Journal: Journal of Innate Immunity

Article Title: Neutrophil Killing of Peptoanaerobacter stomatis Requires TAK1-Driven Degranulation

doi: 10.1159/000553300

Figure Lengend Snippet: Activation of kinases in P. stomatis (Ps)- challenged neutrophils: Human neutrophils (10 × 10 6 /mL) were either left untreated (basal) or challenged with Ps at an MOI 10 for 5, 15, 30, 60, and 90 min. Cell lysates were analyzed by immunoblotting using phospho-specific antibodies and subsequently reprobed for total protein or β-actin as loading controls. Representative immunoblots showing phosphorylation kinetics ( a , e ) and densitometric analysis of ERK1/2, p38, AKT, and Src and Syk ( b , c , d , f , g ) are shown. Densitometric analyses of phosphorylated kinases were normalized to their respective total protein levels, except for p-TAK1, which was normalized to β-actin. Data represents the mean ± SEM of three independent experiments, and statistical significance was determined by one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001). MOI, multiplicity of infection.

Article Snippet: The following inhibitors were used: diphenyleneiodonium (DPI; 10 μ m ; NOX2 inhibitor; Sigma-Aldrich), 5Z-7-oxozeaenol (1 μ m ; TAK1 inhibitor), SB203580 (10 μ m ; p38 MAPK inhibitor; MedChemExpress), U0126 (10 μ m ; ERK inhibitor; Calbiochem), LY294002 (10 μ m ; PI3K inhibitor; Calbiochem), piceatannol (10 μ m ; Syk inhibitor; Cayman Chemical), Src-1 (10 μ m ; Src family kinase inhibitor; Cayman Chemical), and GSK484 (10 μ m ; PAD4 inhibitor; Cayman Chemical).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Infection

Journal: Journal of Innate Immunity

Article Title: Neutrophil Killing of Peptoanaerobacter stomatis Requires TAK1-Driven Degranulation

doi: 10.1159/000553300

Figure Lengend Snippet: TAK1 acts as a critical regulator for kinase activation during P. stomatis (Ps) challenge: Human neutrophils (10 × 10 6 /mL) were either left untreated (basal) or challenged with Ps for 30 min after the indicated inhibitor pre-treatment. Cell lysates were analyzed by immunoblotting using phospho-specific antibodies and subsequently reprobed for total protein or β-actin as loading controls. Representative immunoblots and densitometric analysis of ERK1/2 ( a ), p38 ( b ), AKT ( c ), Src ( d ), and Syk ( e ) are shown. Densitometric analyses of phosphorylated kinases were normalized to β-actin. Data represent the mean ± SEM of three independent experiments, and statistical significance was determined by one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The following inhibitors were used: diphenyleneiodonium (DPI; 10 μ m ; NOX2 inhibitor; Sigma-Aldrich), 5Z-7-oxozeaenol (1 μ m ; TAK1 inhibitor), SB203580 (10 μ m ; p38 MAPK inhibitor; MedChemExpress), U0126 (10 μ m ; ERK inhibitor; Calbiochem), LY294002 (10 μ m ; PI3K inhibitor; Calbiochem), piceatannol (10 μ m ; Syk inhibitor; Cayman Chemical), Src-1 (10 μ m ; Src family kinase inhibitor; Cayman Chemical), and GSK484 (10 μ m ; PAD4 inhibitor; Cayman Chemical).

Techniques: Activation Assay, Western Blot

Journal: Journal of Innate Immunity

Article Title: Neutrophil Killing of Peptoanaerobacter stomatis Requires TAK1-Driven Degranulation

doi: 10.1159/000553300

Figure Lengend Snippet: P. stomatis evades TAK1-dependent ROS- and NET-mediated killing but not degranulation. For the killing assay, neutrophils were pretreated for 15 min with inhibitors followed by P. stomatis incubation for 0 and 3 h. Then, neutrophil cell pellets (for cell-associated) and supernatant (for extracellular) were used to determine and quantify the CFU, which was quantified for percent killing after 1 µ m of 5Z-7-oxozeaenol ( a ), 10 µ m of DPI ( b ), GSK484 ( c ), or 10 µ m SB203580 pre-treatment. d P. stomatis was exposed to conditioned media collected from either unstimulated (US) or latrunculin +fMLF (L+F) stimulated neutrophils for 3 h and CFU enumeration was performed. Percent killing was determined by quantifying P. stomatis exposed to stimulated conditioned media over unstimulated condition. Data represents the mean ± SEM of three independent experiments, and statistical significance was determined by unpaired t -test for each pair (* p < 0.05, ** p < 0.01). ns, non-significant.

