Journal: Journal of cellular signaling

Article Title: Chronic IL-1 Exposed AR + PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles

doi:

Figure Lengend Snippet: Parental MDA-PCa-2b (MDA2b) and chronic IL-1 subline cells (MDA-αs1, MDA-αs2, MDA-βs1, MDA-βs2) were treated acutely for 3 days (A, C) or 6 days (B) with vehicle control (V), 25 ng/ml IL-1α (a), or 25 ng/ml IL-1β (b) and analyzed for cell viability using MTT (A, B) or protein accumulation by western blot (C). Acute IL-1 exposure reduces cell viability and proliferation, reduces full-length PARP (indicative of cell death activation), induces SOD2 and LCN2 protein accumulation (canonical IL-1-induced genes), and reduces AR and NKX3.1 (canonical AR target gene) protein accumulation in MDA-PCa-2b parental cells, but has little to no effect on the chronic IL-1 sublines. Thus, the IL-1 sublines evolved insensitivity to IL-1. Error bars, ± STDEV of 4 biological replicates; p-value, *≤ 0.05, **≤ 0.005, ***≤ 0.005, NS = not significant. Fold MTT optical density (OD) is normalized to treatment control. β-actin is the western blot loading control.

Article Snippet: MDA-PCa-2b cells were maintained in HPC1/20% FB Essence (FBE) containing 0.5 ng/ml IL-1α (Gold Bio, St. Louis, MO; 1110–01A-10) or IL-1β (Gold Bio, St. Louis, MO; 1110–01B-10) for approximately 4 months; and the proliferative colonies that emerged were expanded and termed MDA-PCa-2b IL-1α subline (MDA-αs) and MDA-PCa-2b IL-1β subline (MDA-βs).

Techniques: Western Blot, Activation Assay

Journal: Journal of cellular signaling

Article Title: Chronic IL-1 Exposed AR + PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles

doi:

Figure Lengend Snippet: (A) MDA-PCA-2b parental (MDA2b) and chronic IL-1 subline (MDA-αs1, MDA-αs2, MDA-βs1, MDA-βs2) cells were treated acutely for 3 days with vehicle control, 25 ng/ml IL-1α, or 25 ng/ml IL-1β and analyzed by RT-qPCR for mRNA levels of the IL-1 receptor, IL-1R1 . Acute IL-1 exposure does not increase IL-1 receptor ( IL-1R1 ) mRNA levels in parental cells, suggesting basal IL-1R1 levels are sufficient to mediate IL-1 signaling. Furthermore, IL-1 does not show a differential effect on IL-1R1 mRNA levels in parental versus subline cells, suggesting subline insensitivity is independent of IL-1R1 levels. (B) Vehicle control treated cells were compared for basal mRNA levels of IL-1R1 and of canonical IL-1-induced genes, LCN2 , NOX1 , and SOD2 . IL-1R1, LCN2 , NOX1 , and SOD2 basal mRNA levels are comparable across the parental and subline cells, suggesting chronic IL-1 exposure does not induce constitutive activation of canonical IL-1 intracellular signaling. These data suggest that MDA-PCa-2b cell lines evolve insensitivity to exogenous chronic IL-1 exposure independent of IL-1R1 levels or constitutive activation of intracellular IL-1 signaling. Error bars, ± STDEV of 3 biological replicates; p-value, *≤ 0.05, **≤ 0.005, ***≤ 0.005, NS = not significant. For IL-1-treated cells, mRNA levels are normalized to vehicle control for each cell line. For basal expression, mRNA levels are normalized to the parental cell line.

Article Snippet: MDA-PCa-2b cells were maintained in HPC1/20% FB Essence (FBE) containing 0.5 ng/ml IL-1α (Gold Bio, St. Louis, MO; 1110–01A-10) or IL-1β (Gold Bio, St. Louis, MO; 1110–01B-10) for approximately 4 months; and the proliferative colonies that emerged were expanded and termed MDA-PCa-2b IL-1α subline (MDA-αs) and MDA-PCa-2b IL-1β subline (MDA-βs).

Techniques: Quantitative RT-PCR, Activation Assay, Expressing

Journal: Journal of cellular signaling

Article Title: Chronic IL-1 Exposed AR + PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles

doi:

Figure Lengend Snippet: Parental (MDA-PCA-2b (MDA2b), LNCaP) and subline (MDA-αs1, MDA-αs2, MDA-βs1, MDA-βs2, LNas1, LNbs1) cells were treated acutely for 3 days with vehicle control, 25 ng/ml IL-1α, or 25 ng/ml IL-1β and analyzed by RT-qPCR for mRNA levels (A, B, C). Acute IL-1 exposure increases LCN2 , NOX1 , and SOD2 mRNA levels in parental MDA-PCa-2b and LNCaP cells, but acute IL-1 exposure has attenuated or no effect on mRNA levels in the subline cells. Thus, both LNCaP and MDA-PCa-2b cell lines show conserved intracellular response to acute IL-1-induced changes mRNA levels and evolve chronic IL-1 insensitivity independent of constitutive canonical IL-1 intracellular signaling. Error bars, ± STDEV of 3 biological replicates; p-value, *≤ 0.05, **≤ 0.005, ***≤ 0.005, NS = not significant. For IL-1-treated cells, mRNA levels are normalized to vehicle control for each cell line.

