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: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Schematic diagram of mice feeding and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 . b–n Male AMPKα fl/fl mice and LysM-Cre, AMPKα fl/fl mice with C57BL/6 background at the age of 6 weeks were fed HFD with or without IL-1β neutralizing antibody (1 mg/kg, twice one week) to explore the obesity development. Immunofluorescent staining of IL-1β in BAT and ScWAT ( b ), body weight gain ( c , n = 5 mice), relative fat and lean mass ( d , n = 5 mice), the weight of Liver ( e , n = 5 mice), BAT ( f , n = 5 mice), and ScWAT ( g , n = 5 mice), representative H&E staining of the liver, BAT and ScWAT ( h ), insulin tolerance test ( i , n = 5 mice), the rectal temperature in cold exposure at 4 °C for different times ( j , k , n = 5 mice), immunohistochemical staining of UCP-1 in BAT ( l ), the proinflammatory genes of ScWAT ( m , Il1b, Tnfa, Nos2, Ccl2 and F4/80: n = 5 mice in each group, Il6: n = 4 mice in LysM-Cre, AMPKα fl/fl + IL-1β mAb group and n = 5 mice in other group), and the immunohistochemical staining of F4/80 in BAT and ScWAT ( n ). Data are presented as the mean ± SEM, groups were compared by two-way ANOVA followed by Fisher’s LSD test ( c – g , i – k , m ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Immunohistochemical staining

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a–t Male LysM-Cre, IL-1β fl/fl mice, LysM-Cre, IL-1β fl/fl , AMPKα fl/fl mice, AMPKα fl/fl mice, IL-1β fl/fl mice, and LysM-Cre, AMPKα fl/fl mice with C57BL/6 background at the age of 8 weeks were fed HFD to explore the obesity and related phenotype. Body weight change ( a , n = 8 mice), body weight gain ( b , n = 8 mice), representative mice image ( c ), relative fat and lean mass ( d , n = 8 mice), the representative image of liver, BAT and ScWAT ( e ), the metabolic organ weight of liver ( f , n = 8 mice), BAT ( g , n = 8 mice) and ScWAT ( h , n = 8 mice), representative H&E staining of liver, BAT and ScWAT ( i ), insulin tolerance test ( j , n = 8 mice), the rectal temperature in cold exposure at 4 °C for different times ( k , l , n = 8 mice), immunohistochemical staining of UCP-1 in BAT ( m ), the proinflammatory genes of ScWAT ( n – s , n = 8 mice) and immunohistochemical staining of F4/80 in BAT and ScWAT ( t ). Data are presented as the mean ± SEM, groups were compared by one-way ANOVA followed by Fisher’s LSD test ( b , d , f – h , right of j , l , n – s ) or two-way ANOVA followed by Fisher’s LSD test ( a ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Immunohistochemical staining

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Schematic representation of the potential substrate of AMPK in succinate formation, and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 . Co-immunoprecipitation analysis of the interaction of AMPKα with SUCLA2 ( b ) or SUCLG2 ( c ) in RAW 264.7 cells. d Immunoblot analysis of indicated proteins in BMDMs that are transfected with siRNA for 30 h followed by stimulation with 100 ng/mL LPS for an additional 12 h. e Immunoblot analysis of indicated proteins in AMPKα fl/fl BMDMs (Flox) and LysM-Cre, AMPKα fl/fl BMDMs (MKO) that are transfected with siRNA for 18 h followed by stimulation with 100 ng/mL LPS for an additional 12 h. f Immunoblot analysis of the expression of SUCLG1 and SUCLA2 after AMPKα was knocked down by siRNA for 48 h in RAW 264.7 cells. g The relative enzymatic activity of SUCLA2 (the direction from succinyl-CoA to succinate) was detected after AMPKα was knocked down by siRNA for 36 h followed by stimulation with 100 ng/mL LPS for an additional 12 h in RAW264.7 cells ( n = 3 biological replicates). h In vitro phosphorylation analysis was performed by mixing purified His-CAMKKβ, His-AMPKα1β1γ1 and His-SUCLA2 in the presence of ATP-g-S, and immunoblot analysis of indicated proteins with indicated antibodies. i Venn diagram was used to integrate the phosphorylation site that was detected by mass spectrometry and predicted by GPS 5.0 ( http://gps.biocuckoo.cn/ ) or HPRD . j Protein sequence alignment indicated the conservation of SUCLA2 at Ser60 across multiple species. k Immunoblot analysis of indicated proteins in the in vitro kinase assay that mixed the purified His-CAMKKβ, His-AMPKα1β1γ1 with His-SUCLA2 (WT) or His-SUCLA2 (S60A). l Immunoblot analysis of indicated proteins in BMDMs after incubation with 200 µM A-769662 for 3 h. m In vitro phosphorylation analysis was performed by mixing purified His-CAMKKβ, His-AMPKα1β1γ1 and His-SUCLA2 (WT) or His-SUCLA2 (S60A) in the presence of ATP-g-S, and immunoblot analysis of indicated proteins with indicated antibodies. The enzymatic activity (the direction from succinyl-CoA to succinate) of purified WT-SUCLA2 ( n ) or S60A-SUCLA2 ( o ) after incubation with AMPK in vitro (n = 3 biological replicates). p The enzymatic activity (the direction from succinyl-CoA to succinate) of purified WT-SUCLA2, S60A-SUCLA2, and S60D-SUCLA2 in the same protein content (n = 4 biological replicates). q–r The relative gene expression of IL-1β in RAW264.7 cells that overexpress human WT-SUCLA2, S60A-SUCLA2 and S60D-SUCLA2 through lentivirus infection for 48 h followed the treatment by 100 ng/mL LPS for 6 h in the condition of glutamine replete ( q ) or deprived ( r ) condition (n = 4 biological replicates). The relative gene expression of IL-1β in RAW264.7 cells that overexpress human WT-SUCLA2 with S60A-SUCLA2 ( s ) or S60D-SUCLA2 ( t ) through lentivirus infection for 48 h, the cells were treated with 200 µM A-769662 for 3 h in advance followed by 100 ng/mL LPS for 6 h (n = 4 biological replicates). Data are presented as the mean ± SEM, groups were compared by the unpaired two-tailed Student’s t test (g) or one-way ANOVA followed by Bonferroni’s multiple-comparisons test ( q , r ) or two-way ANOVA followed by Bonferroni’s multiple-comparisons test ( n , o , p , s , t ), representative data are shown from one of the three independent experiments ( b – f , h , k – m ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Activity Assay, In Vitro, Phospho-proteomics, Purification, Mass Spectrometry, Sequencing, Kinase Assay, Incubation, Gene Expression, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Immunofluorescent staining of CD68, pT 172 -AMPK, pS 60 -SUCLA2 and IL-1β in the subcutaneous adipose tissue from lean (BMI = 21.5), overweight (BMI = 25.7) or obese (BMI = 32.0) subjects, representative data are shown from one of the three independent experiments. b Model of how macrophage AMPK responds to glutaminolysis and regulates glutaminolysis-coupled IL-1β expression, and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 .

