Journal: Microbiology

Article Title: Global transcriptional analysis of the stringent response in Enterococcus faecalis

doi: 10.1099/mic.0.060236-0

Figure Lengend Snippet: qRT-PCR primers used to validate the microarray results

Article Snippet: The slides were hybridized to a mixture containing equal amounts of test and reference cDNA for 16 h at 42 °C in a MicroArray User Interface (MAUI) hybridization chamber (BioMicro Systems).

Techniques: Microarray

Journal: Microbiology

Article Title: Global transcriptional analysis of the stringent response in Enterococcus faecalis

doi: 10.1099/mic.0.060236-0

Figure Lengend Snippet: qRT-PCR validation of microarray data

Article Snippet: The slides were hybridized to a mixture containing equal amounts of test and reference cDNA for 16 h at 42 °C in a MicroArray User Interface (MAUI) hybridization chamber (BioMicro Systems).

Techniques: Biomarker Discovery, Microarray

Journal: BMC Microbiology

Article Title: Separation of the bacterial species, Escherichia coli , from mixed-species microbial communities for transcriptome analysis

doi: 10.1186/1471-2180-11-59

Figure Lengend Snippet: Plot of gene expression of sorted/unsorted cells . Plot of one-sample T-test p-values with fold-change in gene expression for all ORFs in microarray study I. Vertical lines show the cutoff of fold-change of 2 (Log 2 ratio of ± 1), while the horizontal line shows the cutoff of p-value 0.05. Genes located in the left-bottom corner (Log 2 ratio <-1 and p-value <0.05) and in the right-bottom corner (Log 2 ratio >1 and p-value <0.05) were considered to have their expressions changed due to dispersion/homogenization and IMS (immuno-magnetic separation) cell sorting. A total of ten genes were selected using these criteria, eight of which also differentially expressed in the independent microarray study II.

Article Snippet: Hybridization was in a Corning Microarray Hybridization Chamber (Corning Inc.) in 42°C water bath.

Techniques: Expressing, Microarray, Homogenization, FACS

Journal: BMC Microbiology

Article Title: Separation of the bacterial species, Escherichia coli , from mixed-species microbial communities for transcriptome analysis

doi: 10.1186/1471-2180-11-59

Figure Lengend Snippet: Genes identified as differentially expressed # between IMS sorted E. coli cells versus unsorted E. coli cells* by the method of cDNA microarray and their differential expression confirmed with another method of qPCR

Article Snippet: Hybridization was in a Corning Microarray Hybridization Chamber (Corning Inc.) in 42°C water bath.

Techniques: Microarray, Expressing

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 1 Regulation of PKN kinase activity. IP-kinase assays with WT and TM mutants of PKN1 (S916A) and PKN2 (T958A). Torin inhibited the PKN kinase activity to about the same extent as mutating the TM in both PKN isoforms. B, The PKN1 TM mutant S916A has reduced kinase activity toward multiple substrates. C, Deletion of the PKN N-terminus results in constitutive histone H3 phosphorylation in vitro and in cells. D and E, The PKN1 TM mutant S916A dramatically reduces autophosphorylation as well as Histone H3 and MARCKS phosphorylation

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, In Vitro

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 3 Torin and rapamycin sensitivity of PKN, AKT, and PKCα. A, Cells stably transduced with WT PKN1 were treated with a range of torin and rapamycin concentrations for 24 h, and analyzed by using pan- and phosphosite-specific antibodies. B, Cells were treated with torin and rapamycin during a time course up to 24 h and subsequently analyzed by using pan- and phosphosite- specific antibodies

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Stable Transfection, Transduction, Phospho-proteomics

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 2 PKN contains a TM phosphorylated by a torin- sensitive kinase. A, Alignment of TM sequences with the predicted phosphorylated residues indicated (bold). B, Transfection of PKN1 bearing mutations in the TM (S916A), activation loop (T774E) and ATP binding pocket (K644E) probed with antibodies specific for phos-S916 and phos-T774. Including nonphospho-TM peptide during the antibody incubation reduces the detection of non- phosphorylated PKN. C, IP-blot of WT PKN1 expressed in cells treated with torin and rapamycin

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Transfection, Activation Assay, Binding Assay, Incubation

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 4 Cell motility functions of PKN. A, Localization of Flag-tagged PKN1 (green) at the cleavage furrow during mitosis, imaged by confocal microscopy. B, Examples of binucleate cells generated in response to depletion of PKN1, PKN2, and Ect2 (positive control), indicative of cytokinesis failure. C, Quantification of cytokinesis failure data as a consequence of PKN1 and PKN2 depletion. D, Expression levels (immunoblotting) of PKN1 and PKN2 after siRNA depletion. E, Stable C4-2b cell lines showing that (E) ectopic expression and (F) knockdown increase and decrease, respectively, cell migration in a Boyden chamber assay (****P < 0.0001). G, Transient depletion of PKN1, PKN2, and the TORC2 subunit Rictor reduces cell invasion of PC-3 cells to a similar extent as torin treatment