Article Snippet: The following inhibitors were used: diphenyleneiodonium (DPI; 10 μ m ; NOX2 inhibitor; Sigma-Aldrich), 5Z-7-oxozeaenol (1 μ m ; TAK1 inhibitor), SB203580 (10 μ m ; p38 MAPK inhibitor; MedChemExpress), U0126 (10 μ m ; ERK inhibitor; Calbiochem), LY294002 (10 μ m ; PI3K inhibitor; Calbiochem), piceatannol (10 μ m ; Syk inhibitor; Cayman Chemical), Src-1 (10 μ m ; Src family kinase inhibitor; Cayman Chemical), and GSK484 (10 μ m ; PAD4 inhibitor; Cayman Chemical).

Techniques: Incubation

Journal: Poultry Science

Article Title: Gut microbiota-derived taurolithocholic acid modulates myofiber-type switching via p38 MAPK/PGC-1α signaling underlying breed differences between Arbor Acres and Taoyuan chickens

doi: 10.1016/j.psj.2026.106914

Figure Lengend Snippet: TLCA regulates mitochondrial biogenesis and altered myofiber type composition through the p38 MAPK/PGC-1α signaling pathway. A Cell viability of cells treated with different concentrations of TLCA for 24 h (n = 10). B & F The mRNA expression of MYH1A and MYH7B (n = 6). C & G Intracellular enzymatic activities of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH), ATP content and mitochondrial DNA (mtDNA) content (n = 6). D & H The mRNA expression of HK1, GPI, PGAM1, PGK1, PYGL, PGC-1α, NRF1 and TFAM (n = 6). E The protein expression of PGC-1α, p38 MAPK and phosphorylation p38 MAPK (n = 3). I The protein expression of PGC-1α (n = 3). Data were shown as mean ± SD; *Represents significant difference when compared with 0 μM TLCA or CON group, and # represents significant difference when compared with TLCA group. *& # P < 0.05, ⁎⁎ & ## P < 0.01, ⁎⁎⁎ & ### P < 0.001.

Article Snippet: The differentiation medium containing TLCA and p38 MAPK inhibitor SB203580 (152121-47-6, MedChemExpress, Shanghai, China) was added to CPMs after 4 days of differentiation, and the cells were collected after 24 h.

Techniques: Expressing, Phospho-proteomics

Journal: Hepatic Oncology

Article Title: RTA‑408 induces p38‑dependent apoptosis and suppresses cell viability in hepatocellular carcinoma cells

doi: 10.1080/20450923.2026.2659967

Figure Lengend Snippet: RTA-408 activates p38–NRF2 signaling and promotes LC3B accumulation in hepatocellular carcinoma cells. HepG2 and PP5 cells were treated with RTA-408 (0, 200, 400, or 600 nM) for 24 h, and protein expression was analyzed by western blotting. Representative immunoblots show phosphorylated p38 (p-p38), total p38, phosphorylated ERK1/2 (p-ERK), total ERK1/2, phosphorylated JNK (p-JNK), total JNK, NRF2, LC3B, p62, cleaved caspase-3 (c-cas3), and β-actin. Densitometric analyses of p-p38/p38, p-ERK/ERK, and p-JNK/JNK, as well as NRF2, LC3B, p62, and c-cas3 normalized to β-actin, are shown in the accompanying bar graphs. RTA-408 increased p38 phosphorylation and NRF2 expression in both cell lines and was accompanied by marked accumulation of LC3B and p62, while ERK and JNK responses differed between HepG2 and PP5 cells. Cleaved caspase-3 increased at higher concentrations of RTA-408. β-Actin was used as a loading control. Data are presented as mean ± SEM from three independent experiments ( n = 3). *P < 0.05 and **P < 0.01 versus control.

