Journal: PLOS Pathogens

Article Title: Glutamine alleviates Staphylococcus aureus -induced mastitis by modulating macrophage polarisation

doi: 10.1371/journal.ppat.1014053

Figure Lengend Snippet: RAW264.7 cells were cultured in αKG-supplemented medium, treated with the STAT3 inhibitor Stattic, and subsequently stimulated with S. aureus . A: Protein expression levels of the JAK1/STAT3 signaling pathway and its negative regulator SOCS3 were detected in the Control, αKG, and αKG + S. au groups. B-I: Relative mRNA expression of M2 and M1 marker genes across the four experimental groups. J-L: Inflammatory cytokine levels and NF-κB signaling pathway activity in the Control, αKG, αKG + S. au , and αKG + S. au +Stattic groups. M: Expression levels of the JAK1/STAT3 signaling pathway across the four experimental groups. The data are presented as mean ± SD. One-way analysis of variance was performed for statistical analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicate significance from each group. ns, no significance.

Article Snippet: The specific primary antibodies used in the experiment included p65 (#8242, Cell Signaling Technology, USA), p-p65 (#3033, Cell Signaling Technology, America), IκB (AF5002, Affinity Biosciences, Jiangsu, China), p-IκB (AF2002, Affinity Biosciences, Jiangsu, China), ZO-1 (AF5145, Affinity Biosciences, Jiangsu, China), Occludin (DF7504, Affinity Biosciences, Jiangsu, China), Claudin-3 (AF0129, Affinity Biosciences, Jiangsu, China), ATG5 (bsm-52596R, Bioss, Woburn, MA, USA), LC3-I/LC3-II (AF5402, Affinity Biosciences, Jiangsu, China), p62 (ab91526, Abcam plc, Cam bridge, UK), SOCS3 (bs-0580R, Bioss, Woburn, MA, USA), JAK1 (bs-1439R , Bioss, Woburn, MA, USA), p-JAK1 (bs-3238R, Bioss, Woburn, MA, USA), STAT3 (AF6294, Affinity Biosciences, Jiangsu, China), p-STAT3 (AF3293, Affinity Biosciences, Jiangsu, China) and β-actin (T0022, Affinity Biosciences, Jiangsu, China).

Techniques: Cell Culture, Expressing, Control, Marker, Activity Assay

Journal: Journal of leukocyte biology

Article Title: Human β-defensin-2 enhances IFN-γ and IL-10 production and suppresses IL-17 production in T cells.

doi: 10.1189/jlb.0111004

Figure Lengend Snippet: Figure 6. hBD-2-induced expression of SOCS3 (A and C) and inhibi- tion of STAT3 phosphorylation (B). (A and B) T cells from a normal donor were transfected with siRNA against SOCS3 or control siRNA and stimulated with CD3/28 in the presence or absence of hBD-2 (20 g/ml) for 2 h; protein levels of SOCS3 in comparison with GAPDH (A) and of tyrosine-phosphorylated and total STAT3 (B) were ana- lyzed. (C) T cells from the same donor were incubated with 0.5 M JNK inhibitor I (JNK in), 10 M PD98059 (PD), or 0.1% DMSO for 30 min and then incubated with hBD-2 (20 g/ml) for 2 h and ana- lyzed for SOCS3 levels. The data represent four separate experiments performed using T cells from two normal donors and two psoriasis patients.

Article Snippet: A control siRNA and siRNA against SOCS3 were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Phospho-proteomics, Transfection, Control, Comparison, Incubation

Journal: Scientific Reports

Article Title: Cancer-Associated fibroblasts regulate the development of cholangiocarcinoma through IL-6/STAT3/AKR1C3 signaling axis

doi: 10.1038/s41598-026-37583-y

Figure Lengend Snippet: The molecular mechanism of STAT3 signaling pathway regulated by AKR1C3 in CCA. (A) After treatment with IL-6, the expression levels of proliferation, invasion, drug resistance, glycolysis, and STAT3 signaling-associated proteins were detected by Western blot in QBC939 cells. IL-6, 30 ng/ml. (B) The mRNA expression levels of the SOCSs family in QBC939 cells after AKR1C3 knockdown were detected by Q-PCR. * P < 0.05 (C) The protein expression levels of SOCS1 and SOCS3 in CCA cells after AKR1C3 knockdown and overexpression were measured by Western blot. (D) Western blot was performed to assess the ubiquitination degradation of SOCS1 in shAKR1C3- or shCtrl-transfected QBC939 cells treated with 50 µM cyclohexane (CHX) at different times. (E) Co-IP assay was conducted to investigate the protein interaction between AKR1C3 and SOCS1 in QBC939 cells.

