Journal: mBio

Article Title: A bacterial family of fatty acid acyltransferases related to the Shigella effector IcsB

doi: 10.1128/mbio.03890-25

Figure Lengend Snippet: A subset of novel k-FATs exhibits yeast toxicity and IcsB-like fatty acid acyltransferase activity. ( A ) Map of the pRS313 Gal1p2 centromeric plasmid used for the galactose-inducible expression of IcsB and its homologs. ( B )The expression of IcsB and seven of its homologs in the presence of galactose inhibits yeast growth compared to the empty vector (EV) control. The glucose plate indicates that there is no growth inhibition in the absence of expression and that similar amounts of each strain were plated. Four dilutions were plated (non-diluted, 1/10, 1/100, and 1/1,000). The graph represents the quantification of growth on three independent replicates of the 1/1,000 dilution. The error bars represent the standard deviation of the mean. A Student’s t-test with unpaired data and a 95% CI was performed (*, P < 0.05; **, P < 0.1, ***, P < 0.01). ( C ) Protein acylation in the membrane protein fraction of yeast expressing IcsB and its homologs according to the in-gel fluorescence acyltransferase assay (upper panels). -Alk IcsB is the IcsB sample prior to the addition of Alk-16. -Alk IcsB, Alk-16 EV, and Alk-16 IcsB were loaded on both gels to serve as controls. This acylation assay was performed using the strains from B that were induced with galactose for six hours in the presence of Alk-16 for the final 4 h. The total protein in these samples were assessed using the TGX stain (bottom panels). ( D ) The expression of IcsB and of its homologs was assessed by immunoblotting with an antibody binding to their 3× FLAG tag and with the GAPDH as a loading control.

Article Snippet: Primary antibodies used were mouse FLAG-tag (Sigma-Aldrich, F3165), mouse Myc-tag (Genescript, A00704), and rabbit GAPDH (Bioss antibodies, BS-8789R) diluted 1/5,000, 1/1,000, and 1/10,000, respectively.

Techniques: Activity Assay, Plasmid Preparation, Expressing, Control, Inhibition, Standard Deviation, Membrane, Fluorescence, Staining, Western Blot, Binding Assay, FLAG-tag

Journal: mBio

Article Title: A bacterial family of fatty acid acyltransferases related to the Shigella effector IcsB

doi: 10.1128/mbio.03890-25

Figure Lengend Snippet: Mutations in the catalytic site of k-FATs abolish their enzymatic activity. ( A ) Yeast growth inhibition tests of catalytic residues mutants H145A (left), D195A (center), and C306A (right) compared to their WT counterpart in IcsB and in toxic homologs. The graph represents the quantification of growth on three independent replicates with the dilution of yeast cultures 1/1,000. The error bars represent the standard deviation from the mean. A Student’s t -test with unpaired data and a 95% CI was performed (*, P < 0.05; **, P < 0.1, ***, P < 0.01). ( B ) In-gel protein acylation of C306A mutants compared to their WT counterparts (upper panels). The total protein in these samples were assessed using the TGX stain (bottom panels). ( C ) The expression of IcsB and toxic homolog C306A mutants was compared to their WT counterparts by immunoblotting with an antibody binding to their 3×FLAG tag and with the GAPDH as a loading control.

Article Snippet: Primary antibodies used were mouse FLAG-tag (Sigma-Aldrich, F3165), mouse Myc-tag (Genescript, A00704), and rabbit GAPDH (Bioss antibodies, BS-8789R) diluted 1/5,000, 1/1,000, and 1/10,000, respectively.

Techniques: Activity Assay, Inhibition, Standard Deviation, Staining, Expressing, Western Blot, Binding Assay, Control

Journal: Nature Communications

Article Title: PTEN expression by an oncolytic herpesvirus directs T-cell mediated tumor clearance

doi: 10.1038/s41467-018-07344-1

Figure Lengend Snippet: HSV-P10 construction and characterization. a Graphical representation of PTEN and PTENα coding sequences showing the mutations engineered in PTENα start codons to enhance translation of full-length PTENα. b – c Graphical representation of the DNA structure of wild-type F-strain HSV1 backbone showing doubly deleted γ34.5 genes, and gene-disrupting insertional ICP6 locus containing an enhanced green fluorescent protein (eGFP) gene with PTENα insertion (HSV-P10) or without PTENα (HSVQ). d PTENα production in infected tumor cell lysates over time. The indicated tumor cells were infected with HSV-P10 at MOI = 0.5 and harvested 0–12 hpi and probed for PTEN by western blot

Article Snippet: Primary (from CST: PTEN (9559), Akt (4691), pAkt-S473 (9271), from abcam: HSV1 ICP4 (ab6514), GAPDH (ab9484)) and secondary antibodies (from abcam: anti-mouse HRP (ab6789), anti-rabbit HRP (ab6721) can be found in Supplementary Data .

Techniques: Infection, Western Blot

Journal: Nature Communications

Article Title: PTEN expression by an oncolytic herpesvirus directs T-cell mediated tumor clearance

doi: 10.1038/s41467-018-07344-1

Figure Lengend Snippet: HSV-P10 induces enhanced immune cell influx towards infected tumors. Tumor-bearing FVB/N mice were treated with 1e5 pfu of HSVQ, HSV-P10, or saline control 8 days post tumor implantation. a Stitched 4X bright field images of H&E stained sagittal sections of mouse brains 3 days post virus injection. b Representative 20X bright field images of H&E, Keratin 8, F4/80, NKp46, CD3, CD8α, PD-L1, and HSV1 (GFP) from sagittal sections of mouse brains 3 days post virus injection. Scale = 0.1 mm. c Ratio of macrophages (CD11b + F4/80 + CD45 bright ) to microglia (CD11b + F4/80 + CD45 dim ). d Percentage of cells in c positive for MHC-II. e Dendritic cell (CD11c + CD80 + MHC-II + ) infiltration. f NK cell (CD49b + NKp46 + ) infiltration. g CD8 + T-cell (CD3 + CD4 + ) infiltration. h CD4 + T-cell (CD3 + CD8 + ) infiltration. Data shown are averages ± s.d ( n = 3/group) Statistical significance was assessed by one-way ANAOVA ( n = 3, * p < 0.05, *** p < 0.001, **** p < 0.0001). Gating strategies for ( c – h ) are decribed in supplementary figure

Article Snippet: Primary (from CST: PTEN (9559), Akt (4691), pAkt-S473 (9271), from abcam: HSV1 ICP4 (ab6514), GAPDH (ab9484)) and secondary antibodies (from abcam: anti-mouse HRP (ab6789), anti-rabbit HRP (ab6721) can be found in Supplementary Data .

Techniques: Infection, Tumor Implantation, Staining, Injection