Journal: Journal of Inflammation Research

Article Title: Therapeutic Potential of TPT-260 in Ischemic Stroke: An Investigation Into Its Anti-Inflammatory Effects and Impact on Microglial Activation

doi: 10.2147/JIR.S497030

Figure Lengend Snippet: Therapeutic effect of TPT-260 on the MCAO mice model of cerebral infarction. TPT-260 treatment improved the Bederson score ( A ), reduced the infarcted brain area ( B and C ), and inhibited the proinflammatory factors IL-1β ( D ) and TNF-α ( E ) levels in brains of MCAO mice compared to that in the control mice injected with saline. Data were analyzed using two-way ANOVA (* p < 0.05, ** p < 0.01, and *** p < 0.001). N=3 in ( A and C ). Representative images from one mouse were shown in ( B ) N=5 in ( D and E ).

Article Snippet: TPT-260 (CAS: 2076–91-7, HY-23769A, MedChemExpress) was initially dissolved in DMSO to a concentration of 20 mm.

Techniques: Control, Injection, Saline

Journal: Journal of Inflammation Research

Article Title: Therapeutic Potential of TPT-260 in Ischemic Stroke: An Investigation Into Its Anti-Inflammatory Effects and Impact on Microglial Activation

doi: 10.2147/JIR.S497030

Figure Lengend Snippet: Effect of TPT-260 treatment on cell viability and membrane integrity in microglia and LPS/Nig-treated microglia. TPT-260 treatment did not affect cell viability ( A ) and did not cause cell damage ( B ). Data were analyzed using one-way ANOVA ( A and B ). The combined administration of LPS/Nig led to diminished microglial viability and damage to cell membrane integrity, whereas TPT-260 treatment effectively alleviated the impairment of cell activity induced by LPS/Nig administration ( C and D ). Data were analyzed using Student’s t -test (* p < 0.05 and *** p < 0.001).

Article Snippet: TPT-260 (CAS: 2076–91-7, HY-23769A, MedChemExpress) was initially dissolved in DMSO to a concentration of 20 mm.

Techniques: Membrane, Activity Assay

Journal: Journal of Inflammation Research

Article Title: Therapeutic Potential of TPT-260 in Ischemic Stroke: An Investigation Into Its Anti-Inflammatory Effects and Impact on Microglial Activation

doi: 10.2147/JIR.S497030

Figure Lengend Snippet: Effect of TPT-260 treatment on the inflammatory response of pro-inflammatory M1 microglia. TPT-260 treatment reduced LPS/Nig-induced inflammation formation (ACS speck) ( A ) and decreased the expression of pro-inflammatory genes Nlrp3, Tnf-α , and Il-1β in M1 microglia ( B and C ). Data were analyzed using two-way ANOVA (** p < 0.01 and *** p < 0.001).

Article Snippet: TPT-260 (CAS: 2076–91-7, HY-23769A, MedChemExpress) was initially dissolved in DMSO to a concentration of 20 mm.

Techniques: Expressing

Journal: Journal of Inflammation Research

Article Title: Therapeutic Potential of TPT-260 in Ischemic Stroke: An Investigation Into Its Anti-Inflammatory Effects and Impact on Microglial Activation

doi: 10.2147/JIR.S497030

Figure Lengend Snippet: Effect of TPT-260 treatment on the nuclear localization of NF-κB p65 in pro-inflammatory M1 microglia. TPT-260 treatment decreased the LPS/Nig-induced nuclear localization of NF-κB p65 in M1 microglia ( A and B ). TPT-260 treatment resulted in decreased levels of NLRP3, TLR4, phosphorylated IKKβ, phosphorylated p65, and IL-1β in LPS/Nig-treated microglia ( C ). Data were analyzed using two-way ANOVA (* p < 0.05).

Article Snippet: TPT-260 (CAS: 2076–91-7, HY-23769A, MedChemExpress) was initially dissolved in DMSO to a concentration of 20 mm.

Techniques:

