Journal: Biomaterials Research

Article Title: Plant-Derived Exosomes as Novel Nanotherapeutics Contrive Glycolysis Reprogramming-Mediated Angiogenesis for Diabetic Ulcer Healing

doi: 10.34133/bmr.0035

Figure Lengend Snippet: HUVECs’ genetic and metabolic alterations to GExos treatment in HG culture. (A1 to A2) Enrichment plots from GSEAs of gene sets for the “Positive regulation of glycolysis signaling”. Expression levels and quantitative analysis of (B1 to B4) PFKM, PGK1, and ENO1, and (C1 to C4) PGLS, ACACA, and PDHA1. (D) Schematic illustration of the major genes and metabolic signaling pathways relevant to angiogenesis, regulated by GExos. (E) Volcano plots of differentially expressed metabolites identified at q < 0.05. (F) KEGG pathway enrichment analyses of the differentially expressed metabolites. (G) Sankey diagram of the differentially expressed metabolites and corresponding enrichment pathways. (H) Mechanism diagram of gene–metabolite interaction. Expression levels (I 1 to I 3 ) and quantitative analysis (J 1 to J 3 ) of G6P, FDP, and Acetyl-CoA. P values are shown: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20, the membranes were then incubated at 4 °C overnight with the following antibodies: rabbit anti-PFKM (Abcam, ab154804, 1:1,000), rabbit anti-PGK1 (Abcam, ab199438, 1:2,000), rabbit anti-ENO1 (Abcam, ab227978, 1:1,000), rabbit anti-PGLS (Abcam, ab229980, 1:2,000), rabbit anti-ACACA (Abcam, ab45174, 1:2,000), rabbit anti-PDHA1 (Abcam, ab168379, 1:2,000), rabbit anti-β-actin (Abcam, ab68477, 1:10,000, internal control), rabbit anti-Cyclin D1 (Abcam, ab134175, 1:10,000), mouse anti-Cyclin D3 (Abcam, ab289546, 1:1,000), and mouse anti-β-actin (Abcam, ab8226, 1:1,000, internal control).

Techniques: Expressing

Journal: Biomaterials Research

Article Title: Plant-Derived Exosomes as Novel Nanotherapeutics Contrive Glycolysis Reprogramming-Mediated Angiogenesis for Diabetic Ulcer Healing

doi: 10.34133/bmr.0035

Figure Lengend Snippet: GExos promote angiogenesis in diabetic ulcers in mice by reprogramming glycolysis. Microvascular imaging (A 1 ) and semi-quantification of the microvascular density (A 2 ) in diabetic healed skins. 8D, 8 days; 16D, 16 days. Immunofluorescence staining (B 1 ) of CD31 (red) and semi-quantification of the area percentage of CD31 (B 2 ) in diabetic healed skins. H&E staining (C) and eNOS staining (D) in diabetic healed skins. Expression levels (E) and quantitative analysis (F 1 to F 3 ) of PFKM, PGK1, and ENO1 on day 8 in diabetic healed skin. Expression levels (G) and quantitative analysis (H 1 to H 3 ) of PFKM, PGK1, and ENO1 on day 16 in diabetic healed skin. Quantitative analysis of G6P (I 1 and I 2 ), FDP (J 1 and J 2 ), and Acetyl-CoA (K 1 and K 2 ) in diabetic healed skins on day 8 and day 16. (L) Schematic diagram of the therapeutic mechanism of GExos promoting angiogenesis in diabetic ulcers. P values are shown: * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20, the membranes were then incubated at 4 °C overnight with the following antibodies: rabbit anti-PFKM (Abcam, ab154804, 1:1,000), rabbit anti-PGK1 (Abcam, ab199438, 1:2,000), rabbit anti-ENO1 (Abcam, ab227978, 1:1,000), rabbit anti-PGLS (Abcam, ab229980, 1:2,000), rabbit anti-ACACA (Abcam, ab45174, 1:2,000), rabbit anti-PDHA1 (Abcam, ab168379, 1:2,000), rabbit anti-β-actin (Abcam, ab68477, 1:10,000, internal control), rabbit anti-Cyclin D1 (Abcam, ab134175, 1:10,000), mouse anti-Cyclin D3 (Abcam, ab289546, 1:1,000), and mouse anti-β-actin (Abcam, ab8226, 1:1,000, internal control).

