Journal: Poultry Science

Article Title: CSF3 enhances the innate immune responses to ALV-J infections via NF-κB and interferon pathways

doi: 10.1016/j.psj.2025.105648

Figure Lengend Snippet: CSF3 enhances the immune response to ALV-J via the NFκB signaling pathway. (A) Western blot analysis of p52, p100, phosphorylated p65 (p-p65), IκBα, and phosphorylated IκBα (p-IκBα) protein following CSF3 overexpression in DF-1 cells. (B) Quantification of protein levels in (A) based on relative grayscale values. (C) Western blot analysis of p52, p100, phosphorylated p65 (p-p65), IκBα, and phosphorylated IκBα (p-IκBα) protein following CSF3 knockdown in DF-1 cells. (D) Quantification of protein levels in (C) based on relative grayscale values. (D, E) RT-qPCR analysis of TNF-α, IL-1β , and IL-6 mRNA expression following CSF3 overexpression (D) or knockdown (E) in DF-1 cells. (F, G) ELISA measurement of TNF-α, IL-1β, and IL-6 protein levels following CSF3 overexpression (F) or knockdown (G) in DF-1 cells. (H, I) RT-qPCR analysis of TNF-α, IL-1β , and IL-6 mRNA expression following CSF3 overexpression (H) or knockdown (I) in CEF cells. (J, K) ELISA measurement of TNF-α, IL-1β, and IL-6 protein levels following CSF3 overexpression (J) or knockdown (K) in CEF cells. (L) Western blot analysis of STAT3, phosphorylated STAT3 (p-STAT3), env, β-actin, IκBα, p-IκBα, p-p65, p52, and p100 after STAT3 phosphorylation inhibition in CSF3-overexpressing DF-1 cells. (M) Quantification of protein levels in (L) based on relative grayscale values. Statistical significance was determined using a two-tailed unpaired Student’s t-test ( p < 0.05). * p < 0.05, **p < 0.01, *** p < 0.001.

Article Snippet: Mouse Anti-ALV-J envelope protein JE9 (kindly provided by Prof. Aijian Qin, Yangzhou University, Yangzhou, China), Rabbit Anti-STAT3 antibody (bs-1141R; Boss, China; 1:1000), Rabbit Anti-phospho-STAT3 (Ser727) antibody (bs-3429R; Boss, China; 1:1000), Rabbit Anti-NFκB2 antibody (10037P, Boss, China; 1:1000), Rabbit Anti-NFκB p-p65 (bs-0982R, Boss, China; 1:1000), Rabbit Anti-IKBα Rabbit (10268-1-AP, Proteintech, USA; 1:1000), Anti-p-IKBα (bs-2513R, Boss, China; 1:1000), and goat anti-rabbit IgG/HRP (bs13278), goat anti-mouse IgG/HRP (bs12478), goat Anti-Mouse IgG ( H + L ) FITC (bs10950) secondary antibody were purchased from Bioss (Beijing, China).

Techniques: Western Blot, Over Expression, Knockdown, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Inhibition, Two Tailed Test

Journal: CNS neuroscience & therapeutics

Article Title: 11,12-Diacetyl-Carnosol Ameliorates Depression-Like Behaviors and Memory Dysfunction in CUMS Mouse Model via Inhibiting HMGB1-Mediated Neuroinflammation.

doi: 10.1111/cns.70406

Figure Lengend Snippet: FIGURE 2 | DACA reverses microglial activation, neuronal damage, and dendritic spine loss, and decreases the expression of HMGB1, NF-κB, and NLRP3 in the hippocampus of CUMS-induced mice. (A and B) Expression of IBA1 and MAP2 in the hippocampal CA1 region of mice. Scale bar, 20 μm. n = 3. (C) Expression levels of IL-6 and TNF-α in mouse serum. n = 3. (D and E) Dendritic spine density in the hippocampus of mice. Scale bar, 20 or 10 μm. n = 3. (F and G) Protein expression of MAP2, PSD95, and BDNF in the hippocampus of mice. n = 4. (H–I) Expression levels of HMGB1, NF-κB, NLRP3, IL-1β, and NRF2 in the hippocampus of mice. n = 4. #p < 0.5, ##p < 0.1, ###p < 0.01, ####p < 0.001, vs. control group; *p < 0.5, **p < 0.1, ***p < 0.01, vs. CUMS group.

