Journal: Cell Reports

Article Title: A Nuclear Export Block Triggers the Decay of Newly Synthesized Polyadenylated RNA

doi: 10.1016/j.celrep.2018.07.103

Figure Lengend Snippet: Nab2p Is Sequestered on Nuclear-Retained RNA in Export-Deficient Cells and Excess Nab2p Can Alleviate mRNA Downregulation (A) SDS-PAGE of eluted samples from an RNA immunoprecipitation (IP) experiment in which endogenous Nab2p was immunoprecipitated from Mex67-AA cells without (“rapamycin –”) or treated with rapamycin for 30 min (“rapamycin +”). Before RNA IP, cells were not subjected to crosslinking (“0 J/cm 2 ”), moderately cross-linked (“300 J/cm 2 ,” ∼2 min cross-linking time), or strongly cross-linked (“700 J/cm 2 ,” ∼5 min cross-linking time). Mock samples were negative control IPs using beads without anti-Nab2p antibody. Shown are IP eluates without (lanes 1–7) or with (lanes 8–14) prior RNaseA/T1 treatment. Top: western blotting analysis of Nab2p levels; bottom: autoradiogram of P 32 -labeled SDS-PAGE gel (representative example of three independent repeats). (B) Quantification of the P 32 -labeled RNA signal from lanes 1–7 in (A). Background was estimated and subtracted on the basis of the mock sample. Single points represent values obtained from three individual repeats. Error bars represent SDs. (C) Quantification of P 32 -labeled RNA signal within the Nab2p contained band of lanes 8–14 from (A). Values were normalized as in Figure 4 B. Error bars represent SDs from three independent repeats, and statistical significance was tested using a two-tailed paired t test. (D) RT-qPCR analysis of selected 10 min 4tU-labeled RNAs purified from cells treated for 15 min with rapamycin as in Figure 3 C. RNA series 2–4 show new RNA production from control Mex67-AA cells grown in glucose (series 3), which have been simultaneously depleted for Nab2p (Mex67-AA/Nab2-AA) (series 2) or containing a Nab2p expressing plasmid (Mex67-AA p2μ_NAB2) (series 4). Values derive from a minimum of four biological replicates, and error bars indicate SDs. Values for RNA series 3 were also included in the mean shown in Figure 3 C. RNA series 5–9 show new RNAs from Mex67-AA cells containing an empty (series 5) or a p Pgal_Nab2 (series 6–9) plasmid and incubated for 2.5, 5, 6, or 8 hr in galactose. Values are a mean of two biological replicates for the p Pgal_Nab2 containing samples and an average of single replicates of all galactose incubation times for the empty plasmid sample. Error bars indicate median absolute deviation. (E) Western blotting analysis of Nab2p levels in non-rapamycin-treated Mex67-AA cells transformed with either an empty 2μ pRS426 plasmid (−) or its counterpart (pNAB2) expressing wild-type Nab2p (+). Rpb3p served as a loading control. (F) Western blotting analysis showing Nab2p and Rpb3p levels in cells containing a NAB2 gene under control of a galactose-inducible promoter (p Pgal_Nab2 ) in glucose (GLU) condition and at the indicated times after galactose (GAL) addition. The image was edited to show only one of the repeats performed in parallel. See also Figure S4 .

Article Snippet: 2μ, ori(f1), ori(pMB1), URA3, Amp r , LacZ, MCS, T7 promoter, T3 promoter , ATCC 77107 , pRS426.

Techniques: SDS Page, RNA Immunoprecipitation, Immunoprecipitation, Negative Control, Western Blot, Labeling, Two Tailed Test, Quantitative RT-PCR, Purification, Control, Expressing, Plasmid Preparation, Incubation, Transformation Assay

Journal: Cell Reports

Article Title: A Nuclear Export Block Triggers the Decay of Newly Synthesized Polyadenylated RNA

doi: 10.1016/j.celrep.2018.07.103

Figure Lengend Snippet:

Article Snippet: 2μ, ori(f1), ori(pMB1), URA3, Amp r , LacZ, MCS, T7 promoter, T3 promoter , ATCC 77107 , pRS426.

