Journal: Cell Death and Differentiation

Article Title: PAK5 promotes the migration and invasion of cervical cancer cells by phosphorylating SATB1

doi: 10.1038/s41418-018-0178-4

Figure Lengend Snippet: PAK5-mediated phosphorylation promotes EMT in cervical cancer cells. a, b Effects of SATB1 and SATB1 Ser47A on the migration and invasion ability in HeLa and SiHa cells. Scale bar, 100μm for a and b. c HeLa and SiHa cells were transfected with pGL3-E, together with the indicated expression plasmids for Dual luciferase reporter assays. d Effects of SATB1 knockdown on EMT markers were analyzed by western blotting in HeLa cells overexpressing PAK5. e Effects of SATB1 overexpression on EMT markers in HeLa cells silencing PAK5. Data are showed as mean ± SD for three independent experiments. **, P < 0.01; ***, P < 0.001. pGL3-E pGL3-E-cadherin promoter, siCtrl siRNA control, siPAK5 siRNA PAK5, ns not significant, GAPDH glyceraldehyde-3-phosphate dehydrogenase

Article Snippet: Briefly, E-cadherin promoter was subcloned into pGL3-Basic luciferase expression vector (Genepharma).

Techniques: Migration, Transfection, Expressing, Luciferase, Western Blot, Over Expression

Journal: The Journal of Experimental Biology

Article Title: Circadian coupling of mitochondria in a deep-diving mammal

doi: 10.1242/jeb.246990

Figure Lengend Snippet: Clock genes expression profile in synchronized hooded seals fibroblasts and tissues. (A) mRNA expression of core clock genes arntl (blue line) and per2 (black line) in hooded seal fibroblasts synchronized by temperature cycle (°C), as represented in the upper panel. Arntl and per2 mRNAs show a 24-h period and are in antiphase. P -values and phases were calculated through JTK cycle analysis ( n =4). (B) mRNA expression of core clock genes arntl (blue line) and per2 (black line) in hooded seal fibroblasts synchronized by temperature cycling and then left at constant temperature (upper panel). Arntl and per2 mRNAs maintain a 24-h period and their antiphasic relationship also in constant conditions. P -values and phases were calculated through JTK cycle analysis ( n =4). (C) Photon multiplier tube recordings of hooded seal skin fibroblasts transfected with arntl :luciferase (blue line) and per2 :luciferase (black line). Cells were synchronized with dexamethasone. (D) Arntl mRNA expression in hooded seal kidney and liver tissue, sampled in mid-light phase (ZT6) and mid-dark phase (ZT18). * P <0.05 (one-way ANOVA; n =3 except liver ZT18, where n =2). Data are expressed as means±s.e.m.

Article Snippet: The latter consisted of the pLV6 backbone and a mouse per2 promoter with adjacent luciferase sequence contained in the pGL3 basic E2 vector (Addgene plasmid 48747).

Techniques: Expressing, Transfection, Luciferase

Journal: Microbial Cell Factories

Article Title: In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation

doi: 10.1186/s12934-015-0195-7

Figure Lengend Snippet: Effect of N-acetylglucosamine on glycoconjugate synthesis. A) Comparison of specific EPA-2a yield in the absence (−) or presence (+N-Ac) of N-acetylglucosamine (4 g/L) during simultaneous induction of PglB and EPA in shake flasks. B) EPA-2a expression in the presence of increasing amounts of N-acetylglucosamine (0 g/L – 10 g/L) in 96-deep well plates. Values were divided by the corresponding final OD 600 before normalization to exclude that the beneficial effect of N-acetylglucosamine is only due to increase of biomass formation. E. coli cells were incubated at 30°C, and PglB and EPA were induced simultaneously for 24 h by addition of 1 mM IPTG and 2 g/L arabinose, respectively. C) . The final OD 600 values in the presence of increasing amounts of N-acetylglucosamine (0 g/L – 10 g/L) in 96-deep well plates. Error bars represent the standard deviations of three biological replicates.

Article Snippet: The PglB sequence was codon-optimized for expression in E. coli by gene synthesis (GenScript, Piscataway, NJ, USA).

Techniques: Comparison, Expressing, Incubation

Journal: Open Biology

Article Title: Increased efficiency of Campylobacter jejuni N -oligosaccharyltransferase PglB by structure-guided engineering

doi: 10.1098/rsob.140227

Figure Lengend Snippet: Effect of amino acid substitution PglB Cj N311V on glycosylation of EPA with two heterologous polysaccharides and natural OSs. Open symbols, wild-type PglB (pGVXN970); closed symbols, PglB N311V (pGVXN1217). ( a ) Host strain and detection antibodies for S. enterica sv. Thyphimurium LT2 polysaccharides, ( b ) host strain and detection antibodies for S. aureus CP5 polysaccharides, and ( c ) host strain and detection antibodies for C. jejuni OSs. Background-corrected ELISA signals for biomass-normalized periplasmic extracts from shake flask cultures, average values and standard deviations of n = 3 biological replicates.

Article Snippet: Low-copy-number, pEXT21-derived plasmids for expression of codon-optimized wild-type PglB and PglB N311V were obtained from GenScript.

Techniques: Glycoproteomics, Enzyme-linked Immunosorbent Assay

Journal: Open Biology

Article Title: Increased efficiency of Campylobacter jejuni N -oligosaccharyltransferase PglB by structure-guided engineering

doi: 10.1098/rsob.140227

Figure Lengend Snippet: Mutation tolerance of PglB Cj residues mutated in second round saturation mutagenesis libraries and amino acid substitutions identified in the 10 clones with the highest LT2-EPA ELISA signals. Clones were counted as active when background-corrected ELISA signals reached more than 10% of the average value of N311V control wells.

Article Snippet: Low-copy-number, pEXT21-derived plasmids for expression of codon-optimized wild-type PglB and PglB N311V were obtained from GenScript.

Techniques: Mutagenesis, Clone Assay, Enzyme-linked Immunosorbent Assay, Control, Residue

Journal: Open Biology

Article Title: Increased efficiency of Campylobacter jejuni N -oligosaccharyltransferase PglB by structure-guided engineering

doi: 10.1098/rsob.140227

Figure Lengend Snippet: Shuffling of neutral and slightly beneficial mutations of PglB Cj . ( a ) Screening results for a representative 96-well library; open circles, library clones; filled triangles, PglB N311V (template plasmid pGVXN1418); shaded triangles, inactive PglBmut (pGVXN408). ( b ) Verification of improvements after retransformation, average values and standard deviations for n = 3 replicate clones/wells per variant plasmid; wt, pGVXN1413; N311V, pGVXN1418. ( c ) SDS-PAGE and western blot analysis of Ni-NTA affinity purified proteins produced with either wild-type PglB (pGVXN970), PglB N311V (pGVXN1217) or PglB S80R-Q287P-N311V (library clone 2B2) in shake flasks; similar loading volumes, total protein concentration (A280) was adjusted to 2 mg ml −1 . Theoretical molecular weight of unglycosylated EPA-6H: 69.4 kDa.

Article Snippet: Low-copy-number, pEXT21-derived plasmids for expression of codon-optimized wild-type PglB and PglB N311V were obtained from GenScript.

Techniques: Clone Assay, Plasmid Preparation, Variant Assay, SDS Page, Western Blot, Affinity Purification, Produced, Protein Concentration, Molecular Weight