Article Snippet: RTA-408 (omaveloxolone) (MCE; 1474034-05-3; USA) and the p38 MAPK inhibitor, SB203580 (MCE; 152121-47-6; USA), were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions (10 mM for RTA-408 and 10 mM for SB203580), which were stored at − 20 °C according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Phospho-proteomics, Control

Journal: Hepatic Oncology

Article Title: RTA‑408 induces p38‑dependent apoptosis and suppresses cell viability in hepatocellular carcinoma cells

doi: 10.1080/20450923.2026.2659967

Figure Lengend Snippet: p38 inhibition attenuates RTA-408-induced p38 signaling, LC3B/p62 accumulation, and apoptotic protein activation in hepatocellular carcinoma cells. HepG2 and PP5 cells were treated for 24 h with vehicle control (Con), RTA-408 (400 nM) alone, or RTA-408 (400 nM) plus SB203580 (10 μM). Protein expression was analyzed by western blotting. Representative immunoblots show phosphorylated p38 (p-p38), total p38, NRF2, LC3B, p62, cleaved caspase-3 (c-cas3), and β-actin. Densitometric analyses of p-p38/p38, as well as NRF2, LC3B, p62, and c-cas3 normalized to β-actin, are shown in the accompanying bar graphs. In both HepG2 and PP5 cells, RTA-408 increased p38 phosphorylation, NRF2 expression, LC3B accumulation, p62 accumulation, and cleaved caspase-3 levels, whereas co-treatment with SB203580 markedly attenuated these RTA-408-induced effects without altering total p38 expression. These findings indicate that p38 activity contributes to RTA-408-induced stress signaling, LC3B/p62-associated responses, and apoptotic protein activation in hepatocellular carcinoma cells. Data are presented as mean ± SEM from three independent experiments ( n = 3). *P < 0.05 and **P < 0.01 versus control; #P < 0.05 and ##P < 0.01 versus RTA-408 alone.

Article Snippet: RTA-408 (omaveloxolone) (MCE; 1474034-05-3; USA) and the p38 MAPK inhibitor, SB203580 (MCE; 152121-47-6; USA), were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions (10 mM for RTA-408 and 10 mM for SB203580), which were stored at − 20 °C according to the manufacturer’s instructions.

Techniques: Inhibition, Activation Assay, Control, Expressing, Western Blot, Phospho-proteomics, Activity Assay

Journal: Hepatic Oncology

Article Title: RTA‑408 induces p38‑dependent apoptosis and suppresses cell viability in hepatocellular carcinoma cells

doi: 10.1080/20450923.2026.2659967

Figure Lengend Snippet: Pharmacological inhibition of p38 MAPK partially restores cell viability and attenuates RTA-408-induced apoptosis in hepatocellular carcinoma cells. (A) HepG2 and PP5 cells were treated for 24 h with vehicle control (Con), RTA-408 (400 nM) alone, or RTA-408 (400 nM) plus SB203580 (10 μM), and cell viability was assessed by the MTT assay. Bar graphs show the percentage of viable cells relative to the control group. (B) Under the same treatment conditions, apoptosis was evaluated by Annexin V/7-AAD flow cytometry. Representative dot plots show the distribution of viable (Annexin V − /7-AAD − ), early apoptotic (Annexin V + /7-AAD − ), late apoptotic (Annexin V + /7-AAD + ), and necrotic (Annexin V − /7-AAD + ) cell populations. The accompanying bar graphs summarize total apoptosis (early + late apoptosis). In both cell lines, RTA-408 reduced cell viability and increased apoptosis, whereas co-treatment with SB203580 partially reversed the loss of viability and attenuated the pro-apoptotic effect of RTA-408. Data are presented as mean ± SEM from three independent experiments ( n = 3). Statistical significance was determined by one-way analysis of variance with appropriate post hoc testing. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control; ##P < 0.01 versus RTA-408 alone.

Article Snippet: RTA-408 (omaveloxolone) (MCE; 1474034-05-3; USA) and the p38 MAPK inhibitor, SB203580 (MCE; 152121-47-6; USA), were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions (10 mM for RTA-408 and 10 mM for SB203580), which were stored at − 20 °C according to the manufacturer’s instructions.

Techniques: Inhibition, Control, MTT Assay, Flow Cytometry