Techniques: Recombinant, SYBR Green Assay, Purification, Software, Selection, Real-time Polymerase Chain Reaction, Microscopy

Journal: Molecular Cancer

Article Title: Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis

doi: 10.1186/1476-4598-9-258

Figure Lengend Snippet: Establishment of C4-2B/Rx2 dox sub-line with conditional Runx2 expression . A, Schematic diagram of the pSLIK-based lentiviral vector. Initially developed for expression of shRNAs the pSLIK vector was used in the present study to express Runx2. The Hygromycin resistance marker ( Hyg ) and the Dox-dependent activator protein rtTA3 are constitutively expressed under the control of the Ubi-c promoter. Upon treatment with Dox, rtTA binds to its tetracycline responsive elements ( TRE ) and drives expression of the inserted cDNA (Flag-Runx2). The Runx2 block diagram depicts its glutamine/alanine-rich QA domain, the DNA-binding Runt domain, and the proline/serine/threonine-rich PST domain. Arrowhead indicates the position of the R265D and R268D mutations in Runx2-M, which eliminate Runx2's DNA binding function. B, Whole cell extracts, prepared from C4-2B/Rx2 dox and C4-2B/Rx2-M dox cells treated with the indicated concentrations of Dox, were subjected to western blot analysis using anti-Flag antibodies. C, Total RNA was extracted from C4-2B/Rx2 dox cells treated with Dox or vehicle, as well as from PC3 high cells, and the mRNA levels of Runx2 (and GAPDH as control) were measured by RT-qPCR. D, Whole cell extracts were prepared from C4-2B/Rx2 dox cells treated with Dox as indicated and from ROS 17.8/2 osteoblastic cells , and subjected to western blot analysis using anti-Runx2 antibodies. The same blot was re-probed with anti-Tubulin antibodies as loading control. E, C4-2B/Rx2 dox and C4-2B/Rx2-M dox cells were transiently transfected with the 6XOSE2-luciferase reporter plasmid and subjected to luciferase assay. Dotted line represents the background luciferase activity with no cell extract. F, C4-2B/Rx2 dox cells were treated with Dox and levels of the indicated transcripts were measured by RT-qPCR and corrected for that of GAPDH. In Figure 1, bars represent Mean ± SEM (n = 3) from a representative experiment, which was repeated at least three times with similar results. Abbreviations used: Dox, Doxycycline; Veh, vehicle; BSP, Bone Sialoprotein; MMP9, Matrix Metalloprotein-9; OC, Osteocalcin.

Article Snippet: The Flag-Runx2 cDNA was initially cloned into the SpeI/MfeI-digested lentiviral entry vector pEN_TmiRc3 (ATCC ® catalog: MBA-248), and the resulting plasmid was recombined using Gateway ® LR Clonase ® II enzyme mix (Invitrogen, Carlsbad, CA, USA) with the pSLIK (single lentivector for inducible knockdown) destination vector carrying a hygromycin resistance gene (ATCC ® catalog: MBA-237).

Techniques: Expressing, Plasmid Preparation, Marker, Control, Blocking Assay, Binding Assay, Western Blot, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay

Journal: ACS chemical biology

Article Title: Fluorescence Assessment of the AmpR-Signaling Network of Pseudomonas aeruginosa to Exposure to β ‑Lactam Antibiotics