Techniques: Imaging, Immunofluorescence, Staining, Expressing

Journal: Journal of biochemistry

Article Title: The protein N-terminal acetyltransferase A complex contributes to yeast mitophagy via promoting expression and phosphorylation of Atg32.

doi: 10.1093/jb/mvab068

Figure Lengend Snippet: Fig. 1. Loss of the NatA complex affects Atg32 phosphorylation profiles. Wild-type, ard1D and nat1D cells transformed with a plasmid encod- ing Atg32-3HA (pATG32-3HA) were grown in non-fermentable glycerol medium (Gly), collected at the indicated OD600 points, and sub- jected to western blotting. All strains are pep4- and prb1-null derivatives (defective for vacuolar degradation) lacking the endogenous ATG32 gene (atg32D (1)) or expressing Atg32-3HA from the chromosomal locus (ATG32-3HA (2)). Atg32 is phosphorylated at the early stages of respiratory growth, and phosphorylated Atg32 molecules are detected as multiple upper bands. Arrowheads indicate putative phosphory- lated Atg32. Pgk1 was monitored as a loading control.

Article Snippet: After treatment with the horseradish peroxidase-conjugated rabbit anti-mouse IgG (HþL) secondary antibody for mCherry, GFP, HA and Pgk1 (1:10,000; Jackson ImmunoResearch, 315-035- 003) followed by the enhanced chemiluminescence reagent ChemiLumi One L (Nacalai, 07880-70) or Western Lightning Plus-ECL (PerkinElmer, NEL105001EA), proteins were detected using a luminescent image analyzer (FUSION Solo S; Vilber).

Techniques: Phospho-proteomics, Transformation Assay, Plasmid Preparation, Western Blot, Expressing, Control

Journal: Journal of biochemistry

Article Title: The protein N-terminal acetyltransferase A complex contributes to yeast mitophagy via promoting expression and phosphorylation of Atg32.

doi: 10.1093/jb/mvab068

Figure Lengend Snippet: Fig. 2. Hyperphosphorylation of Atg32 partially rescues mitophagy defects in NatA-deficient cells. (A, B) Wild-type, ppg1D, ard1D, ard1D ppg1D, nat1D, nat1D ppg1D, and atg32D cells expressing mitochondria-targeted DHFR-mCherry (mito-DHFR-mCherry) were pregrown to mid-log phase in glucose medium (Gly 0 h), cultured in glycerol medium (Gly), collected at the indicated time points, and subjected to west- ern blotting. Generation of free mCherry indicates transport of mitochondria to the vacuole. Free mCherry signals in cells at the indicated time points were quantified more than three times in independent experiments. The signal intensity of free mCherry in wild-type cells at the 48 h time point was set to 100%. Data represent the averages of all experiments, with bars indicating standard deviations. (C, D) Wild-type, ard1D, and nat1D cells expressing full-length Atg32-3HA (ATG32-3HA), an Atg32 deletion mutants (D151–200) fused with 3HA ((D151– 200)-3HA) or not expressing Atg32 were pregrown to mid-log phase in glucose medium (Gly 0 h), cultured in glycerol medium (Gly), col- lected at the indicated time points, and subjected to western blotting. This deletion mutant is highly phosphorylated to strongly promote mitophagy. All strains are atg32-null derivatives (atg32D) expressing mito-DHFR-mCherry. Free mCherry signals in cells at the indicated time points were quantified more than three times in independent experiments. The signal intensity of free mCherry in wild-type cells at the 48 h time point was set to 100%. Data represent the averages of all experiments, with bars indicating standard deviations. (E) Wild-type, ppg1D, ard1D, ard1D ppg1D, nat1D, nat1D ppg1D, and atg32D cells expressing Atg32-3HA were grown in glycerol medium (Gly), collected at the indicated OD600 points, and subjected to western blotting. All strains are derivatives lacking Atg7, a protein essential for all autophagy- related processes, to avoid degradation of Atg32-3HA via mitophagy. Atg32-3HA signals normalized with Pgk1 (loading control) signals were quantified more than three times in independent experiments. Data represent the averages of all experiments, with bars indicating standard deviations. *Non-specific bands. (F) Wild-type, ard1D, and nat1D cells expressing full-length Atg32-3HA (ATG32-3HA), an Atg32 deletion mutants (D151–200) fused with 3HA ((D151–200)-3HA), or not expressing Atg32 were grown in glycerol medium (Gly), collected at the indicated OD600 points, and subjected to western blotting. All strains are atg7-null derivatives. Atg32-3HA signals normalized with Pgk1 (loading control) signals were quantified more than three times in independent experiments. Data represent the averages of all experiments, with bars indicating standard deviations. *Non-specific bands.