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Confocal Microscopy, Generated, Positive Control, Expressing, Western Blot, Knockdown, Migration, Boyden Chamber Assay

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 5 Analysis of PKN isoform expression in human prostate cancer. A, Representative IHC showing PKN1 protein levels in normal, primary tumor, and lymph node metastasis. B, PKN1 and PKN2 expression (using microarray data from reference 47) in normal prostate, primary tumor, and metastases. C, RNA expression (using RNAseq data from TCGA) of PKN1-3 isoforms, PTEN, PKCα, AKT, and select mTOR components. **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Expressing, Microarray, RNA Expression

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 6 Pkn2 is required for embryonic development. A, Embryos from Pkn1 and Pkn2 lacZ reporter mice were stained for β- galactosidase activity, and are shown as whole mount images. Upper row: E10.5, E11.5, E11.5. Scale bars: 1.0 mm. Bottom row: E6.5, E8.5 (side and dorsal view), E9.5, E9.5. Scale bars: 0.2 mm, 0.5 mm, 1.0 mm. B, Whole mount images of Pkn2 heterozygotes and homozygous null embryos at E7.0, E7.75, and E9.5. Scale bars 0.2 mm (upper four panels) 1.0 mm. C, Whole mount images of wild-type and Pkn2 null embryos analyzed by whole mount in situ hybridization for Otx2 (E7.5) and Bra (E7.25) are shown. Scale bars: 0.2 mm

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Staining, Activity Assay, In Situ Hybridization

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 7 Analysis of PKN1 overexpression in prostate. A, Immunoblots showing transgenic expression of full-length (Tg-PKN1) and constitutively active (Tg-PKN1ΔN) proteins in anterior, dorsal, lateral, and ventral lobes (AP, DP, LP, VP). B-E, H&E stained images of sections through the ventral prostates from mice of the indicated genotypes are shown. The ages of the mice are as follows: WT, 53 weeks; Tg-PKN1, 58 weeks; Tg-PKN1ΔN, 58 weeks; Tg-AKT1, 52 weeks; Tg-AKT1;Tg-PKN1, 41 weeks; Tg-AKT1;Tg-PKN1ΔN, 52 weeks; TRAMP and TRAMP;Tg-PKN1, 16 weeks (showing HGPIN); TRAMP and TRAMP;Tg-PKN1, 17 weeks (showing small cell carcinoma). All images were captured at 200× magnification. Lower magnification views of the same samples are also provided (Supplemental Figure S3)

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Over Expression, Western Blot, Transgenic Assay, Expressing, Staining

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 8 Analysis of PKNs in Pten null prostate tumors. H&E stained images of sections through the prostates from mice of the indicated genotypes are shown. All images were captured at 200× magnification and are of the ventral prostate, except for the right-most image in panel D, which shows squamous differentiation from the anterior prostate. The ages of the mice (panels A–C) are as follows: Ptenr/r, 12 and 45 weeks; Ptenr/r;Tg-PKN1, 12 and 43 weeks; Ptenr/r;Pkn1r/r;Pkn2r/r, 26 and 45 weeks. D, The images of invasive cancer (left and middle) are from 53-week ventral prostates, the squamous differentiation shown to the right is from the anterior prostate of a 53-week animal. Lower magnification views of the same samples are also provided (Supplemental Figure S4)

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Staining

Journal: Cell Death & Disease

Article Title: MicroRNA-221-3p, a TWIST2 target, promotes cervical cancer metastasis by directly targeting THBS2

doi: 10.1038/s41419-017-0077-5

Figure Lengend Snippet: a SiHa and HeLa cells were transfected with miR-221-3p mimic, miR-221-3p inhibitor, miR-221-3p mimic-nc, and miR-221-3p inhibitor-nc. Western blotting analysis of E-cadherin, N-cadherin, and Vimentin was performed of the five groups in SiHa and HeLa cells. β-actin was used as loading control. b qRT-PCR analysis of E-cadherin, N-cadherin, and Vimentin of the five groups in SiHa and HeLa cells. c Wound-healing assay of the five groups in SiHa and HeLa cells. d Boyden chamber assay of the five groups in SiHa and HeLa cells. e Cell migration was quantified as percentage of wound-healed area. f Average number of invading cells per field from three independent experiments. Data represent means ± SD of five randomly selected areas. * p < 0.05

Article Snippet: Human CC cell lines SiHa and HeLa, and human embryonic kidney 293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transfection, Western Blot, Control, Quantitative RT-PCR, Wound Healing Assay, Boyden Chamber Assay, Migration

Journal: Cell Death & Disease

Article Title: MicroRNA-221-3p, a TWIST2 target, promotes cervical cancer metastasis by directly targeting THBS2

doi: 10.1038/s41419-017-0077-5

Figure Lengend Snippet: a Correlation of bioluminescence intensity with cell quantity in SiHa-luc-RFP-221-3p, SiHa-luc-RFP-nc, HeLa-luc-RFP-221-3p, and HeLa-luc-RFP-NC cells in vitro . b In vivo bioluminescence images of lymphatic metastasis. Primary tumour (P) in claw pad of mice and metastases (M). Lymph nodes in popliteal and inguinal regions were measured by detecting bioluminescence signals. The ratio of metastasis-positive lymph nodes was calculated. * p < 0.05. c In situ hybridization of miR-221-3p expression in primary tumors of mice injected with SiHa-luc-RFP-221-3p, SiHa-luc-RFP-nc, HeLa-luc-RFP-221-3p or HeLa-luc-RFP-NC cells. d Primary tumour site and lymph nodes with tumour cells were identified by tumour cell-expressed RFP

