Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice

doi: 10.1016/j.omtn.2017.05.007

Figure Lengend Snippet: Evaluation of ISIS 486178 and ISIS 445569 ASOs upon Systemic Administration in DMSXL Mouse (A) FISH quantification of foci per nucleus in DMSXL mice quadriceps (n = 3; treated n = 9 per ASO; multiple sections throughout the muscle for each mice). **p < 0.01 by Kryskall Wallis test. (B) DMPK mRNA in transgenic DMSXL mice treated with ISIS 486178 (25 mg/kg) and ISIS 445569 (50 mg/kg) (DMSXL, n = 11; ISIS 486178, n = 17; 445569, n = 7). (C) Mice forelimb strength after treatment with by ASOs (wild-type [WT], n = 24; DMSXL, n = 15; ISIS 486178, n = 15; ISIS 445569, n = 7). (D) DMSXL mice serum chemistry after ISIS 486178 injection regiment. (E) Muscle fibers type 1/2a in mice soleus (WT, n = 3; DMSXL, n = 12; ISIS 486178, n = 5; ∼4,500 fibers/mouse). (F) Cell death, necrosis, and inflammation genes downregulated by a 2-fold change in DMSXL mice treated with ISIS 486178 determined by microarray (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001 by unpaired two-tailed t test.

Article Snippet: Sections were then incubated consecutively for 1 hr each at 37°C with two mouse monoclonal antibodies with different isotypes directed against myosin heavy chain type 1 (BA-D5, IgG2b; DSHB) and type 2a (SC-71, IgG1; DSHB).

Techniques: Transgenic Assay, Injection, Microarray, Two Tailed Test

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: Figure 1 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 BclxL (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: Figure 2 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 BclxL (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: Figure 3 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. CDK11p110, 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 BclxL (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: Figure 4 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 Materials and methods. 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 BclxL (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: Figure 5 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 BclxL (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: Figure 6 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 Materials and methods 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 BclxL (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: Figure 7 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 BclxL (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: Nature communications

Article Title: Genome-wide Transcriptome Profiling of Homologous Recombination DNA Repair

doi: 10.1038/ncomms4361

Figure Lengend Snippet: ( a ) MCF-10A cells were infected by the indicated lentiviral particles targeting the indicated genes. Control cells and knockdown cells were subjected to microarray analysis. The presence of the HRD gene signature was analyzed by supervised clustering analysis. ( b ) Modified HR repair assay was performed in MCF-10A cells by transfecting cells with DRGFP DSB substrate plasmid and I-SceI plasmid through electroporation, followed by analysis of GFP-positive cells by flow cytometry 48 to 72 hours later. Student’s t -test was performed from results of three independent experiments as mean + SD. ( c ) The rate of cell survival in response to olaparib was determined by colony formation assay. Each value was relative to control cells without treatment and represents the mean ± SD from three independent experiments. Student’s t -test showed that treatment response differed between BRCA1-PTEN double knockdown cells and single knockdown cells (P<0.001). ( d ) Heat map of the HRD gene signature with the 26 genes most significantly changed in BRCA1-PTEN double knockdown cells compared to single-knockdown-cells. ( e ) Microarray data from 295 breast cancers were clustered into basal-like, Her2-positive (Her2), luminal A, luminal B, and normal breast-like. Gene expression levels of TTK among the different breast cancer subtypes are indicated. ( f ) Quantitative analysis of HR repair assay in cells transfected with TTK plasmids. Results are shown as mean + SD from three independent experiments. Student’s t -test showed that over-expression of TTK significantly increased HR repair efficiency (P<0.05). BRCA1 SMARTpool siRNA was used as a positive control. Western blots demonstrating effective knockdown are shown to the bottom.

Article Snippet: BRCA1 (D-9) monoclonal and TTK polyclonal antibodies were purchased from Santa Cruz (SC-6954, 1:1000) and Cell Signaling (#3255, 1:1000), respectively.

Techniques: Infection, Control, Knockdown, Microarray, Modification, Plasmid Preparation, Electroporation, Flow Cytometry, Colony Assay, Gene Expression, Transfection, Over Expression, Positive Control, Western Blot

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 expression is up-regulated in osteosarcoma cells. (A) , mRNA levels of NRP2 in normal osteoblast (NHOst) and osteosarcoma cell lines (Saos-LM7, 143B, 143.98.2, MG-63, MNNG/HOS, Saos-2, and U2-OS) were determined by real time PCR. (B) , the protein levels of NRP2 in NHOst and seven osteosarcoma cell lines were detected by western blot using NRP2 antibody. The relative protein levels were determined by densitometry and normalized with β-actin level. (C) , Kaplan-Meier survival curves of disease–specific mortality for patients whose osteosarcoma expressed or didn’t express NRP2. The log-rank test was used to compare differences between two groups. NRP2 expression was predictive of poor overall survival. Significant difference is indicated by (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. Column: mean value; Error bars: SEM