Article Snippet: Antibodies against SOCS3 (68631 S) and PFK-1 (55028-1-AP) were obtained from Proteintech. (Hubei, China).

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modulation of AgRP-neuronal function by SOCS3 as an initiating event in diet-induced hypothalamic leptin resistance

doi: 10.1073/pnas.1218284110

Figure Lengend Snippet: HFD-induced SOCS3 up-regulation occurs in AgRP neurons before POMC and other hypothalamic cells. (A) Mice were fed with a LFD or HFD for 2 d or 2 wk. Fed animals were perfused and immunofluorescence analysis was performed to examine SOCS3 expression in the hypothalamus. POMC neurons are identified by ACTH immunoreactivity, and AgRP neurons are identified by GFP signals. (B) SOCS3 intensity was increased in AgRP neurons, but not in POMC neurons, in mice after 2 d of HFD compared with mice fed with a LFD. n = 4–5 mice per group. (C) The numbers of SOCS3-positive AgRP or POMC neurons were not altered in mice fed with a HFD for 2 d compared with mice fed LFD. However, the number of SOCS3-positive POMC neurons was increased significantly after 2 wk of HFD. n = 4–5 mice per group. (D) Number of SOCS3-positive cells outside the arcuate nucleus (ARC) was increased in mice fed with a HFD for 2 wk. n = 4–5 mice per group. *P ≤ 0.05 and **P ≤ 0.01 as determined by a one-way ANOVA with a Bonferroni post hoc test in C and D, and a Student t test was used to compare LFD and 2-d HFD in B. Data represent mean ± SEM. 3V: third ventricle.

Article Snippet: Sections were incubated with primary antibody against pSTAT3 (1:200; Cell Signaling), ACTH (1:400; National Hormone & Peptide Program), or albumin (1:250; Thermo Scientific Pierce) overnight at 4 °C or against SOCS3 (1:120; Novus Biologicals) for 36 h at 4 °C, washed, and incubated for 1 h at room temperature with a secondary goat anti-rabbit or goat anti-guinea pig IgG antibody (1:200; Invitrogen).

Techniques: Immunofluorescence, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modulation of AgRP-neuronal function by SOCS3 as an initiating event in diet-induced hypothalamic leptin resistance

doi: 10.1073/pnas.1218284110

Figure Lengend Snippet: Expression of SOCS3 is reversibly regulated in the AgRP neurons in response to acute changes of dietary conditions. (A) Female mice with Npy-GFP reporter were fed with a LFD, a HFD for 2 d, or a HFD for 2 d followed by a LFD for 1 d. Fed animals were perfused and immunofluorescence analysis was performed to examine SOCS3 expression in the hypothalamus. AgRP neurons are identified by GFP signals. (B) SOCS3 intensity was increased in AgRP neurons in mice fed a HFD for 2 d compared with mice fed a LFD. In mice fed a HFD for 2 d followed by 1 d of LFD, the SOCS3 intensity returned to levels comparable to in mice fed a LFD. n = 3–4 mice per group.*P ≤ 0.05 as determined by a one-way ANOVA with a Bonferroni post hoc test. n.s., not significant. Data represent mean ± SEM. 3V: third ventricle.

Article Snippet: Sections were incubated with primary antibody against pSTAT3 (1:200; Cell Signaling), ACTH (1:400; National Hormone & Peptide Program), or albumin (1:250; Thermo Scientific Pierce) overnight at 4 °C or against SOCS3 (1:120; Novus Biologicals) for 36 h at 4 °C, washed, and incubated for 1 h at room temperature with a secondary goat anti-rabbit or goat anti-guinea pig IgG antibody (1:200; Invitrogen).

