Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 1. Biological activity of hLMP2 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#2 (T type) clone and flat revertants of the SKN-LMP2#122 (F type) clone (magnification 100). Changes in human uterine LMS cell line, SKN-transfectants, SKN-CEM9 (T type) clone, and SKN-LMP2wt (F type) clone xenograft volumes in mice (n = 8). Representative photographs of xenografts in mice (Left). Tumor growth of SKN-LMP2 was markedly reduced in comparison with that of the control transfectant SKN-CEM9 (T type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-CEM9 (T type) clone and SKN-LMP2 (F type) clone (Right). (B) RT-PCR experiments revealed hLMP2, hLMP7, Calponin h1, SRF, cyclin B and b-actin mRNA expression in tumors. Precursor LMP2 or LMP7 (pre-LMP2, pre- LMP7) and mature LMP2 or LMP7 (LMP2, LMP7) are shown. (C) Western blotting revealed LMP2, LMP7, calponin h1, SRF, cyclin B, and b-actin in SKN-transfectant clones. (D) The luciferase reporter vectors containing the hCalponin h1 promoter with wild type SRF binding sites (Calponin-wt-Luc.), mutant SRF binding sites (Calponin-mut-Luc.), or empty luciferase reporter vector (Basic-Luc.) [23] were transiently co-transfected with pSV-b-galactosidase in SKN-transfectants, SKN-CEM9#2, SKN-LMP2#121, or SKN- LMP2#122 clones for the final 48 h, and then luciferase activities were measured. Values were normalized to those obtained with the co-transfected pSV-b-galactosidase expression vector. Each assay was performed at least three times and in triplicate. Luciferase reporter assays showed that LMP2 expression markedly induced calponin h1 promoter activation. Data are presented as the mean from three independent experiments (⁄S.D.). The experiments were performed four times with similar results. SKN transformantsa, CEM9 SKN-CEM9#2; LMP2, SKN-LMP2wt#121, SKN-LMP2wt#122. Detail is shown in SFig. 2, SFig. 3 and STable 3. RT-PCRb, total RNA samples were isolated from the individual xenografted-tumors, which were removed at 5 weeks after xenografting. W.B.c, W.B. are performed with the total cell lysates from SKN transformants.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, Comparison, Control, Transfection, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Clone Assay, Luciferase, Binding Assay, Mutagenesis, Plasmid Preparation, Activation Assay, Isolation

Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 2. Biological activity of calponin h1 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#1Scr.shRNA (T type) clone, SKN-CEM9#2 calponin h1shRNA (T type) clone, SKN-CEM9#2 (T type) clone, SKN-LMP2#1Scr.shRNA (F type) clone, and SKN-LMP2#2Calponin h1shRNA (T type) clone of the SKN-LMP2 (F type) clone (magnification 60). The growth rates of the SKN-transfectant clones were measured as population doubling time (PDT). (B) Western blotting and RT-PCR experiments revealed calponin h1, precursor LMP2 (pre-LMP2), mature LMP2 (LMP2), and b-actin in SKN-transfectant clones. SKN transformantsa, CEM9#3 Scr.shRNA, CEM9#4 Calponin h1shRNA, LMP2#1 Scr.shRNA, LMP2#2 Calponin h1shRNA, Detail is shown in Table 1 and SFig. 5 and STable 3. (C) Changes in the human uterine LMS cell line, SKN-transfectant, SKN-CEM9#2 (T type) clone, SKN-LMP2wt#2/Calponin h1shRNA (T type) clone, and SKN-LMP2wt#1/ Scr.shRNA (F type) clone xenograft volumes in mice (n = 3). Representative photographs of xenografts in mice (Left). Tumor growth of the SKN-LMP2wt#2/Calponin h1shRNA (T type) clone is mildly increased in comparison with that of the SKN-LMP2wt#1/Scr.shRNA (F type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-transfectant clones (Right). RT-PCR experiments revealed hCalponin h1, hLMP2 and b-actin mRNA expression in tumors (Bottom). Experiments were performed three times with similar results. SKN-CEM9c, SKN- CEM9#2; LMP2wt+Calponin h1shRNAd, SKN-LMP2wt#2/ CalponinshRNA; LMP2wt/Scr.shRNAe, SKN-LMP2wt#1/Scr.shRNA. Details of SKN transfectants are shown in Table 1, SFig. 5 and STable 3. RT-PCRf, total RNA samples were isolated from the individual xenografted-tumors, which were removed from BALB/c nu/numice at 5 weeks after xenografting. Xenograftsg, BALB/c nu/nu mice were inoculated with SKN-CEM9#2, SKN-LMP2wt#2/CalponinshRNA or SKN-LMP2wt#1/Scr.shRNA.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, shRNA, Transfection, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Comparison, Injection, Expressing, Isolation

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Known CHD risk genes

Article Snippet: ACP6 , ACID PHOSPHATASE 6, LYSOPHOSPHATIDE , 1q21.1 , 145585791 , 23467 , Hs00320736_cn , , 611471 , 15117819, 19597493.