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Expressing

Journal: Biology of Sex Differences

Article Title: Age and sex dependent effects of early overnutrition on metabolic parameters and the role of neonatal androgens

doi: 10.1186/s13293-016-0079-5

Figure Lengend Snippet: The limit of detection and intra- and inter-assay coefficients (CV) of variation for the hormone and cytokine assays

Article Snippet: Assay-on-demand kits (Applied Biosystems) were used to assess the mRNA levels of leptin (Rn00565158_m1), adiponectin (Rn00595250_m1), IL6 (Rn01410330_m1), IL1β (Rn01336189_m1), and TNFα (Rn01525859_g1) quantitative real-time PCR according to the manufacturer’s protocol and analyzed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

Techniques: Inter Assay, Intra Assay

Journal: Biology of Sex Differences

Article Title: Age and sex dependent effects of early overnutrition on metabolic parameters and the role of neonatal androgens

doi: 10.1186/s13293-016-0079-5

Figure Lengend Snippet: Glycemia and serum hormone and cytokine levels at postnatal days (PND) 10, 21, 30, 50, 85, and 150 in male (M) and female (F) rats raised in litters (L) 4 or 12 pups

Article Snippet: Assay-on-demand kits (Applied Biosystems) were used to assess the mRNA levels of leptin (Rn00565158_m1), adiponectin (Rn00595250_m1), IL6 (Rn01410330_m1), IL1β (Rn01336189_m1), and TNFα (Rn01525859_g1) quantitative real-time PCR according to the manufacturer’s protocol and analyzed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

Techniques:

Journal: Biology of Sex Differences

Article Title: Age and sex dependent effects of early overnutrition on metabolic parameters and the role of neonatal androgens

doi: 10.1186/s13293-016-0079-5

Figure Lengend Snippet: Relative mRNA levels of cytokines in inguinal and perigonadal adipose tissue at postnatal days (PND) 10, 21, 30, 50, 85, and 150 in male (M) and female (F) rats raised in litters (L) 4 or 12 pups

Article Snippet: Assay-on-demand kits (Applied Biosystems) were used to assess the mRNA levels of leptin (Rn00565158_m1), adiponectin (Rn00595250_m1), IL6 (Rn01410330_m1), IL1β (Rn01336189_m1), and TNFα (Rn01525859_g1) quantitative real-time PCR according to the manufacturer’s protocol and analyzed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

Techniques:

Journal: Biology of Sex Differences

Article Title: Age and sex dependent effects of early overnutrition on metabolic parameters and the role of neonatal androgens

doi: 10.1186/s13293-016-0079-5

Figure Lengend Snippet: Metabolic changes in males, females, and females that were androgenized (AF) on postnatal day (PND) 1. At PND10, there were significant differences in a the percentage of inguinal adipose tissue (IngAT) and serum b leptin, c adiponectin, and d interleukin (IL) 6 levels. At PND90, there were significant differences in e body weight, f glycemia and serum, g insulin, h leptin and i IL1β levels. The mRNA levels of j leptin, k adiponectin, l IL6, and m TNFα in perigonadal adipose tissue at PND90 were also affected. # p < 0.0001, ## p < 0.002, ### p < 0.0002, * p < 0.01, *** p < 0.005, @ p < 0.05. PND10: inguinal adipose tissue: N = 10; serum leptin and IL6 levels: N = 6; serum adiponectin: M and F: N = 8, AF: N = 9. PND90: body weight and glycemia: M: N = 8, F: N = 12, AF: N = 11; serum insulin, leptin, and Il1β levels: N = 6; mRNA levels for leptin, adiponectin, IL6, and TNFα: N = 6

Article Snippet: Assay-on-demand kits (Applied Biosystems) were used to assess the mRNA levels of leptin (Rn00565158_m1), adiponectin (Rn00595250_m1), IL6 (Rn01410330_m1), IL1β (Rn01336189_m1), and TNFα (Rn01525859_g1) quantitative real-time PCR according to the manufacturer’s protocol and analyzed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

Techniques: