protein coding partners. ( A and B ) Promoter targeting of active XH lncCARs detected by ChOP. A) qPCR analysis of ChOP pull-downs, performed using FOXD3-AS1 and HOXC13-AS antisense probes, with primers over TSS (transcription start sites), regions spanning upstream and downstream of TSS and over transcription termination sites (TTS) of FOXD3 and HOXC13 genes in BT-549 cells. The ChOP pull-down with LacZ antisense oligo, used as a negative control (grey bars). Specific enrichment pattern for each of the XH lncCARs is depicted in green bars. The schematic in the right of the bar graph represents the genomic locations of the respective primers (grey: not enriched and rosetta: showing enrichment). Data are shown as mean ± SD (n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( B ) Similar qPCR analysis of ChOP pull-downs using FOXD3-AS1 and HOXC13-AS antisense probes upon ActD treatment in BT-549 cells. Data are shown as mean ± SD ( n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( C ) RT-qPCR analysis to detect relative levels of nascent FOXD3 and HOXC13 transcripts by Click-iT at 48 hours after the knockdown of FOXD3-AS1 and HOXC13-AS . Bar graph depicts the level of nascent protein coding transcripts upon removal of their respective active XH lncCARs. Data represent the mean ± SD of two independent biological experiments. * if P ≤ 0.05 and ** if P ≤ 0.01. ( D and E ) ChIP-qPCR analysis of the enrichment of H3K4me2 and H3K4me3 at the promoter region of FOXD3 (D) and HOXC13 (E) upon down regulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of H3K4me2 and H3K4me3 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA.The locations of ChIP primers used are depicted in schematic in the bottm of each bar graph. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( F and G ) ChIP-qPCR analysis of enrichment of WDR5 at the promoter region of FOXD3(F) and HOXC13 (G) upon downregulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of WDR5 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA. The ChIP primers used in the experiment are same as described in panel D–E. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( H and I ) FOXD3-AS1 and HOXC13-AS overexpression: RT–qPCR analysis of FOXD3 expression, upon ectopic overexpression of FOXD3-AS1 (H) and HOXC13-AS transcript (I) respectively, at Day 2 and Day 5 post transfection in BT-549 cell line. pcDNA empty vector transfection used as a control. Data represent the mean ± SD. of two independent biological experiments. P -values has been denoted as * if P ≤ 0.05, ** if P ≤ 0.01. ns denotes non-significant. ( J ) Cell fractionation in BT-549 cells show distribution of the overexpressed FOXD3-AS1 and HOXC13-AS transcripts. Data represent the mean ± SD of two independent biological experiments. GAPDH serves as positive control for cytoplasmic fraction, U6 and KCNQ1OT1 serves as positive control for nuclear fraction.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Negative Control, Quantitative RT-PCR, IF-P, Over Expression, Expressing, Transfection, Plasmid Preparation, Cell Fractionation, Positive Control

Journal: Journal of the American College of Cardiology

Article Title: Deficiency of GATA3-Positive Macrophages Improves Cardiac Function Following Myocardial Infarction or Pressure Overload Hypertrophy

doi: 10.1016/j.jacc.2018.05.061

Figure Lengend Snippet: (A) Representative immunostaining of LV sections from control Cre mice. Serial sections of LVs from control Cre mice at 8 days post-MI were stained with anti–Mac-2 or anti-GATA3 antibodies. GATA3-positive staining is clearly visible in the infarcted region of the myocardium, which contains large numbers of macrophages, but not in the healthy region. Sections were also stained with fluorescent-labeled antibodies to demonstrate the nuclear colocalization of GATA3 in macrophages located in the infarcted region of the myocardium. (B) Representative M-mode echocardiograms from the 2 groups of mice before (BL) and 1 month after MI (1M). (C) Representative photographs of Masson Trichrome staining from sections from the base to the apex of hearts from the 2 mice genotype groups. Photographs of all of the hearts are shown in Online Figure 3. (D) Morphometric analysis of each heart section. The number of animals in each group is shown in Table 1. LV = left ventricle; mGATA3KO = myeloid-specific GATA3 knockout mouse; MI = myocardial infarction.

Article Snippet: The anti-GATA3 antibody (clone D-16; sc-22206, Santa Cruz Biotechnology, Dallas, Texas) was used for GATA3 staining.

Techniques: Immunostaining, Control, Staining, Labeling, Knock-Out

Journal: Journal of the American College of Cardiology

Article Title: Deficiency of GATA3-Positive Macrophages Improves Cardiac Function Following Myocardial Infarction or Pressure Overload Hypertrophy

doi: 10.1016/j.jacc.2018.05.061

Figure Lengend Snippet: (A) Representative M-mode echocardiographic data from the 2 mouse genotypes at 2 months after transverse aortic constriction. (B) Harvested hearts were analyzed to determine the size of cardiomyocytes and collagen content. N = 5 mice/genotype. The number of animals in each group is shown in Table 2. mGATA3KO = myeloid-specific GATA3 knockout mouse.

Article Snippet: The anti-GATA3 antibody (clone D-16; sc-22206, Santa Cruz Biotechnology, Dallas, Texas) was used for GATA3 staining.

Techniques: Knock-Out

Journal: Journal of the American College of Cardiology

Article Title: Deficiency of GATA3-Positive Macrophages Improves Cardiac Function Following Myocardial Infarction or Pressure Overload Hypertrophy

doi: 10.1016/j.jacc.2018.05.061

Figure Lengend Snippet: (A) Macrophages were isolated from the peritoneum or the bone marrow of control Cre mice and cultured. Cultured cells were treated with IL-4 at the indicated times, and GATA3 expression was evaluated by qPCR. GATA3 gene expression data were normalized against GAPDH. Results are the mean of triplicate determinations ± SD. (B) Proteins were extracted from cells treated with 20 ng/ml of IL-4 at the indicated times. (C) Dose-response analysis of GATA3 expression was determined by qPCR using cultured macrophages isolated from the bone marrow of control mice. (D) Proteins were analyzed by western blot analysis of lysates from cells treated with 10 or 20 ng/ml of IL-4. (E) Bone marrow-derived macrophages were plated onto chamber slides and treated with 20 ng/ml IL-4 for 4 h, followed by staining with the indicated fluorescent antibodies. Isotypematched antibodies were used as controls. (F) Total RNA was isolated from cultured bone marrow-derived macrophages from Cre mice treated with IL-4 at the indicated times and analyzed by qPCR using primers specific to the proximal (E1b) or distal (E1a) GATA3 promoter. GATA3 gene expression data were normalized against the GAPDH gene. (G) Cultured bone marrow-derived macrophages were treated with the indicated agonists for 2 and 24 h. Total RNA was extracted and analyzed by qPCR, and the resulting GATA3 data were normalized against GAPDH. (H) Cultured bone marrow-derived macrophages from the indicated mouse genotype were treated with IL-4 or IFNγ for 2 h, followed by isolation of total RNA. qPCR data for Arg-1 expression were normalized against GAPDH gene expression. In addition, proteins were extracted from the treated cells and analyzed by western blot using an antibody to Arg-1. All qPCR results are means of triplicate determinations ± SD. *Indicates a significant difference between control and treated cells, p < 0.05. (I) Representative immunostaining data for IL-4 or IL-33 expression in LV sections from control mice at 8 days post-MI. (J) Representative immunostaining of the LV section from the mGATA3KO mice stained with antibodies to Mac-2, IL-4, or IL-33. The upper panels show low magnification and the lower panels show high magnification. Arg-1 = arginase-1; GAPDH = glyceraldehyde 3-phosphate dehydrogenase IFN = interferon; IL = interleukin; qPCR = quantitative real-time polymerase chain reaction. Other abbreviations as in Figure 1.

Article Snippet: The anti-GATA3 antibody (clone D-16; sc-22206, Santa Cruz Biotechnology, Dallas, Texas) was used for GATA3 staining.

Techniques: Isolation, Control, Cell Culture, Expressing, Gene Expression, Western Blot, Derivative Assay, Staining, Immunostaining, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A. Prostate cancer cell lines were lysed and subjected to SDS-PAGE, followed by immunoblotting with antibodies against PG and GAPDH. PG levels were determined by normalizing to GAPDH levels for each cell line using Image J. The immunoblot shows that the expression of PG in prostate cancer cell lines inversely correlates with the degree of aggressiveness of the cell line. B. Prostate cancer cell lines were plated onto coverslips and allowed to attach and spread. Immunofluorescence was performed using antibodies against PG to demonstrate the localization of PG in ARCaP E , ARCaP M , LNCaP, C4-2B, DU145, PC-3, and PC3-M cell lines. The immunofluorescence shows weaker staining and a loss of cell-cell border localization of PG in the more aggressive cell lines (ARCaP M , C4-2B, and PC3-M). Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized PG protein levels for the blot shown. All the PG protein levels were normalized to ARCaP E levels within each blot. The average level and standard deviation of PG protein levels in panel A is as follows: ARCaP E 1.0, ARCaP M 0.3+/−0.2, LNCaP 1.5+/−0.5, C4-2B 0.5+/−0.2, DU145 3.4+/−1.2, PC-3 1.0+/−0.4, PC3-M 0.5+/−0.2.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: SDS Page, Western Blot, Expressing, Immunofluorescence, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A. Representative image of a dispase assay in PC3-M cells after transduction with PG OE adenovirus or control GFP adenovirus. B. Quantitations of dispase assays performed in triplicate; LNCaP, C4-2B, or PC3-M cells were plated in 6-well plates, and allowed to attach and spread. Cultures were transduced with GFP-containing adenovirus (GFP) or PG-containing adenovirus (PG OE), and after 24 hours the dispase assay was performed. Overexpression of PG in all 3 cell lines strengthens cell-cell adhesion. C. LNCaP cells were plated in 6-well plates and allowed to attach and spread. The cells were then transfected with control siRNA or PG siRNA pool. The dispase assay was performed 96 hours after transfection. Suppression of PG expression results in a weakening of cell-cell adhesion in LNCaP cells. D. Representative image of a hanging drop assay in DU145 cells after transfection with control or PG siRNA pool. E–F. Quantitation of hanging drop assays performed in triplicate; C4-2B and PC3-M cells were transduced with GFP- or PG-containing adenoviruses (E), ARCaP E, LNCaP, DU145 and PC3 cells were transfected with control siRNA or PG siRNA pool (F). Aggregation assays were done 24 h (E), or 96 h (F) after the treatment. Graphs represent averages +/− SEM. *P<0.06; **P<0.005; ***P<0.0007, by paired Student t test.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transduction, Control, Over Expression, Transfection, Expressing, Quantitation Assay

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A. PC3-M and ARCaP M were plated in 24-well plates and allowed to attach and spread. The cells were then transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours a scratch wound was made. Images were taken at 0 and 24 hours and the % wound closure was calculated by comparing the size of the wound at 24 hours to the 0 hour time point for each condition. Overexpression of PG suppresses motility in these two cell lines. B. ARCaP E and C4-2B cells were plated in 24-well plates and allowed to attach and spread. The cells were then transfected with control siRNA or PG siRNA pool, and 72 hours after transfection, the scratch wounds were made. Suppression of PG leads to an increase in motility in ARCaP E and C4-2B cells. Scratch wound assay done in triplicate in prostate cancer cell lines after overexpression or knockdown of PG. C. ARCaP M , C4-2B, DU145 and PC3-M cells were transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours plated onto Matrigel coated transwell membranes for an invasion assay. Overexpression of PG suppresses invasion in these four cell lines. D. ARCaPE, LNCaP, C4-2B, DU145, PC-3 and PC3-M cells were transfected with control siRNA or PG siRNA pool, and 72 hours after transfection, plated onto Matrigel coated transwell membranes for an invasion assay. Suppression of PG leads to an increase in invasion in these six cell lines. Invasion assays were done in triplicate. Average of the total number of invading cells is presented. Graphs represent averages +/− SEM. *P<0.1; **P<0.05; ***P<0.001, by paired Student t test.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transduction, Over Expression, Transfection, Control, Scratch Wound Assay Assay, Knockdown, Invasion Assay

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A–B. PC3-M, C4-2B (A), and LNCaP cells (B) were plated in 6 well plates and allowed to attach and spread. LNCaP cells were transfected with control siRNA or PG siRNA (a pool of 4 sequences). PC3-M, C4-2B, and LNCaP cells were treated with medium containing PP2 (10 µM) or DMSO (solvent control) for 24 hours prior to performing the dispase assay. Inhibition of Src strengthens cell-cell adhesion in PCa cells in general and is able to rescue cell-cell adhesion in PG-deficient cells. C. PC3-M and ARCaP M cells were plated in 24-well plates and allowed to attach and spread. The cells were treated with medium containing the selective Src- family kinase inhibitor PP2 (10 µM) or DMSO (solvent control) for 24 hours prior to performing the scratch wound assay. D. ARCaP E cells were plated in 24-well plates and allowed to attach and spread. The cells were transduced with GFP-containing adenovirus or caSrc-containing adenovirus, and after 24 hours a scratch wound was made. Activity of Src is directly correlated with motility of PCa cells. E. ARCaP M , PC3-M, and LNCaP cells were plated in 6-well plates and allowed to attach and spread. LNCaP cells were transfected with control siRNA or PG siRNA pool and after 96 hours, the cells were lysed. ARCAP M and PC3-M cells were transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours the cells were lysed. The lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against PG, pSrc, Src, and GAPDH. Levels of pSrc were determined by normalizing to Src levels for each cell line using Image J. Phosphorylation (activation) of Src is inversely correlated with PG levels. Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized pSrc/Src ratio for the blot shown. The average ratio and standard deviation of pSrc/Src normalized for loading by GAPDH is as follows: ARCaP M 0.6+/−0.2, LNCaP 1.7+/−0.2, PC3M 0.7+/−0.1. Graphs represent averages +/− SEM. *P<0.04; **P<003; ****P<0.0001, by paired Student t test.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transfection, Control, Solvent, Inhibition, Scratch Wound Assay Assay, Transduction, Activity Assay, SDS Page, Western Blot, Phospho-proteomics, Activation Assay, Standard Deviation

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A–E. ARCaP E , ARCaP M , LNCaP, C4-2B, and PC3-M cells were plated in 6-well plates and allowed to attach and spread. The cells were either transduced with GFP- or PG-containing adenovirus and were lysed after 24 hours (PC3-M, C4-2B, ARCaP M ); or the cells were transfected with control siRNA or PG siRNA pool and after 96 hours lysed (ARCaP E , LNCaP, C4-2B). The lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against VN (A), FN (B), LN (C), Col. I (D), Col IV (E) and GAPDH. Levels of ECM proteins were determined by normalizing to GAPDH levels for each cell line using Image J. Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized PG siRNA/Con siRNA or PG OE/GFP ratio for the blot shown. The average ratio and standard deviation of GAPDH normalized PG siRNA/Con siRNA and PG OE/GFP is as follows: (A) ARCaP E 3.1+/−1.3, LNCaP 2.0+/−0.9, PC3-M 0.3+/−0.1; (B) LNCaP 0.7+/−0.05, C4-2B KD 0.8+/−0.1, C4-2B OE 1.2+/−0.2, PC3-M 1.5+/−0.05; (C) ARCaP M 0.6+/−0.1, PC3-M 0.4+/−0.3; (D) 0.3+/−0.1; (E) 0.4+/−0.2.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transduction, Transfection, Control, SDS Page, Western Blot, Standard Deviation

Journal: Cancers

Article Title: Knockdown of microRNA-214-3p Promotes Tumor Growth and Epithelial-Mesenchymal Transition in Prostate Cancer

doi: 10.3390/cancers13235875

Figure Lengend Snippet: Downregulation of miR-214 in PCa cell lines using CRISPR/Cas9 leads to increased cell survival. ( A ) miR-214 pre-miRNA hairpin structure. ( B ) Two sgRNAs were designed for miR-214-3p depletion by using CRISPR DESIGN. ( C ) DNA cleavage by CRISPR/Cas9 is detected by PCR assay. ( D ) The relative expression of miR-214 and PTK6 was determined by qRT-PCR in parental (wild type-WT) PCa cells compared to miR-214KO PCa cells. MiRNA levels were normalized to that of U44 and Gapdh RNA. ( E ) Western blot analysis of PTK6 protein expression in PC3 and MDA-PCa-2b miR-214KO cells compared to WT cells. Original blots see . ( F ) Cell proliferation was evaluated using an MTT assay in PC3 and MDA-PCa-2b cells at 24 h, 48 h, and 72 h after seeding. ( G ) Cell colony formation assays showed the effect of knocking down miR-214 on PCa growth. The number of colonies was counted by ImageJ analysis (U.S. National Institutes of Health, Bethesda, MD, USA). Two-way ANOVA followed by Šídák’s multiple comparisons test was used to determine statistical significance. Data are presented as mean ± SEM, ** p < 0.005, *** p < 0.0005.

Article Snippet: Clonal populations were also generated by Synthego for PC3 miR-214KO cells and the D2 clone was chosen for use in experiments, as noted.

Techniques: CRISPR, Expressing, Quantitative RT-PCR, Western Blot, MTT Assay

Journal: Cancers

Article Title: Knockdown of microRNA-214-3p Promotes Tumor Growth and Epithelial-Mesenchymal Transition in Prostate Cancer

doi: 10.3390/cancers13235875

Figure Lengend Snippet: Inhibition of miR-214 expression increases PC3 cell anoikis resistance, migration, and invasion. PC3 cells transfected with miR-214 mimic or CRISPR/Cas9-deleted miR-214 were inoculated in a Poly-HEMA coated 24-well plate for 48 h. ( A ) Quantification of anoikis after 48 h using MTT assay of miR-214 mimic compared to NC mimic and miR-214KO normalized to WT PC3 cells. ( B ) Images and quantification of relative fluorescent staining of living cells (C-AM) and dead cells (EthD-1). ( C ) Transwell migration and invasion assays were used to determine the effect of miR-214 downregulation on PC3 cell migration and invasion (left panel). The percent of miR-214KO cells that migrated or invaded through the membrane was calculated with the formula (miR-214KO/WT) × 100 compared to WT (right panel). ( D ) Changes in E-Cadherin, N-Cadherin, and Vimentin mRNA in miR214-deleted PC3 cells normalized to its WT control were analyzed by qRT-PCR. ( E ) Western blot analysis revealed that knockdown of miR-214 significantly decreased E-Cadherin and increased N-Cadherin and Vimentin expression in PC3 cells. Original blots see . GAPDH was used as the loading control. One-way ANOVA and two-way ANOVA followed by Tukey’s and Šídák’s multiple comparisons test were used to determine statistical significance for anoikis and qRT-PCR assay, respectively. Data are presented as mean ± SEM, * p < 0.05, *** p < 0.0005, NS = not significant.

Article Snippet: Clonal populations were also generated by Synthego for PC3 miR-214KO cells and the D2 clone was chosen for use in experiments, as noted.

Techniques: Inhibition, Expressing, Migration, Transfection, CRISPR, MTT Assay, Staining, Membrane, Control, Quantitative RT-PCR, Western Blot, Knockdown

Journal: Cancers

Article Title: Knockdown of microRNA-214-3p Promotes Tumor Growth and Epithelial-Mesenchymal Transition in Prostate Cancer

doi: 10.3390/cancers13235875

Figure Lengend Snippet: Overexpression of miR-214 partially reverses the silencing of miR-214 induced proliferative, migratory, and invasive effects. WT and miR-214KO PCa cells were transfected with NC mimic or miR-214 mimic. ( A ) After 48 h, the effect of the miR-214 expression on cell proliferation was determined by MTT assay. Migration ( B ) and invasion ( C ) ability were analyzed by transwell invasion assay in WT and miR-214KO miR-214 transfected PC3 cells, left panel, and quantified in the right panel. Pairwise normalization of WT vs miR-214KO; miR-214KO NC mimic vs miR-214KO miR-214 mimic; and WT NC mimic vs WT miR-214 mimic. Data are presented as mean ± SEM one-way ANOVA using Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.005, *** p < 0.0005.

Article Snippet: Clonal populations were also generated by Synthego for PC3 miR-214KO cells and the D2 clone was chosen for use in experiments, as noted.