doi: 10.1021/acschembio.9b00875

Figure Lengend Snippet: Schematic depiction of reporter plasmid and fluorescence gene induction by the β-lactam antibiotic. (A) The plasmid map for pCN61_AmpRtdT is shown to the left. Ampicillin (AP, Tem-1 β-lactamase) or streptomycin (STR) is used for selection. The ampRC operon (top right) and the reporter operon where tdTomato is positioned downstream of the ampC promoter (denoted PampC) are depicted. (B) We constructed GFP-labeled P. aeruginosa and transformed this bacterium with pCN61_AmpRtdT. The bacteria were inoculated on an agar plate in proximity to the β-lactam antibiotic CAZ (spotted at the red dot). The antibiotic diffuses outward from the spot. The interface (white arrow) of CAZ and the GFP-labeled P. aeruginosa without the plasmid shows only green fluorescence (top row). In contrast, the GFP-labeled P. aeruginosa transformed by pCN61_AmpRtdT shows green fluorescence (constituitively) and the red fluorescence induced by the cell-wall-acting antibiotic (bottom row). The top row confirms the absence of red backgroud fluorescence from P. aeruginosa. The bottom row demonstrates the functionality of our reporter plasmid. A 10 μM scale bar is given in panel two of the top row.

Article Snippet: The fluorescent-reporter systems were excised from pUC57 with restriction enzymes Bam HI and Pst I and ligated into an E. coli - P. aeruginosa shuttle vector, pCN61 (ATCC).

Techniques: Plasmid Preparation, Fluorescence, Selection, Construct, Labeling, Transformation Assay

Journal: ACS chemical biology

Article Title: Fluorescence Assessment of the AmpR-Signaling Network of Pseudomonas aeruginosa to Exposure to β ‑Lactam Antibiotics

doi: 10.1021/acschembio.9b00875

Figure Lengend Snippet: Correlation of the β-lactamase and fluorescence assays. (A) Nitrocefin hydrolysis by AmpC β-lactamase shifts λmax from 390 nm (yellow) to 486 nm (red). (B) Nitrocefin assay performed for P. aeruginosa wild-type and dacB::Tn is plotted as the slope of the absorbance at 486 nm over time for two concentrations of FOX (1/8 MIC and 1/4 MIC) and no antibiotic. (C) Fluorescent response expressed as relative fluorescence (A.U.) of P. aeruginosa wild-type and dacB::Tn under the same experimental conditions as used for the nitrocefin assay. The error bars represent the standard deviation of three biological replicates.

Article Snippet: The fluorescent-reporter systems were excised from pUC57 with restriction enzymes Bam HI and Pst I and ligated into an E. coli - P. aeruginosa shuttle vector, pCN61 (ATCC).

Techniques: Fluorescence, Standard Deviation

Journal: ACS chemical biology

Article Title: Fluorescence Assessment of the AmpR-Signaling Network of Pseudomonas aeruginosa to Exposure to β ‑Lactam Antibiotics

doi: 10.1021/acschembio.9b00875

Figure Lengend Snippet: GFP-labeled P. aeruginosa harboring pCN61_AmpRtdT was imaged on a swarm plate in proximity of a second bacterium, either (A) P. mesacidophila, (B) B. licheniformis, (C) E. coli, or (D) M. xanthus. Each panel depicts the bacteria after 15 h of growth. P. aeruginosa is to the left of each plate, while the second bacterium is to the right (see plate panels). The white arrow (top plate) points to a representative site where the two bacteria encounter one another. The second column from left shows bright-field images of comingled bacteria, where the strains meet, as uniform continuous lawns. The GFP panel shows the green fluorescence displayed by P. aeruginosa in this lawn. The black voids are the locations of the second bacterium in this same lawn. The RFP panel shows the red-fluorescent signal from P. aeruginosa that results from contact with an antibiotic-producer strain. Red fluorescence is seen for P. mesacidophila and B. lichemiformis as antibiotic producers. No red fluorescence is seen for E. coli and M. xanthus (not antibiotic producers). The far-right column merges the green and red fluorescent images. A 10-μm black scale bar is given in the bright-field image of panel A.

Article Snippet: The fluorescent-reporter systems were excised from pUC57 with restriction enzymes Bam HI and Pst I and ligated into an E. coli - P. aeruginosa shuttle vector, pCN61 (ATCC).

Techniques: Labeling, Fluorescence