Article Snippet: Human CC cell lines SiHa and HeLa, and human embryonic kidney 293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, In Vivo, In Situ Hybridization, Expressing, Injection

Journal: Cell Death & Disease

Article Title: MicroRNA-221-3p, a TWIST2 target, promotes cervical cancer metastasis by directly targeting THBS2

doi: 10.1038/s41419-017-0077-5

Figure Lengend Snippet: a Bioinformatic prediction and screening of potential transcription factors of miR-221-3p. b Correlation analysis of TWIST2 and miR-221-3p expression detected by qRT-PCR in 28 additional SCC tissues. The expression levels of TWIST2 were plotted against the expression levels of miR-221-3p. c The certified result of microarray analysis. Hierarchical clustering of eight significantly dysregulated miRNAs expression profiles of the three groups, including SiHa, SiHa-shtw2, and SiHa-tw2 cells. d qRT-PCR analysis of the eight significantly dysregulated miRNAs expressed in SiHa, SiHa-shtw2, and SiHa-tw2 cells, and in HeLa cells transfected with TWIST2 expression vector or TWIST2 siRNA. MiR-221-3p emerged as a highly upregulated miRNA. Data represent means ± SD of five randomly selected areas (* p < 0.05)

Article Snippet: Human CC cell lines SiHa and HeLa, and human embryonic kidney 293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Microarray, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: MicroRNA-221-3p, a TWIST2 target, promotes cervical cancer metastasis by directly targeting THBS2

doi: 10.1038/s41419-017-0077-5

Figure Lengend Snippet: a Schematic structure of the miR-221-3p upstream promoter containing a TWIST2-binding site. b Luciferase activity of PGL3-221-3p construct after transfection of TWIST2 plasmid in SiHa and 293 T cells. c Western blotting analysis of expression of E-cadherin, N-cadherin, and Vimentin proteins in SiHa, SiHa-tw2, SiHa-shwt2, or SiHa-shtw2 cells transfected with miR-221-3p mimic or miR-221-3p inhibitor; and HeLa cells transfected with TWIST2 expression vector with/without miR-221-3p mimic or TWIST2 siRNA with/without miR-221-3p inhibitor. β-actin was used as loading control. d Cell migration was quantified as the percentage of wound-healed area; average number of invaded cells per field from three independent experiments. Data represent means ± SD of five randomly selected areas (* p < 0.05; ** p < 0.001). e IHC analysis of TWIST2 and in situ hybridization analysis of miR-221-3p in matched cervical specimens. Human CC tumors with high TWIST2 expression and had strong miR-221-3p expression. Normal cervical tissues with low TWIST2 expression and had weak miR-221-3p expression. Representative images are shown at ×400 magnification. The HSCOREs of miR-221-3p and TWIST2 in different cervical specimens are also shown (* p < 0.05) (upper-right panel). Correlation of miR-221-3p staining and TWIST2 staining was analyzed (lower-right panel)

Article Snippet: Human CC cell lines SiHa and HeLa, and human embryonic kidney 293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Transfection, Plasmid Preparation, Western Blot, Expressing, Control, Migration, In Situ Hybridization, Staining

Journal: Cell Death & Disease

Article Title: MicroRNA-221-3p, a TWIST2 target, promotes cervical cancer metastasis by directly targeting THBS2

doi: 10.1038/s41419-017-0077-5

Figure Lengend Snippet: a Bioinformatic prediction and screening of potential miRNAs targeting miR-221-3p; incomplete complementation of the base of miR-221-3p to the 3′UTR region of THBS2 mRNA. Also shown are nucleotides mutated in THBS2 mutant 3′UTR. b Luciferase activity of wild-3′UTR-THBS2-luc and mutant 3′UTR-THBS2-luc constructs in SiHa or 293 T cells after transfection of miR-221-3p mimic. c Western blotting analysis of the expression of THBS2 in SiHa and HeLa cells transfected with miR-221-3p mimic, miR-221-3p inhibitor, miR-221-3p mimic-nc, and miR-221-3p inhibitor-nc. β-actin was used as loading control. d The HSCOREs of THBS2 in different cervical specimens (left), and correlation of THBS2 staining and miR-221-3p was analyzed (right). e Normal cervical tissues with high THBS2 expression. Human CC tumors with low THBS2 expression. * p < 0.05

Article Snippet: Human CC cell lines SiHa and HeLa, and human embryonic kidney 293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Mutagenesis, Luciferase, Activity Assay, Construct, Transfection, Western Blot, Expressing, Control, Staining