Techniques: Over Expression, Transfection, Expressing, MTT Assay, Migration, Transwell Invasion Assay

Journal: Cancers

Article Title: Knockdown of microRNA-214-3p Promotes Tumor Growth and Epithelial-Mesenchymal Transition in Prostate Cancer

doi: 10.3390/cancers13235875

Figure Lengend Snippet: Identification and modulation of differentially expressed genes in miR-214-deleted PCa cells. Gene expression analysis was performed on the cells to compare the expression level of genes between WT and miR-214KO PCa cells. The differential miRNA expressional profiles from PC3 ( A ) and MDA-PCa-2b ( B ) cells miR-214KO compared with that in normal WT cells determined using RNA-Seq assays. Green represents relatively lower expression, and black/red indicates relatively higher expression. Volcano plot of the differentially expressed genes in PC3 ( C ) and MDA-PCa-2b ( D ) miR-214KO cells. Overexpressed genes are demonstrated in red and downregulated genes are demonstrated in blue. GO enrichment analysis of the differentially expressed mRNAs in PC3 ( E ) and MDA-PCa-2b ( F ) miR-214KO cells (Top 40 GO enrichment are presented, red box= enriched cell proliferation and migration processes).

Article Snippet: Clonal populations were also generated by Synthego for PC3 miR-214KO cells and the D2 clone was chosen for use in experiments, as noted.

Techniques: Gene Expression, Expressing, RNA Sequencing, Migration

Journal: Cancers

Article Title: Knockdown of microRNA-214-3p Promotes Tumor Growth and Epithelial-Mesenchymal Transition in Prostate Cancer

doi: 10.3390/cancers13235875

Figure Lengend Snippet: Modulation of overlapping target genes in miR-214-deleted PCa cells. ( A ) Venn diagram analysis of the top 400 aberrantly expressed mRNAs between WT and miR-214KO cells, as well as the overlapping genes between PC3 and MDA-PCa-2b groups. Normalized read counts of commonly regulated genes between PC3 ( B ) and MDA-PCa-2b ( C ) WT and miR-214KO groups. Relative expression levels of up/downregulated target genes were determined by qRT-PCR in PC3 ( D ) and MDA-PCa-2b ( E ) WT and miR-214KO cells. The mRNA ( F , G ) and protein expression ( H , I ) of CXCR4, ALK, SESN3, PD-L1, and SAA1 upon overexpression or knockdown of miR-214 was determined by qRT-PCR and Western blot analysis. Original blots see . Data are presented as mean ± SEM using two-way ANOVA with Šídák’s multiple comparisons test, ** p < 0.005, *** p < 0.0005.

Article Snippet: Clonal populations were also generated by Synthego for PC3 miR-214KO cells and the D2 clone was chosen for use in experiments, as noted.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Knockdown, Western Blot

Journal: Cancers

Article Title: Knockdown of microRNA-214-3p Promotes Tumor Growth and Epithelial-Mesenchymal Transition in Prostate Cancer

doi: 10.3390/cancers13235875

Figure Lengend Snippet: The effects of miR-214 on tumor growth, angiogenesis, and EMT in tumor xenografts. WT or miR-214KO PC3 cells were injected in nude male mice to form xenograft tumors. ( A ) Representative images of xenograft tumors at day 42 after injection. ( B ) Tumor volume in nude mice subcutaneously injected with miR-214 WT or KO PC3 cells. Data are presented as means ± SEM of the tumor volumes, calculated with the formula, V = length × width 2 /2. n = 6, * p < 0.05, *** p < 0.0005 by two-way ANOVA with Šídák’s multiple comparisons test between WT and KO groups. ( C ) Tumor growth of nude mice. ( D ) Quantification of expression of miR-214 by qRT-PCR as a percentage of U44 expression in micro-dissected xenograft prostate cancer cells. N = 6, *** p < 0.0005 ( E ) Representative IHC staining of hematoxylin and eosin, Ki67, PCNA, VEGFA, PTK6, and CD31 staining in xenograft prostate tumor tissues. Original magnification, ×400 (PTK6, Ki67, PCNA, VEGFA) and ×200 (CD31, red arrows = blood vessels). ( F ) Western blot expression of proliferative, angiogenesis, and EMT markers in PC3 WT and miR-214KO xenograft tumors. GAPDH was used as the loading control. Original blots see .

Article Snippet: Clonal populations were also generated by Synthego for PC3 miR-214KO cells and the D2 clone was chosen for use in experiments, as noted.

Techniques: Injection, Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Western Blot, Control