Journal: bioRxiv

Article Title: Boosting Proteasome Activity: A Novel Mechanism of NMDAR Blockers Against Neurodegeneration

doi: 10.1101/2024.08.20.608787

Figure Lengend Snippet: (A) Effects of RPN13 inhibition by RA190 on p21 levels and the corresponding influence of ketamine in RA190 (1µM, 12 h incubation) pre-treated conditions. (B and C) Role of RA190 in chymotrypsin-like activity and ketamine’s intervention in these conditions. (D) RA190’s elevation of ubiquitinated proteins contrasted with ketamine’s reduction effect. (E) IU1’s effect on p21 protein levels and the modifying role of ketamine alongside USP14 inhibition. (F) Changes in USP14 protein levels with overexpression or silencing techniques. (G) How USP14 expression levels affect p21 and p53 proteins and ketamine’s influence under these conditions. (H, I) Impact of USP14 expression alterations on chymotrypsin-like activity and modifications by ketamine. (J) Dose-dependent effects of the PSMD14 inhibitor gliotoxin on p21 levels were examined, alongside the effects of ketamine on p21 with and without gliotoxin pre-treatment, with evidence that gliotoxin inhibits 20S proteasome chymotrypsin activity at high doses. p21 levels were analyzed via western blot at two time points of signal exposure. (K and L) Ketamine’s effects on chymotrypsin activity were evaluated with and without 5 µM gliotoxin. ( M) At 25 µM gliotoxin, ketamine fails to decrease p21 levels while reducing p53 levels. Due to this discrepancy, the Western blot was repeated multiple times and the result was ultimately confirmed by using p21 and p53 antibodies concurrently on the same membrane (N and O) Chymotrypsin activity in response to ketamine was assessed with and without 25 µM gliotoxin. Results are presented as the mean ± SEM, based on 3 to 5 technical replicates from each of n = 3 independent experiment. *Asterisks denote significance levels: *p < 0.05, **p < 0.01, ***p < 0.001. P values were calculated using a two-tailed unpaired t-test to compare the control group with the individual chemical effect or USP14 knockdown cells.

Article Snippet: We measured proteasome activity with the Enzo Life Sciences’ 20S assay kit, applying isolated and purified proteasomes from human erythrocytes.

Techniques: Inhibition, Incubation, Activity Assay, Over Expression, Expressing, Western Blot, Membrane, Two Tailed Test, Control, Knockdown

Journal: The Biochemical journal

Article Title: N-terminal acetylation and methylation differentially affect the function of MYL9

doi: 10.1042/BCJ20180638

Figure Lengend Snippet: (A) WT MYL9 (SSK) has been found in Nα-methylated and Nα-acetylated forms. The S3P (SPK) MYL9 mutant is Nα-methylated, and the K4Q (SSQ) MYL9 mutant is Nα-acetylated. In vivo, the initiator methionine of WT MYL9 is cleaved to reveal the N-terminal sequence shown. (B–G) in vitro methyltransferase and acetyltransferase assays were performed to determine the KM of NRMT1 or NAA10 (the catalytic subunit of NatA) when using peptides corresponding to the 14 N-terminal amino acids (after Met cleavage) of WT and mutant MYL9 as substrates. (B) The KM of NRMT1 with WT (SSK) MYL9 was determined to be 28.8 μM. (C) NAA10 with WT (SSK) MYL9 had a KM of 0.7 μM. (D) SPK MYL9 was confirmed to be a preferred substrate of NRMT1 with a KM of 0.7 μM. (E) NAA10 showed no activity with SPK MYL9 up to 40 μM. (F) SSQ MYL9 Figure 1. N-terminal mutants of MYL9 select for Nα-methylation or Nα-acetylation. Part 2 of 2 was not a substrate of NRMT1, showing no activity up to 160 μM. (G) The KM of NAA10 with SSQ MYL9 was 5.0 μM. n = 3 for all experiments. All error bars represent standard deviation.

Article Snippet: In vitro acetyltransferase experiments were performed using the Enzo Acetyltransferase Activity Kit (Enzo Life Sciences, Farmingdale, NY) following manufacturer guidelines.