Techniques: Retroviral, Sequencing, Virus, Reverse Transcription

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Case reports of likely causal CNVs

Article Snippet: ACP6 , ACID PHOSPHATASE 6, LYSOPHOSPHATIDE , 1q21.1 , 145585791 , 23467 , Hs00320736_cn , , 611471 , 15117819, 19597493.

Techniques:

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: CHD-associated gene regions significantly enriched with large, rare CNVs

Article Snippet: ACP6 , ACID PHOSPHATASE 6, LYSOPHOSPHATIDE , 1q21.1 , 145585791 , 23467 , Hs00320736_cn , , 611471 , 15117819, 19597493.

Techniques:

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Enriched syndrome genes

Article Snippet: ACP6 , ACID PHOSPHATASE 6, LYSOPHOSPHATIDE , 1q21.1 , 145585791 , 23467 , Hs00320736_cn , , 611471 , 15117819, 19597493.

Techniques:

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Known CHD risk genes

Article Snippet: TFAP2B , TRANSCRIPTION FACTOR AP2-BETA , 6p12.3 , 50894397 , 28888 , Hs01355864_cn , NS, S , 601601 , 10802654.

Techniques: Retroviral, Sequencing, Virus, Reverse Transcription

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: EGF induces translocation of EGFR to the Golgi. (a) HeLa cells were transfected with pDsRed-syntaxin 6. Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. EGFR was labeled with the indicated antibodies. The boxed areas are shown in detail in the insets. Insets 2–1 and 2–2 show representative colocalizations of EGFR and syntaxin 6. Scale bar, 10 μm. (b) Cells were serum starved overnight and then treated without or with EGF (50 ng/ml) for 20 min. Endogenous EGFR and syntaxin 6 were labeled with a primary antibodies and secondary fluorescein isothiocyanate (donor, green) and Texas-Red (acceptor; red) antibody. An Fc image was obtained using the Zeiss ZEN software. Scale bar, 20 μm. Quantitation of the FRET intensity is shown in the right. (c) Cell lysate was loaded onto the 0–30% OptiPrep density gradient medium and subjected to ultracentrifugation, and fractions were separated using the Gradient Station. The early endosome, the Golgi and ER markers were used to analyze fractions. S, short expose; L, long expose. (d) HeLa cells were treated with or without EGF (50 ng/ml) for 20 min after starvation overnight. The EGFR levels in the Golgi-enriched fraction (fraction 9) were analyzed using immunoblotting. (e) Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. One cell was used for z-stack scanning. Representative images were shown. The boxed areas are shown in detail in the insets. Scale bar, 10 μm. (f) Cells were transfected with GalNac T2 for 48 h or direct staining of endogenous marker, GM130. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for indicated time and analyzed using confocal microscope. Scale bar, 20 μm. Quantitation of colocalization of EGFR and endosomal markers is shown in the bottom. (g) HeLa cells were transfected with EGFP-GalNac T2. Cells were exposed to serum-free media overnight following treatment without or with EGF (50 ng/ml) for indicated time. Scale bar, 20 μm. The boxed areas are shown in the insets. Quantitation of colocalization of phospho-EGFR and total EGFR with the GalNac T2 is shown in the bottom. (h) HeLa cells were serum-starved overnight before EGF stimulation for indicated time. Total lysate and the Golgi-enriched fractions were performed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot to examine the phospho-1086 of EGFR and total EGFR levels.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Labeling, Software, Quantitation Assay, Western Blot, Staining, Marker, Microscopy, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for the Golgi translocation of EGFR. (a) Cells were first transfected with syntaxin 6 or control (Ctrl) siRNAs for 24 h and then transfected with GalNac T2 for 48 h. Cells were then maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (b) Cells were transfected with syntaxin 6 or control siRNAs. After 72 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. The EGFR levels in the Golgi-enriched fraction were analyzed using immunoblotting. (c) Cells were transfected with CCD domain of syntaxin 6 or control vector. After 48 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. Cells were analyzed by confocal microscope. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (d) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and was restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated without or with EGF (50 ng/ml) for 20 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (e) Cells were transfected with syntaxin 6 or control siRNAs. After 24 h transfection, cells were transfected with GalNac T2 for 48 h. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and then analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Quantitation of colocalization of EGFR and endosomal markers is shown in the right. (f) HeLa cells were serum-starved overnight and stimulated without or with EGF (50 ng/ml) for 20 min. Cell lysates were immunoprecipitated with the indicated antibodies and subjected to immunoblot analysis as indicated. (g) In vitro transcribed and translated biotin-labeled syntaxin 6 was incubated with recombinant GST-fused EGFR fragments, pulled down using glutathione-Sepharose beads and visualized with horseradish peroxidase (HRP) conjugated streptavidin. CT, c-terminal domain; IB, immunoblot; KD, kimase domain fragment; TM, transmembrane domain fragment.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Confocal Microscopy, Quantitation Assay, Marker, Western Blot, Plasmid Preparation, Microscopy, shRNA, Immunoprecipitation, In Vitro, Labeling, Incubation, Recombinant