Techniques: Expressing, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modulation of AgRP-neuronal function by SOCS3 as an initiating event in diet-induced hypothalamic leptin resistance

doi: 10.1073/pnas.1218284110

Figure Lengend Snippet: Transgenic SOCS3 up-regulation in AgRP neurons results in metabolic phenotypes mimicking those seen after short-term consumption of a high-fat diet. (A) Caloric intake during the light and dark cycle in 12-wk-old control mice (n = 16) and AgRP-SOCS3-OE mutant mice (n = 5) as measured by CLAMS. (B) Oxygen consumption was measured in 12-wk-old mice and the values were normalized to lean body mass of each mouse. Control mice n = 16, mutant mice n = 5. (C) Body weight of control mice (n = 20–74) and AgRP-SOCS3-OE mutant mice (n = 14–39) on chow diet at indicated ages. (D) Lean mass and fat mass as analyzed by DEXA in 25- to 35-wk-old control mice (n = 36) and AgRP-SOCS3-OE mutant mice (n = 21). (E) Weight of dissected fat pads from 30- to 40-wk-old control mice (n = 18) and AgRP-SOCS3-OE mutant mice (n = 9). (F) Plasma leptin levels under fed conditions for 30- to 42-wk-old control mice (n = 11) and AgRP-SOCS3-OE mutant mice (n = 9). (G) Eight-week-old control (n = 7) and mutant (n = 5) mice were injected with vehicle (saline) or leptin (2.5 mg/kg body weight), and 24-h food intake was measured. (H) Liver triglyceride in 30- to 40-wk-old control mice (n = 8) and AgRP-SOCS3-OE mutant mice (n = 8). (I and J) Blood glucose and plasma insulin after 6 h fast in 20- to 30-wk-old control mice (n = 9) and AgRP-SOCS3-OE mutant mice (n = 6). (K) AgRP-SOCS3-OE (n = 11) and control mice (n = 22) at 12 wk of age were fasted for 6 h, and injected with 2.5 g/kg glucose. Glucose levels were measured from tail blood using a glucometer. (L) Thirteen-week-old control mice (n = 17) and AgRP-SOCS3-OE mutant mice (n = 11) were fasted overnight, and injected with 1 U/kg insulin. Glucose levels were measured from tail blood using a glucometer. Data represent mean± SEM *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001 between control and mutant mice as determined by two-way repeated measures ANOVA (A–C, K, and L) and Student t tests (D–J).

Article Snippet: Sections were incubated with primary antibody against pSTAT3 (1:200; Cell Signaling), ACTH (1:400; National Hormone & Peptide Program), or albumin (1:250; Thermo Scientific Pierce) overnight at 4 °C or against SOCS3 (1:120; Novus Biologicals) for 36 h at 4 °C, washed, and incubated for 1 h at room temperature with a secondary goat anti-rabbit or goat anti-guinea pig IgG antibody (1:200; Invitrogen).

Techniques: Transgenic Assay, Control, Mutagenesis, Clinical Proteomics, Injection, Saline

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modulation of AgRP-neuronal function by SOCS3 as an initiating event in diet-induced hypothalamic leptin resistance

doi: 10.1073/pnas.1218284110

Figure Lengend Snippet: AgRP neurons situated outside the BBB are primary targets of SOCS3 up-regulation after short-term consumption of a HFD. (A) Transgenic mice expressing Npy-GFP were fed a HFD for 2 d or a matched LFD. Fed animals were perfused and immunofluorescence analysis was performed to examine SOCS3 (green) and albumin (red) expression in the hypothalamus. Npy-GFP (blue) was used to identify AgRP neurons, and albumin was used to identify cells outside the BBB. (B) Two days of HFD increased the SOCS3 intensity in AgRP neurons situated outside the BBB. In contrast, feeding a HFD for 2 d did not increase the SOCS3 intensity in AgRP neurons situated inside the BBB. n = 4–5 mice per group. *P ≤ 0.05 between LFD and 2 d HFD as determined by a two-way ANOVA with a Bonferroni post hoc test. n.s., not significant. Data represent mean ± SEM. 3V: third ventricle.

Article Snippet: Sections were incubated with primary antibody against pSTAT3 (1:200; Cell Signaling), ACTH (1:400; National Hormone & Peptide Program), or albumin (1:250; Thermo Scientific Pierce) overnight at 4 °C or against SOCS3 (1:120; Novus Biologicals) for 36 h at 4 °C, washed, and incubated for 1 h at room temperature with a secondary goat anti-rabbit or goat anti-guinea pig IgG antibody (1:200; Invitrogen).

Techniques: Transgenic Assay, Expressing, Immunofluorescence