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

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

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 knockdown inhibited both in vitro and in vivo tumor growth. (A & B) , NRP2 expression was knocked down by NRP2 shRNA in 143B cells. Knockdown efficiency was determined by real-time PCR (A) and Western blotting (B) . (C) , By MTT assay, NRP2 knockdown suppressed anchorage-dependent growth of osteosarcoma 143B cells. (D) Soft agar assay. NRP2 knockdown did not reduce the number of colony formed by 143B cells. 143B cells transfected with ShNRP2 formed smaller colony than control vector transfected cells as shown in the representative images of soft agar (insert). (E) & (F) NRP2 knockdown inhibited the in vivo tumor growth in xenograft nude mice model. 1 × 10 6 143B cells were inoculated in NCR nu-nu nude mice. Tumor size was measured every 3 days and a tumor growth curve was created (E) . Points, mean tumor volume; Bars, SEM. (F) , Representative picture of tumor harvested at day 21. (G) , the knockdown of NRP2 expression in tumor samples was confirmed by immunofluorescence staining using NRP2 antibody. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01. Column: mean value; Error bars: SEM.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, In Vitro, In Vivo, Expressing, shRNA, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, Soft Agar Assay, Transfection, Control, Plasmid Preparation, Immunofluorescence, Staining

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: Knockdown of NRP2 inhibited the tumor invasion, migration and lung metastasis of osteosarcoma. (A) , Migration assay. The BD chamber system without Matrigel coating was used to evaluate the migration of shNRP2 and control vector transfected osteosarcoma 143B cells. The migration of 143B cells was significantly inhibited by NRP2 knockdown. (B) , Matrigel invasion assay was performed in BD chamber system coated with Matrigel, using shNRP2 and control vector transfected osteosarcoma 143B cells. There were less NRP2 depleted 143B cells invaded through the matrigel coated porous membrane. (C) , Knockdown of NRP2 in 143B cells reduced the lung metastasis of osteosarcoma in an orthotopic lung metastasis mouse model. Mouse lungs were fixed in Bouin’s solution, and the number of lung surface metastatic nodules was counted and graphed. Each group contained 10 mice and the experiment was repeated 3 times (D) , Representative photographs of lungs with metastatic nodules of osteosarcoma. (E) , NRP2 knockdown significantly reduced both 143B and Saos-2 cells adherence to the endothelial monolayer. The mean cell number was calculated from 10 fields (×100). (F) , Representative images of GFP transfected tumor cells adhering to the endothelial monolayer. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. Column: mean value; Error bars: SEM.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, Migration, Control, Plasmid Preparation, Transfection, Invasion Assay, Membrane

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: Knockdown of NRP2 resulted in decreased blood vessel density of OS in vivo . (A) , Blood vessels (top panel) and capillaries (bottom panel) in tumor samples were visualized by immunohistochemistry with CD31 antibody. Tumor cell nuclear was stained with DAPI. Number of blood vessels per field (100×) was calculated and graphed (Right). Column, mean number of blood vessel per field (x100); Error bars, SEM. (B & C) , Matrigel tube formation assay: no difference was found in the number of tubules formed by HUVEC in conditioned medium from shNRP2 OS cells and shRNA control cells. (B) Representative photographs of tubules formed by HUVEC cells on Matrigel. (C) The tubular number was calculated and graphed, Column: mean number of tubules; Error bars: SEM. (D) , tumor-endothelial co-culture tube formation assay: The HUVEC cells were stained with CellTracker Red CMTPX dye and the tumor cells were transfected with shNRP2 vector with GFP expression. Left panel: tubules formed by HUVEC (red); middle panel: tumor cells (green) attached on tubules; right panel: merged image (right) of HEVEC tubules (red) and attached tumor cells (green). NRP2 depleted tumor cells sustained distinct morphologic changes compared to control cells (bottom panel).

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, In Vivo, Immunohistochemistry, Staining, Tube Formation Assay, shRNA, Control, Co-Culture Assay, Transfection, Plasmid Preparation, Expressing

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 knockdown suppressed endothelial recruitment by osteosarcoma cells. (A) , in a trans-well co-culture migration model, less endothelial cells migrated through the porous insert membrane of the Chamber in shNRP2 group compared to control group. shNRP2 depleted osteosarcoma cells vs control cells were seeded in the lower chamber. HUVECs were seeded in the top insert chamber. Insert picture: Representative photograph of migrated HUVECs. (B) , in a trans-well co-culture invasion model, less endothelial cells invaded through the matrigel coated porous insert membrane of the Chamber in shNRP2 group compared to control group. shNRP2 depleted osteosarcoma cells vs control cells were seeded in the lower chamber. HUVECs were seeded in the top insert chamber. Insert picture: Representative photograph of invaded HUVECs. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01. Column: mean migrated/invaded cells per field (40×); Error bars: SEM.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Knockdown, Co-Culture Assay, Migration, Membrane, Control

