Journal: bioRxiv

Article Title: Stem cell-derived cranial and spinal motor neurons reveal proteostatic differences between ALS resistant and sensitive motor neurons

doi: 10.1101/629295

Figure Lengend Snippet: (A) Experimental outline: mass spectrometry was performed in Day 2 neurons and proteasome activity tests were performed in Day 2 and Day 5 living neurons. (B) Unbiased mass spectrometry revealed that iCrMNs contain higher levels of all proteasome 20S core subunits but not 19S regulatory subunits (n=2). (C) iCrMNs have a higher proteasome activity than iSpMNs measured by hydrolysis rate of Suc-LLVY-AMC in living cells (n=6, mean ± SEM). n = biological replicates; statistical analysis was performed by student’s t-test, * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: The measurement was carried out in a total volume of 100 μL in 96 well plates with 50 μM Suc-LLVY-AMC (BML-P802, Enzo Lifesciences) in assay buffer (25 mM HEPES, 0.5 mM EDTA, 0.05% NP-40 Tergitol and 0.01% SDS) using 5, 15 and 25 μg of protein supernatant.

Techniques: Mass Spectrometry, Activity Assay

Journal: Cell stem cell

Article Title: Vimentin coordinates protein turnover at the aggresome during neural stem cell quiescence exit

doi: 10.1016/j.stem.2020.01.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Each well received the same amount of protein, 2 mM adenosine triphosphate (Fisher ICN15026605), 0.37 mM proteasome substrate (caspase – Z-Leu-Leu-Glu-AMC (Enzo BML-ZW9345), chymotrypsin – Suc-Leu-Leu-Val-Tyr-AMC (Enzo BML-P802), trypsin – Boc-Leu-Arg-Arg-AMC (Enzo BML-BW8515)), and then diluted to a final volume of 100 μL per well with proteasome activity buffer.

Techniques: Immunofluorescence, Western Blot, Proximity Ligation Assay, Virus, Recombinant, cDNA Synthesis, SYBR Green Assay, RNA Sequencing, Clone Assay, Control, Over Expression, Software

Journal: PLoS Pathogens

Article Title: Latency reversal agents modulate HIV antigen processing and presentation to CD8 T cells

doi: 10.1371/journal.ppat.1008442

Figure Lengend Snippet: A. Aminopeptidase hydrolytic activity in primary CD4 T cells of healthy donors (n = 8–10) at 6, 24 and 48 h after treatment with DMSO (open circles), Bryostatin (green triangles) or Panobinostat (blue squares). B. Aminopeptidase hydrolytic activity in paired DMSO- (open circles) and Panobinostat-treated (blue circles, left) or DMSO- and Bryostatin-treated (green circles, right) primary CD4 T cells at 48 h post-treatment (n = 8 healthy donors). Wilcoxon matched-pairs signed-rank t tests analysis. C. Fold change in aminopeptidase, proteasomal chymotryptic, caspase-like, tryptic-like hydrolytic activities in LRA-treated over DMSO-treated cells at 48 hours: Panobinostat- (blue), Romidepsin- (red), Disulfiram- (orange), Bryostatin- (green), Ingenol- (purple) or aCD3/CD28-treated samples (n = 6–16). One-Way ANOVA Kruskal-Wallis tests on the 6 groups and p-values are reported. D. CD25 and CD69 surface levels were monitored by flow cytometry in primary CD4 T cells at 6, 24 and 48 h post treatment with DMSO (black crosses), Panobinostat (inverted blue triangles), Romidepsin (red squares), Disulfiram (orange circles), Bryostatin (green triangles), Ingenol (purple diamonds) or aCD3/CD28 (grey circles). n = 4–12 healthy donors. E. Unsupervised visualization with t-SNE on Z score centered and normalized data, which included the percentages of CD25-, CD69-, CD38 and HLA-DR-positive CD4 T cells in each sample and the values for aminopeptidase and proteasome chymotryptic, caspase-like and tryptic-like hydrolytic activities. Panobinostat and Romidepsin were grouped as HDACi (green, n = 13) and Bryostatin and Ingenol were grouped as PKCa (red, n = 30), also displayed are mock-treated DMSO (blue, n = 33) and aCD3/CD28-treated (black, n = 14). Ellipses indicate 75% confidence interval.

Article Snippet: The proteasome hydrolytic activities caspase-like (50 μM Z-LLE-AMC; EMD Millipore), tryptic (50 μM Boc-LRR-AMC; Bachem), chymotryptic (50 μM Suc-LLVY-AMC; Bachem), post-proteasomal aminopeptidases (50 μM H-Leu-AMC; Bachem) were measured in 25x10 3 live CD4 T cells at given time points after drug treatment with peptidase-specific fluorogenic substrates as in [ , ].

Techniques: Activity Assay, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Latency reversal agents modulate HIV antigen processing and presentation to CD8 T cells

doi: 10.1371/journal.ppat.1008442

Figure Lengend Snippet: A. Aminopeptidase (left) and proteasomal chymotryptic (right) hydrolytic activities in paired DMSO- (open circles) and Panobinostat+Bryostatin-treated (filled circles) primary CD4 T cells measured at 48 h post-treatment. n = 12 healthy donors. Wilcoxon matched-pairs signed-rank t tests. ns: not significant. B. The surface expression of CD3 (right) and CD25 and CD69 (left) was monitored by flow cytometry in primary CD4 T cells at 48 h post treatment with DMSO (open circles), Panobinostat (blue squares), Bryostatin (green triangles), a combination of Panobinostat+Bryostatin (black inverted triangles) (n = 7 healthy donors). One-Way ANOVA Kruskal-Wallis tests were performed on the 4 groups and the overall p-value is reported, Dunn’s Multi-comparison test was calculated and the pairs that statistically differed are indicated with a bar. * p < 0.05, ** p < 0.01, *** p < 0.001. C. Cleavage patterns of p24-10-35m after degradation in extracts from DMSO- (open bars), Panobinostat- (blue), Bryostatin- (green) and combination Panobinostat+Bryostatin-treated (black) primary CD4 T cells extracts at 120 min, showing the relative amount of fragments starting (N-terminus cleavage site) at each residue. Results shown for a representative experiment. D. Heat map representing the relative amount of 8–11 aa peptides containing each aa. Each row represents an LRA-treatment. One representative experiment is shown.

Article Snippet: The proteasome hydrolytic activities caspase-like (50 μM Z-LLE-AMC; EMD Millipore), tryptic (50 μM Boc-LRR-AMC; Bachem), chymotryptic (50 μM Suc-LLVY-AMC; Bachem), post-proteasomal aminopeptidases (50 μM H-Leu-AMC; Bachem) were measured in 25x10 3 live CD4 T cells at given time points after drug treatment with peptidase-specific fluorogenic substrates as in [ , ].

Techniques: Expressing, Flow Cytometry, Comparison, Residue