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Microtubules and dynein are required for EGF-induced Golgi transport of EGFR. (a) Serum-starved cells were treated with EGF. Double staining of EGFR and α-tubulin were subjected to confocal microscopy assay. Scale bars, 20 μm. (b) HeLa cells were transfected with GFP-GalNac T2, treated with microtubules or dynein inhibitors and then stimulated with EGF. The Golgi-enriched fractions were purified and subjected to immunoblot analysis with the indicated antibodies. (c) Serum-starved HeLa cells were treated as shown in (b) and then stimulated with EGF and analyzed by a confocal microscope. Scale bars, 20 μm. The boxed areas are shown in detail in the insets. Representative colocalization of EGFR and GalNac T2 is shown in inset 2–1. Quantitation of cells with Golgi-localized EGFR is shown in the lower panel. (d) HeLa cells were transfected with GFP-GalNac T2 expression plasmid and then transfected with control (ctrl) vector or CDK1 and cyclin B plasmids, respectively. Cells were then serum starved overnight, stimulated with EGF and further analyzed under a confocal microscope. Scale bar, 20 μm. Quantitative results are shown in the right. (e) Representative frames of time-lapse confocal microscopic image of cells treated with or without nocodazole. HeLa cells were transfected with EGFP–EGFR (green) and DsRed–syntaxin 6 (red) plasmids. After serum starvation overnight and EGF stimulation, images were collected at 30-s intervals as indicated. Scale bar, 5 μm. (f) Serum-starved HeLa cells were transfected with dynein shRNAs and then stimulated with EGF. Golgi-enriched fractions were purified and subjected to immunoblot analysis with indicated antibodies. DMSO, dimethyl sulfoxide; Noc, nocodazole; PT, paclitaxel; Van, vanadate.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Double Staining, Confocal Microscopy, Transfection, Purification, Western Blot, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for EGFR nuclear translocation. (a) HeLa cells were transfected with syntaxin 6 or control siRNAs and maintained in a serum-free media overnight and treated with EGF (50 ng/ml) for 30 min. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (b) Cells were transfected with syntaxin 6 or control siRNA and maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (c) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and vector control were restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (d) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (e) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Nuclear and non-nuclear fractions were subjected to immunoblot analysis with the indicated antibodies. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Quantitation Assay, Western Blot, shRNA, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Nuclear function of EGFR requires syntaxin 6 and microtubules. (a) After overnight serum starvation, cells were pretreated with the indicated inhibitors for 30-min treatment and then stimulated with EGF for 30 min, followed by chromatin-IP assay. For IgG control, lysate of cells without EGF stimulation was used. (b) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for 30 min, followed by chromatin-IP assy. For IgG control, lysate of cells without EGF stimulation was used. (c) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for indicated time. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) was used to analyze the mRNA level. (d) HeLa cells transfected with control siRNAs and siRNAs for syntaxin 6 were transfected with reporter plasmids containing CCND1 promoter. Then, after 24 h transfection, cells were maintained in serum-free media overnight and treated with EGF for indicated time. Total lysates were used for luciferase assay. Error bars were derived from three independent experiments. (e) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After transfection, 4 × 105 cells were seeded in a six-well plate, incubated for 72 h and then counted. (f) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 48 h transfection, cells were treated with BrdU (100 μm) for 1 h. Cells were assayed for BrdU incorporation by flow cytometry. (g) BT20 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (h) OVCAR3 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (i) A schematic model of syntaxin 6- and microtubule-mediated Golgi and nuclear transport of EGFR.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Chromatin Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Derivative Assay, Incubation, BrdU Incorporation Assay, Flow Cytometry