Journal: Molecular Cancer

Article Title: Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma

doi: 10.1186/s12943-015-0359-4

Figure Lengend Snippet: NRP2 expression is regulated by Wnt signaling pathway. A , Genechip® microarray showed the down-regulated expression of NRP2 in Wnt antagonist sLRP5 transfected osteosarcoma Saos-2 cells. VEGF expression is intact. Real time PCR (B) and western blot (C) with accompanying densitometric assay (D) confirmed the down-regulated mRNA and protein level of NRP2 in Wnt antagonist sLRP5 transfected Saos-2 cells, while VEGF level remains intact. E , western blot and accompanying densitometry showed the down-regulated NRP2 expression in additional Wnt antagonist DKK3 transfected Saos-2 cells, 143B cells and U2-OS cells, and Wif-1 transfected 143B cells. F , Chromatin immunoprecipitation (ChIP) assay verified the binding of TCF4 on the five binding sites in NRP2 promoter region. G , schematic representation of the binding sites of TCF/LEF on the 3-kb promoter region of NRP2 genes.

Article Snippet: Antigen retrieval was done using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 min. After blocking with PBS containing 3% bovine serum albumin for 1 h, cells were incubated with an NRP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, Catalog# sc-13117) for 12 h. After washing, cells were incubated with an Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, Catalog # A-11059) and mounted in Vectashield with 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Microarray, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Binding Assay

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: mRNA expression levels in nontransformed and malignant breast cells a

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

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: 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: mRNA expression levels in small interfering RNA transfected cells

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, Transfection

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: Veterinary Research

Article Title: Live and inactivated Salmonella enterica serovar Typhimurium stimulate similar but distinct transcriptome profiles in bovine macrophages and dendritic cells

doi: 10.1186/s13567-016-0328-y

Figure Lengend Snippet: Summary of the differentially expressed genes identified by analysis of the microarray results

Article Snippet: The second novel sequence investigated was identified as a novel transcript of TNFAIP3, which is not represented by any annotated probe-set on the Affymetrix bovine microarray.

Techniques: Microarray

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Molecular events for CRBP1 gene in cervical epithelium samples. A: In order to know the gain of copy number of the CRBP1 gene, DNA of healthy cervix and CC samples, were subjected to real time PCR with specific Taqman probes. White bar (healthy cervix samples) represents the mean of the normal cervices (n = 26) without extra copies of CRBP1 gene. Black bars show CC samples with gain of copy number (2-20X); while gray dotted line bars are showing CC samples that do not change in the copies number. Values above the cut-off line (as 1), being assigned as increased gene copy number compared with normal cervical epithelium. CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [26]. In X-axis represents cervical samples, Y-axis relative copies fold change of CRBP1 gene. B: CRBP1 expression was observed as positive immunostaining result on tissue microarray as mentioned in Methods section The DNAs used for gain of copy number (panel A) were also used for the methylation assay. Methylation result represents the methylation of the CRBP1 promoter. In this case, each healthy or CC sample, correspond to each column for CRBP1 expression and methylation status. Interestingly, in most of the cases, there was an association between the lack of expression of the CRBP1 gene and its methylation status.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

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

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: CRBP1 immunodetection in the uterine cervix samples. A: (1) Cytoplasmic CRBP1 expression is present in cells of the basal layer of normal cervical epithelium (healthy tissue); (2) the immunodetection in the transformed cells of a cervical cancer (CC03) tissue harboring gain of CRBP1 gene. (3) CC samples without gain CRBP1 gene showing negative immunostaining (CC16 sample). A kidney tissue section (4) was used as positive control, while a heart tissue section for negative control (5). B: cervical progression spectrum. The tissue section shows a brownish reaction (positive reaction) in the basal cell layer of the “normal” region, in the high-grade lesion, and also in the invasive region. All tissue sections were hematoxylin counterstained, 200X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunodetection, Expressing, Transformation Assay, Immunostaining, Positive Control, Negative Control, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Association between CRBP1 gene gain copy number and its expression in cervical cancer samples

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Immunodetection

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Correlation between CRBP1 expression and clinic pathological variables in cervical cancer

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Activity Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Immunolocalization of CRBP1 by immunofluorescence in cervical cells. Nuclei were Dapi stained in blue color (A-C). The immunodetection of CRBP1 was observed in green color (D-F). Cytoplasmic immunodetection of CRBP1 in the merge imaging (G-I). 100X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunofluorescence, Staining, Immunodetection, Imaging, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Methylation promoter of CRBP1 gene in cervical cancer samples. Example of CRBP1 gene promoter methylation analysis. Lanes: Healthy cervix sample, CC03 and CC06 samples with un-methylated status; lanes CC 10 and CC 16 with methylated status; HeLa cells as un-methylated control (109 bp), or MCF-7 cells as methylated control (99 bp). MW: molecular weight marker of 100 bp.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Methylation, Molecular Weight, Marker