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: Advanced Science

Article Title: Polyplex Nanomicelle‐Mediated Pgc‐1α4 mRNA Delivery Via Hydrodynamic Limb Vein Injection Enhances Damage Resistance in Duchenne Muscular Dystrophy Mice

doi: 10.1002/advs.202409065

Figure Lengend Snippet: Expression levels of PGC‐1α and associated genes in dystrophic muscles. A) Western blotting of PGC‐1α1 and PGC‐1α4 protein levels ( n = 3 mice). B) Quantitative analysis of the western blot bands normalized to GAPDH. C) Relative mRNA expression levels of total Pgc‐1α (Pgc‐1α1,2,3, and 4), Pgc‐1α1, and Pgc‐1α4. D–K) Relative mRNA levels of genes associated with PGC‐1α in various aspects including muscle hypertrophy (D), myogenic induction (E), mitochondrial activity (F), angiogenesis (G), neuromuscular junction (NMJ) (H), myofiber type (I), metabolism (J), and inflammation (K). All analyses were performed using plantar flexor muscles. Data are presented as mean ± SEM ( n = 6 mice), and Welch's t ‐test was used ( *** p < 0.001, ** p < 0.01, and * p < 0.05).

Article Snippet: Membranes were blocked with 5% non‐fat dry milk and incubated with primary antibodies against PGC‐1α (sc‐518025, Santa Cruz; ST1202, Calbiochem) and loading controls GAPDH (D16H11, Cell Signaling Technology) and α‐tubulin (T6199, Sigma–Aldrich).

Techniques: Expressing, Muscles, Western Blot, Activity Assay

Journal: Advanced Science

Article Title: Polyplex Nanomicelle‐Mediated Pgc‐1α4 mRNA Delivery Via Hydrodynamic Limb Vein Injection Enhances Damage Resistance in Duchenne Muscular Dystrophy Mice

doi: 10.1002/advs.202409065

Figure Lengend Snippet: General upregulation of dysregulated genes after nanomicelle‐delivered Pgc‐1α4 mRNA treatment. A,B) Western blotting images of PGC‐1α4 protein expression on day 2 (A) and day 7 (B). C) Quantification of western blotting bands normalized to GAPDH (for day 2, n = 5 for the LNP + α4 group, n = 6 for all other groups; for day 7, n = 4 for the LNP + α4 group, n = 5 for the micelle + Fluc group, and n = 6 for the micelle + α4 group). D–L) Relative mRNA levels of genes associated with PGC‐1α on day 2 and day 7. All analyses were performed using plantar flexor muscle samples. Data are presented as mean ± SEM ( n = 6 mice), and one‐way ANOVA followed by Tukey's post hoc test was used ( *** p < 0.001, ** p < 0.01, and * p < 0.05).

Article Snippet: Membranes were blocked with 5% non‐fat dry milk and incubated with primary antibodies against PGC‐1α (sc‐518025, Santa Cruz; ST1202, Calbiochem) and loading controls GAPDH (D16H11, Cell Signaling Technology) and α‐tubulin (T6199, Sigma–Aldrich).

Techniques: Western Blot, Expressing