Article Snippet: MDA-PCa-2b cells were maintained in HPC1/20% FB Essence (FBE) containing 0.5 ng/ml IL-1α (Gold Bio, St. Louis, MO; 1110–01A-10) or IL-1β (Gold Bio, St. Louis, MO; 1110–01B-10) for approximately 4 months; and the proliferative colonies that emerged were expanded and termed MDA-PCa-2b IL-1α subline (MDA-αs) and MDA-PCa-2b IL-1β subline (MDA-βs).

Techniques: Quantitative RT-PCR

Journal: iScience

Article Title: A double-edged sword role of IFN-γ-producing iNKT cells in sepsis: Persistent suppression of Treg cell formation in an Nr4a1-dependent manner

doi: 10.1016/j.isci.2024.111462

Figure Lengend Snippet:

Article Snippet: Mouse IL-4 Recombinant Protein , MedChemExpress , Cat# HY-P70653.

Techniques: Control, Virus, Recombinant, Staining, Activation Assay, Cell Isolation, Software

Journal: Investigative ophthalmology & visual science

Article Title: The Effect of A2A Receptor Antagonist on Microglial Activation in Experimental Glaucoma.

doi: 10.1167/iovs.15-18024

Figure Lengend Snippet: FIGURE 5. Changes of microglial morphology and inflammatory mediators in the rat retinae with or without ZM241385 injection 2 weeks after the induction of COH. (A) The morphologic and quantity change of the microglia in different groups. In the COH rats, the amount of round or ameboid microglia in the retina decreased following intravitreal injection of ZM241385 (COHþZM) than that following the vehicle injection (COHþvehicle) and that without any injection (COH). The microglia extended 1 to 2 thick and short processes in the group of COHþZM, but still less ramified than that in the CTRL. White arrows pointed to microglia. Scale bar: 50 lm. (B) Changes of the mRNA expressions of TNF-a and IL-1b in retinae of the different groups. (C) Changes of the protein expressions of TNF-a, IL-1b, and Iba-1 in retinae of the different groups. *P < 0.05 represents comparison to CTRL group; #P < 0.05 represents comparison to COHþvehicle group. Each panel represents the average value. Error bar: mean 6 SD.

Article Snippet: Conditioned media collected from microglia treated with glutamate and ZM241385 for 24 hours was assayed for soluble TNF-a and IL-1b using ELISA kit (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China) according to the manufacturer’s instructions.

Techniques: Injection, Comparison

Journal: Investigative ophthalmology & visual science

Article Title: The Effect of A2A Receptor Antagonist on Microglial Activation in Experimental Glaucoma.

doi: 10.1167/iovs.15-18024

Figure Lengend Snippet: FIGURE 7. Tumor necrosis factor-a and IL-1b secretion of the cultured microglia cells after treatment with glutamate and ZM241385. *P < 0.05 represents comparison to ctrl group; #P < 0.05 represents comparison to Glu1 group. Ctrl, control. Glu0.1, Glu1, Glu10 represent the culture media containing 0.1, 1, and 10 mM glutamate, respectively, Glu1þzm0.5 represents the culture media containing 1 mM glutamate and 0.5 lM ZM241385; zm0.5 represents the culture media containing 0.5lM ZM241385. Each panel represents the average. Error bar: mean 6 SD.

Article Snippet: Conditioned media collected from microglia treated with glutamate and ZM241385 for 24 hours was assayed for soluble TNF-a and IL-1b using ELISA kit (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China) according to the manufacturer’s instructions.

Techniques: Cell Culture, Comparison, Control

Journal: Experimental Biology and Medicine

Article Title: Mechanisms of AGE-induced VSMC phenotypic switching and macrophage modulation in human abdominal aortic aneurysms

doi: 10.3389/ebm.2025.10527

Figure Lengend Snippet: AGEs Activate the NF-κB Pathway and NLRP3 Inflammasome via the RAGE/RhoA/ROCK Pathway. (A) The TransAM NF-κB Transcription Factor Assay detected the effect of the RAGE/RhoA/ROCK inhibitor on the nuclear translocation of c-Rel, p52, and p65; (B) The NF-κB dual luciferase reporter gene system assessed the impact of RAGE/RhoA/ROCK inhibitors on AGE-induced NF-κB signaling; (C) Western blot analysis showed that AGEs regulate the expression of NLRP3 via the RAGE/RhoA/ROCK/NF-κB signaling pathway; (D) The effects of the inhibitors on caspase-1 activation were assessed using a caspase-1 assay kit; (E) An LDH release assay showed that AGE promoted VSMC cell pyroptosis, but it could be reversed by RAGE, RhoA, and ROCK inhibitors; (F) An ELISA assay demonstrated that AGE promoted the release of the inflammatory factor IL-1β through RAGE/RhoA/ROCK. Data are presented as the mean ± SD from three independent experiments and were analyzed with a one-way ANOVA followed by a Dunnett’s multiple comparisons test. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) between the treatment group and the control group. Pound signs indicate a significant difference (#P < 0.05, ##P < 0.01, ###P < 0.001) between the treatment group and the AGE group.

Article Snippet: The enzymatic activity of caspase-1 was assayed using a Caspase-1 Colorimetric Assay Kit (MCE, Cat. No. HY-K2610-100T) according to the manufacturer’s protocol.

Techniques: Transcription Factor Assay, Translocation Assay, Luciferase, Western Blot, Expressing, Activation Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Control