Journal: PLOS Pathogens

Article Title: The viral packaging motor potentiates Kaposi’s sarcoma-associated herpesvirus gene expression late in infection

doi: 10.1371/journal.ppat.1011163

Figure Lengend Snippet: (A) TurboID was tethered to the C-terminus of ORF18. ORF18 directly contacts ORF30, ORF31, ORF66, and ORF34 and is proximal to ORF24 and RNA polymerase II. (B) Infectious virion production from reactivated iSLKs was measured by viral supernatant transfer onto HEK293T cells using flow cytometry for the GFP-expressing virus. Data are from three independent biological replicates and statistics are calculated using an unpaired t test. * = P<0.05. (C) Overlap between proteins identified by mass spectrometry with ≥2 unique peptides that demonstrated >2-fold increase in the ORF18-TurboID + biotin test condition compared to the three negative controls, across 2 biological replicates. Forty-five high confidence hits were specifically enriched in ORF18-TurboID + biotin sample using these filtering criteria. (D) STRING protein-protein interaction network of high-confidence cellular proteins (above) and KSHV proteins (below). ORF18 is shown in dark blue. Known interactors of ORF18 are in light blue, and all other proteins are shown in grey. Proteins with nuclear localization annotated in UniProt are outlined in gold. (E) Gene ontology enrichment analysis of cellular ORF18-TurboID hits. All major enriched functional classes are shown.

Article Snippet: Plasmids pcDNA4/TO-ORF18-2xStrep (Addgene 120372), pcDNA4/TO-ORF24-2xStrep (Addgene 129742), pcDNA4/TO-ORF30-2xStrep (Addgene 129743), pcDNA4/TO-ORF31-2xStrep (Addgene 129744), pcDNA4/TO-ORF66-2xStrep (Addgene 130953) and pcDNA4/TO-2xStrep-ORF34 (Addgene 120376) have been previously described [ , ].

Techniques: Flow Cytometry, Expressing, Virus, Mass Spectrometry, Functional Assay

Journal: Molecular Cancer

Article Title: Regulation of Sox2 and stemness by nicotine and electronic-cigarettes in non-small cell lung cancer

doi: 10.1186/s12943-018-0901-2

Figure Lengend Snippet: a Depletion of E2F1, YAP1 or α7 nAChR by siRNA prevents the nicotine-mediated induction of Sox2. Depletion of Oct4 or TEAD2 did not have any impact on the induction. b A schematic showing the location of the E2F binding sites that were tested by ChIP assays; the location of forward and reverse primers for each site are indicated by arrows. c A ChIP assay shows the association of E2F1 with all the tested binding sites; E2Fs 2 and 3 were mainly associated with the binding site spanned by primers F2 and R2. There was no E2F associated with c-Fos promoter. d Nicotine stimulation induces the association of E2F1 with sites spanned by primers F1R1 and F2R2; surprisingly, Rb could also be detected on site F1R1. e A transient transfection experiment conducted on A549 cells showing the induction of Sox2-Luc reported by E2F family members. The data represented is with ± standard deviation (SD) values derived from three independent experiments. The statistical comparison was carried out by unpaired two tailed Student’s t-test. f YAP1 and E2F1 can induce the Sox2 promoter, and can show an additive effect in transient transfection experiments. The data represented is with ± standard deviation (SD) values derived from three independent experiments. The statistical comparison was carried out by one-way ANOVA. g Nicotine and E-cigarette extracts induce YAP1 levels in A549 cells and human mesenchymal stem cells, as seen by an immunofluorescence experiment

Article Snippet: The expression vectors used were pcDNA3-HA-E2F1, pcDNA3-E2F2, pcDNA3-E2F3, pcDNA3-E2F4, pcDNA3-E2F5, and Yap1 (Addgene #18978).

