Journal: Nucleic acids research

Article Title: Metabolically-targeted dCas9 expression in bacteria.

doi: 10.1093/nar/gkac1248

Figure Lengend Snippet: Figure 2. Repression of GusA activity in E. coli with dCas9. (A) Schematic of the arabinose-regulated pBAD-dCas9. Large right-facing arrows represent genes and small right-facing arrows represent promoters. CmR, chloramphenicol resistance gene; pTet, promoter from the tetracycline resistance gene; pBAD, arabinose-inducible promoter. (B) Schematic of pGreg-dCas9 with the E. coli gus operon (not to scale) depicted on top with dashed line indicating the gusA regulatory region cloned upstream of dCas9. O1 and O2, GusR operator binding sites; pGusA, promoter from the gusA regulatory region. (C) Expression of dCas9 from E. coli harbouring pBAD-dCas9 under the indicated conditions assessed by western blot with an anti-Cas9 antibody. (D) Expression of dCas9 under the indicated conditions with E. coli harbouring pBad-dCas9 or pGreg-dCas9 assessed by western blots with an anti-Cas9 antibody. The positive control is 5 ng of purified Streptococcus pyogenes Cas9. (E) Impact of tiling sgRNAs along the length of the gusA gene on GusA activity. Plot of GusA activity (left) in control strains expressing dCas9 without an sgRNA and in the absence or presence of 1 mM pNPG. Plot of GusA activity (right) in dCas9-sgRNA strains for 74 individual sgRNAs targeted to the upstream or coding region of GusA on either the template (orange circles) or non-template (purple diamonds) strand. sgRNAs 349, 373, 451 and 980 used in later experiments are indicated. (F) Schematic of sgRNA multi-array to express sgRNA349, sgRNA451, sgRNA373 and sgRNA980. Right-facing coloured arrows represent different promoters for each sgRNA. H, sgRNA handle sequence; T, sgRNA terminator sequence. Note that promoter, handle and terminator sequences are different for each sgRNA. (G) Knockdown of E. coli GusA activity with pGreg-dCas9 and four different sgRNAs, or an array consisting of the same four sgRNAs. (H) Western blot of dCas9 and GusA expression in pGreg-dCas9 strains without (NG) or with the indicated sgRNAs. Uncropped images for gel images in panels (C), (D) and (H) are in Supplementary Figures S3, S4 and S5, respectively. For panels (E) and (G) data points are mean values from three biological replicates with whiskers indicating the mean plus or minus the standard deviation. *P < 0.05 calculated by Welch’s t-test.

Article Snippet: Anti-Cas9 rabbit polyclonal primary antibody (Rockland, 600-401-GK0S) was used for dCas9 blots, anti- -glucuronidase N-terminal rabbit polyclonal primary antibody (Sigma-Aldrich, G5420) was used for GusA blots, and anti-HA-tag rabbit monoclonal primary antibody (Invitrogen, MA5-27915) was used for GusR blots.

Techniques: Activity Assay, Clone Assay, Binding Assay, Expressing, Western Blot, Positive Control, Purification, Control, Sequencing, Knockdown, Standard Deviation

Journal: Nucleic acids research

Article Title: Metabolically-targeted dCas9 expression in bacteria.

doi: 10.1093/nar/gkac1248

Figure Lengend Snippet: Figure 5. dCas9 expression with pGreg-GusR-dCas9.7 is limited to GUS-positive bacteria. (A) Strategy to examine dCas9 expression in GUS-positive or GUS-negative bacteria by conjugation of pGreg-GusR-dCas9. CmR, chloramphenicol resistance. The dap- donor strain has a knockout of the dapA gene that is used for counter selection during conjugation. (B) Western blots of dCas9 expression in exconjugants with pGreg-GusR-dCas9.7 in the indicated strains grown in liquid media supplemented with 0.2% D-glucose (Glu), 0.2% L-arabinose (Ara) or 1 mM MetGluc (MG). The Cas9+ lane is 5 ng of purified Cas9. (C) As for panel (B) but with exconjugants harbouring pBAD-dCas9. (D) GusA activity assays with the indicated strains harbouring either pBAD-dCas9 (pBAD) or pGreg-GusR-dCas9.7 (pGreg) without (NG) or with sgRNA349. Open and filled circles represent data for pBAD, and open and filled triangles represent data for pGreg. (E) SN-38G processing by E. coli OBEAV1 cell extracts harbouring pGreg-GusR-dCas9 with (orange circles) or without (black circles) sgRNA349. SN-38G hydrolysis in the E. coli gusA strain is plotted for comparison. (F) Plot of SN-38G hydrolysis for the indicated cell extracts calculated using the data shown in panel (D). For panels (D), (E) and (F), data points are the mean of three biological replicates with the whiskers indicating the mean plus or minus the standard deviation. N.S. not significant, *P < 0.05, **P < 0.005 calculated by Welch’s t-test. Uncropped gel images in panels (B) and (C) are in Supplementary Figures S11 and S12, respectively, and uncropped gel images of the native microbiome species (to ensure no cross-reactivity occurred with the anti-Cas9 antibody) are in Supplementary Figure S13.

Article Snippet: Anti-Cas9 rabbit polyclonal primary antibody (Rockland, 600-401-GK0S) was used for dCas9 blots, anti- -glucuronidase N-terminal rabbit polyclonal primary antibody (Sigma-Aldrich, G5420) was used for GusA blots, and anti-HA-tag rabbit monoclonal primary antibody (Invitrogen, MA5-27915) was used for GusR blots.

Techniques: Expressing, Bacteria, Conjugation Assay, Knock-Out, Selection, Western Blot, Purification, Activity Assay, Comparison, Standard Deviation

Journal: Nutrients

Article Title: Multifaceted Protective Effects of Hesperidin by Aromatic Hydrocarbon Receptor in Endothelial Cell Injury Induced by Benzo[a]Pyrene

doi: 10.3390/nu14030574

Figure Lengend Snippet: Anti-inflammatory effect of Hesperidin in EA.hy926 induced by BaP. ( A ) Representative Immunofluorescence of interleukin-1β (IL-1β) level in EA.hy926 ( n = 3 per group). ( B ) mRNA levels of IL-1β in EA.hy926 ( n = 3 per group). ** p < 0.01 vs. normal control group, # p < 0.05, vs. BaP control group. ( C ) mRNA levels of IL-1β in EA.hy926( n = 3 per group). ** p < 0.01 vs.LDL group, # p < 0.05, vs. LDL + BaP group. ( D ) Representative immunocytochemistry of TNF-α level in EA.hy926. ( n = 3 per group, Scale bars, 100 μm). The data are presented as means ± SD ( n = 3) and were analyzed using one-way ANOVA.

Article Snippet: For immunohistochemistry experiments, the cells were incubated with anti-AHR mouse monoclonal primary antibodies (1:200, SC133088, Santa Cruz, CA, USA), anti-ABCA1 mouse monoclonal primary antibodies (1:200, SC53482, Santa Cruz, CA, USA), anti-AGEs rabbit polyclonal primary antibodies (1:200, bs-1158R, BIOSS, Beijing, China), and anti-TNF-α rabbit polyclonal primary antibodies (1: 200, GTX110520, Gentex, MI, USA) overnight at 4 °C.

Techniques: Immunofluorescence, Control, Immunocytochemistry