Article Snippet: After treatment with the horseradish peroxidase-conjugated rabbit anti-mouse IgG (HþL) secondary antibody for mCherry, GFP, HA and Pgk1 (1:10,000; Jackson ImmunoResearch, 315-035- 003) followed by the enhanced chemiluminescence reagent ChemiLumi One L (Nacalai, 07880-70) or Western Lightning Plus-ECL (PerkinElmer, NEL105001EA), proteins were detected using a luminescent image analyzer (FUSION Solo S; Vilber).

Techniques: Expressing, Cell Culture, Western Blot, Mutagenesis, Control

Journal: Frontiers in Endocrinology

Article Title: Quantitative Acetylomics Revealed Acetylation-Mediated Molecular Pathway Network Changes in Human Nonfunctional Pituitary Neuroendocrine Tumors

doi: 10.3389/fendo.2021.753606

Figure Lengend Snippet: MS/MS spectrum of the tryptic peptide. (A) The tryptic peptide ALMDEVVK*ATSR from PGK1. (B) The tryptic peptide TATPQQAQEVHEK*LR from triosephosphate isomerase. K* = acetylated lysine residue.

Article Snippet: The negative control IP experiment was performed with the use of the normal mouse IgG antibody (6 μg; B900620, Proteintech) to replace the anti-PGK1 antibody, which tested the specificity of anti-PGK1 antibody.

Techniques: Tandem Mass Spectroscopy

Journal: Frontiers in Endocrinology

Article Title: Quantitative Acetylomics Revealed Acetylation-Mediated Molecular Pathway Network Changes in Human Nonfunctional Pituitary Neuroendocrine Tumors

doi: 10.3389/fendo.2021.753606

Figure Lengend Snippet: Semiquantitative analysis of acetylated PGK1 between NF-PitNETs and controls. PGK1 in protein samples extracted from NF-PitNET and control tissues was immunoprecipitated (IP) with anti-PGK1 antibody. A negative control immunoprecipitation experiment was performed with the normal mouse IgG antibody but not anti-PGK1 antibody to test the specificity of anti-PGK1 antibody. The IP products (PKG1 and IgG), anti-PGK1 antibodies (Ab), and total protein samples (tumor; control) were simultaneously immunoblotted with anti-acetyl-lysine antibody. T = NF-PitNETs. N = controls. M = markers.

Article Snippet: The negative control IP experiment was performed with the use of the normal mouse IgG antibody (6 μg; B900620, Proteintech) to replace the anti-PGK1 antibody, which tested the specificity of anti-PGK1 antibody.

Techniques: Immunoprecipitation, Negative Control