Techniques: Binding Assay, Transfection, Standard Deviation, Derivative Assay, Comparison, Two Tailed Test, Immunofluorescence

Journal: Molecular Cancer

Article Title: Regulation of Sox2 and stemness by nicotine and electronic-cigarettes in non-small cell lung cancer

doi: 10.1186/s12943-018-0901-2

Figure Lengend Snippet: a Nicotine and E-cigarette extracts promote the co-localization of E2F1 and YAP1 in A549 cells, as seen by a double immunofluorescence experiment followed by confocal microscopy. b An immunoprecipitation-western blot experiment showing the association of YAP1 with E2F1 in A549 and H1650 cells; the immunoprecipitation was conducted by an E2F1 antibody followed by western blotting with a YAP1 antibody. c An IP-western blot experiment in the reverse direction, where a YAP1 antibody was used for IP followed by western blotting with an E2F1 antibody, further confirms the association of YAP1 with E2F1. IP with an antibody to Rb was used as a positive control in this experiment. d A proximity ligation assay showing the enhanced association of YAP1 with E2F1 in A549 cells upon treatment with nicotine or E-cigarette extracts. The interaction was maximal at 24 h

Article Snippet: The expression vectors used were pcDNA3-HA-E2F1, pcDNA3-E2F2, pcDNA3-E2F3, pcDNA3-E2F4, pcDNA3-E2F5, and Yap1 (Addgene #18978).

Techniques: Immunofluorescence, Confocal Microscopy, Immunoprecipitation, Western Blot, Positive Control, Proximity Ligation Assay

Journal: Molecular Cancer

Article Title: Regulation of Sox2 and stemness by nicotine and electronic-cigarettes in non-small cell lung cancer

doi: 10.1186/s12943-018-0901-2

Figure Lengend Snippet: a Treatment with selected inhibitors prevents nicotine-mediated induction of Sox2; the Src family kinases appear to be especially vital for the induction. b Depletion of Src, Yes or β-arrestin-1 reduces the nicotine-mediated induction of Sox2 in A549 cells ( c ) Treatment with a siRNA to Src reduces YAP1 levels and its interaction with E2F1; similar results were obtained upon treatment with a Src inhibitor ( d ). e Proximity ligation assay showing that depletion of Src abrogates the interaction of YAP1 with E2F1; similar results were obtained upon treatment with a Src inhibitor ( f ). g An IP-western blot experiment showed that depeltion of Yes and perhaps Src reduces the icotine-mediated interaction of YAP1 with E2F1

Article Snippet: The expression vectors used were pcDNA3-HA-E2F1, pcDNA3-E2F2, pcDNA3-E2F3, pcDNA3-E2F4, pcDNA3-E2F5, and Yap1 (Addgene #18978).

Techniques: Proximity Ligation Assay, Western Blot

Journal: Molecular Cancer

Article Title: Regulation of Sox2 and stemness by nicotine and electronic-cigarettes in non-small cell lung cancer

doi: 10.1186/s12943-018-0901-2

Figure Lengend Snippet: a Nicotine can induce the association of Oct4 with YAP1 in A549 cells, as seen by a proximity ligation assay. b Depletion of Oct4, E2F1 or YAP1 prevents the nicotine-mediated induction of Sox2-Luc in A549 and H1650 cells. c Similarly, mutating Oct4 or E2F binding sites on the Sox2 promoter prevents its induction by nicotine in A549 cells, as seen in a transient transfection experiment. d Mutating Oct4 binding sites prevents the induction of Sox2-Luc by Oct4, but the promoter is responsive to E2F1; conversely, mutating E2F binding site 1 or site 2 prevents the induction of the Sox2-Luc reporter by E2F1, but conserves the response to Oct4 in a transient transfection experiment. The graphical data represented in this figure has ± standard deviation (SD) values derived from three independent experiments. The statistical comparisons between the representative groups were carried out by one-way ANOVA to determine the statistical significance. e Schematic representing the nicotine mediated upregulation of Sox2. Nicotine binds to the α7 nAChR receptor and activates Src in a βArr1 dependent manner which promotes the binding of to Oct4 and E2F1, resulting in the induction of Sox2

Article Snippet: The expression vectors used were pcDNA3-HA-E2F1, pcDNA3-E2F2, pcDNA3-E2F3, pcDNA3-E2F4, pcDNA3-E2F5, and Yap1 (Addgene #18978).

Techniques: Proximity Ligation Assay, Binding Assay, Transfection, Standard Deviation, Derivative Assay