Journal: Journal of Cell Science

Article Title: Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane

doi: 10.1242/jcs.218610

Figure Lengend Snippet: A pharmacological chaperone stimulates NHE3 trafficking to the apical membrane of human enteroids and increases NHE3 activity. (A) Differentiated human enteroid monolayers (5 days post Wnt3A removal) with higher TER than undifferentiated enteroids (1458±110 Ω cm2 vs 451±19 Ω cm2, ***P<0.001) were used. Na+/H+ exchange in human enteroid monolayers was measured using the pH-sensitive dye BCECF. A single donor was used for all experiments. Basal NHE3 transport activity was measured as the initial rates of Na+-dependent intracellular alkalinization in R55- (5 μM for 48 h) or vehicle-treated monolayers. A single experiment is shown above and below are shown means±s.e.m., n=4. P values are for the comparison between control and R55 treatment. (B) A differentiated human enteroid monolayer was treated with R55 (5 μM for 48 h) and the amount of NHE3 protein in the PM was determined by surface biotinylation. GAPDH was used as a loading control. A representative experiment is shown above and below are shown results as mean±s.e.m. (n=3 separate studies on monolayers each taken from a separate passage of the same cell line). *P<0.05. (C) NHE3 expression in human enteroid monolayers was detected by immunofluorescence (confocal microscopic) analysis. A differentiated monolayer was treated with R55 as above and stained for NHE3 (Alexa Fluro 488) and phosphorylated ezrin (Alexa Fluro 568). Each panel displays an xz projection; top panels are an xy projection, bottom panels are at the level of the apical PM. The quantification of the NHE3 intensity in control and R55 treated monolayers was performed with Volocity Software. The total NHE3 amount was not increased upon R55 treatment (lower left). The percentage of apical NHE3 in the microvillar region (stacks 1–5, each stack was 1 μm in the xz dimension and 20–25 μm total cell height) normalized to total NHE3 (whole image) expression was increased with R55 treatment (lower right). At least four separate images and ten random individual areas (z sections) were analyzed for each group. Scale bar: 10 µm. Results are mean±s.e.m., n=3. P values are comparison with untreated control.

Article Snippet: R55 TPT-260 (a thiophene thiourea derivative) was from Medkoo Biosciences (Morrisville, NC).

Techniques: Membrane, Activity Assay, Comparison, Control, Expressing, Immunofluorescence, Staining, Software

Journal: Journal of Cell Science

Article Title: Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane

doi: 10.1242/jcs.218610

Figure Lengend Snippet: A pharmacological chaperone rescues CT-dependent retromer destabilization and stimulates fluid absorption by intestinal epithelial cells. (A) Caco-2/bbe cells were treated with either CT (8 h or 48 h) or R55 (5 μM for 48 h) or both, in pre- (CT treatment before R55 treatment) or post-combinations (CT treatment after R55 treatment) for indicated periods of time, and levels of retromer proteins VPS26 and VPS35 were analyzed using western blot analysis. A representative blot is shown. (B) Quantitative analysis of the immunoblots in A. Levels of VPS26 and VPS35 were normalized to total levels of actin (n=4 per group). *P<0.05 versus control, #P<0.05 versus CT for 8 h; $P<0.05 versus CT for 48 h. (C) Polarized Caco-2/bbe-HA-NHE3 cells were treated with CT for indicated periods of time and the effect of R55 (5 μM for 48 h) on NHE3 surface expression was analyzed in a surface biotinylation assay. S-NHE3, surface NHE3; T-NHE3, total NHE3. Results from a single experiment are shown. (D) Quantitative analysis of western blots from four separate experiments, as in C, are shown as a bar graph. The amount of surface NHE3 was normalized to total NHE3 and expressed as percentage of control. Results are means±s.e.m. P values are in comparison with the control conditions related to the individual R55 experiments. (E) FIS assay in Calcein Green-labeled human duodenal enteroids exposed to forskolin and/or R55. Upper panel, a single example of a forskolin-stimulated enteroid. Lower panel, statistical evaluation of results from four separate experiments. Surface areas, expressed as percentage increase relative to that at t=0 (100%) are indicated on the y-axis. Forskolin-stimulated (5 μM) enteroids were pre-incubated with DMSO (0.2%) or R55 (5 μM for 48 h). The surface areas of 30 enteroids were measured in each group. Results are mean±s.e.m. with average from each experiment used as n=1. *P<0.05 versus control, #P<0.05 versus forskolin. (F) Role of NHE3 in the FIS assay response to forskolin and change mediated by R55. Upper panel, a representative blot showing expression of CFTR and NHE3 in 3D enteroids used for swelling assay. Arrows indicate B and C bands of CFTR. Lower panel, normalized forskolin-induced swelling of enteroids pretreated for 1 h with DMSO or the NHE3 inhibitor S3226 (20 μM) with or without R55 (5 μM for 48 h). Contribution of NHE3 was calculated as the change induced by S3226. Results are means±s.e.m. of at least three separate experiments. *P<0.05 versus control; #P<0.05 versus forskolin.

Article Snippet: R55 TPT-260 (a thiophene thiourea derivative) was from Medkoo Biosciences (Morrisville, NC).

Techniques: Western Blot, Control, Expressing, Surface Biotinylation Assay, Comparison, Labeling, Incubation