Techniques: Methylation, Mutagenesis, In Vivo, Sequencing, In Vitro, Activity Assay, Standard Deviation

Journal: Journal of Personalized Medicine

Article Title: Therapeutic Effects of Engineered Exosomes from RAW264.7 Cells Overexpressing hsa-let-7i-5p against Sepsis in Mice—A Comparative Study with Human Placenta-Derived Mesenchymal Stem Cell Exosomes

doi: 10.3390/jpm14060619

Figure Lengend Snippet: ( A ) The 48-h survival rates, as determined by calculating the number of mice that survived the 48 h observational duration in each group after normal saline or lipopolysaccharide administration. Data were derived from 6 mice in the Sham, MExo, and EExo groups and 12 mice from the LPS, LMExo, LMExoi, LEEXo, and LEExoi groups. * p < 0.05, the LPS group versus the Sham group. # p < 0.05, the LEExo group versus LPS group. ˄ p < 0.05, the LEExoi group versus the LEExo group. ( B ) Plasma concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, measured using enzyme-linked immunosorbent assay. Data were obtained from 5 mice in each group. All assays were measured at 48 h after normal saline or lipopolysaccharide administration. Data represented as mean ± standard deviations. * p < 0.05, versus the Sham group. # p < 0.05, versus the LPS group. † p < 0.05, the LMExoi group versus the LMExo group. ˄ p < 0.05, the LEExoi group versus the LEExo group. Sham: the normal saline group. MExo group: the normal saline plus hpMSC exosome group. EExo group: the normal saline plus engineered exosome group. LPS: the lipopolysaccharide (LPS) group. LMExo: the LPS plus hpMSC exosome group. LMExoi: the LPS plus inhibitor-treated hpMSC exosome group. LEExo: the LPS plus engineered exosome group. LEExoi: the LPS plus inhibitor-treated engineered exosome group.

Article Snippet: The levels of cytokines, including TNF-α, IL-1β, and IL-6, in lung tissues were assessed using ELISA kits for TNF-α, IL-1β, and IL-6 (all from Enzo Life Science).

Techniques: Saline, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Personalized Medicine

Article Title: Therapeutic Effects of Engineered Exosomes from RAW264.7 Cells Overexpressing hsa-let-7i-5p against Sepsis in Mice—A Comparative Study with Human Placenta-Derived Mesenchymal Stem Cell Exosomes

doi: 10.3390/jpm14060619

Figure Lengend Snippet: ( A ) Representative gel photography of phosphorylated nuclear factor-kB (p-NF-kB) and Actin (internal standard), assayed via the Simple Western method and the relative band density of p-NF-kB/Actin ratio in lung tissues. Data were obtained from 5 mice in each group. ( B ) The concentrations of tumor necrosis factor-α (TNF-α), interleukin 1-β (IL-1β), and IL-6 in lung tissues, measured via the enzyme-linked immunosorbent assay. Data were obtained from 5 mice in each group. All assays were measured at 48 h after normal saline or lipopolysaccharide administration. Data represented as mean ± standard deviations. * p < 0.05, versus the Sham group. # p < 0.05, versus the LPS group. † p < 0.05, the LMExoi group versus the LMExo group. ˄ p < 0.05, the LEExoi group versus the LEExo group. Sham: the normal saline group. MExo group: the normal saline plus hpMSC exosome group. EExo group: the normal saline plus engineered exosome group. LPS: the lipopolysaccharide (LPS) group. LMExo: the LPS plus hpMSC exosome group. LMExoi: the LPS plus inhibitor-treated hpMSC exosome group. LEExo: the LPS plus engineered exosome group. LEExoi: the LPS plus inhibitor-treated engineered exosome group.

Article Snippet: The levels of cytokines, including TNF-α, IL-1β, and IL-6, in lung tissues were assessed using ELISA kits for TNF-α, IL-1β, and IL-6 (all from Enzo Life Science).

Techniques: Simple Western, Enzyme-linked Immunosorbent Assay, Saline