Article Snippet: For western blot analysis and immunofluorescence, we used the following antibodies: anti- Nrf2 antibody (1: 3000, Proteintech, 66,009- 1- IG), anti- HMGB1 antibody(1: 1000, Abcam, ab18256), anti- P- NF- κB antibody(1: 1000, Cell Signaling Technology, 3033S), anti- NF- κB p65 (D14E12) antibody (1: 1000, Cell Signaling Technology, 8242), anti- NLRP3 antibody (1: 1000, Cell Signaling Technology, 15,101), anti- IL- 1β antibody (1: 1000, Affinity, AF5103), anti- BDNF antibody (1: 1000, Cell Signaling Technology, 47,808), anti- Nrf2 antibody (1: 3000, Proteintech, 16,396- 1- AP), anti- IBA- 1 antibody (1: 1000, Abcam, ab178846), anti- MAP2 antibody (1: 1000, Abcam, ab11267), anti- PSD95 antibody (1: 1000, Abcam, ab18258), goat anti- rabbit IgG H&L (HRP) (1: 5000, Abcam, ab6721), goat anti- mouse IgG H&L (HRP) (1: 5000, Abcam, ab6789), goat anti- mouse IgG (H+L) cross- adsorbed secondary antibody, Alexa Fluor 488 (1: 500, nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cns.70406 by IN A SP - N E PA L , W iley O nline L ibrary on [25/05/2025].

Techniques: Activation Assay, Expressing, Control

Journal: CNS neuroscience & therapeutics

Article Title: 11,12-Diacetyl-Carnosol Ameliorates Depression-Like Behaviors and Memory Dysfunction in CUMS Mouse Model via Inhibiting HMGB1-Mediated Neuroinflammation.

doi: 10.1111/cns.70406

Figure Lengend Snippet: FIGURE 3 | DACA reverses cell injury, inhibits the release of inflammatory factors, prevents HMGB1 nuclear translocation, and downregulates the expression of HMGB1, NF-κB, NLRP3, and IBA1 induced by LPS+ATP in BV2 cells. (A) Schematic timeline of BV2 cell experimental protocol. (B and C) CCK8 assay to assess BV2 cell viability. n = 5. (D) NO levels in BV2 cell supernatants measured by NO assay kit. n = 5. (E) ELISA detection of IL-6 and TNF-α levels in BV2 cell supernatants. n = 3. (F and G) Western blot analysis of HMGB1, P-P65/NF-κB, NLRP3, IL-1β, BDNF, and Nrf2 expression in BV2 cells. n = 4. (H and I) Immunofluorescence staining to detect IBA1 expression and HMGB1 nuclear translocation in BV2 cells. Scale bar, 20 μm. n = 3. #p < 0.5, ##p < 0.1, ###p < 0.01, ####p < 0.001, vs. control group; *p < 0.5, **p < 0.1, ***p < 0.01, ****p < 0.001, vs. LPS+ATP group.

Article Snippet: For western blot analysis and immunofluorescence, we used the following antibodies: anti- Nrf2 antibody (1: 3000, Proteintech, 66,009- 1- IG), anti- HMGB1 antibody(1: 1000, Abcam, ab18256), anti- P- NF- κB antibody(1: 1000, Cell Signaling Technology, 3033S), anti- NF- κB p65 (D14E12) antibody (1: 1000, Cell Signaling Technology, 8242), anti- NLRP3 antibody (1: 1000, Cell Signaling Technology, 15,101), anti- IL- 1β antibody (1: 1000, Affinity, AF5103), anti- BDNF antibody (1: 1000, Cell Signaling Technology, 47,808), anti- Nrf2 antibody (1: 3000, Proteintech, 16,396- 1- AP), anti- IBA- 1 antibody (1: 1000, Abcam, ab178846), anti- MAP2 antibody (1: 1000, Abcam, ab11267), anti- PSD95 antibody (1: 1000, Abcam, ab18258), goat anti- rabbit IgG H&L (HRP) (1: 5000, Abcam, ab6721), goat anti- mouse IgG H&L (HRP) (1: 5000, Abcam, ab6789), goat anti- mouse IgG (H+L) cross- adsorbed secondary antibody, Alexa Fluor 488 (1: 500, nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cns.70406 by IN A SP - N E PA L , W iley O nline L ibrary on [25/05/2025].

Techniques: Translocation Assay, Expressing, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Control

Journal: CNS neuroscience & therapeutics

Article Title: 11,12-Diacetyl-Carnosol Ameliorates Depression-Like Behaviors and Memory Dysfunction in CUMS Mouse Model via Inhibiting HMGB1-Mediated Neuroinflammation.

doi: 10.1111/cns.70406

Figure Lengend Snippet: FIGURE 5 | The HMGB1/NF-κB/NLRP3 pathway is involved in the regulation of DACA on CUMS-induced depressive-like behavior, memory impairment, microglial activation, and neuronal damage. (A) Schematic timeline of the animal experiment procedure. (B) Tracking plots of exper- imental animals in the OFT and MWM. (C) Sucrose preference percentage in the SPT, total distance traveled (mm) in the OFT, immobility time (s) in the TST and FST, escape latency (s), and number of platform area crossings in the MWM. n = 9. (D and E) Expression of IBA1 and MAP2 in the hippocampal CA1 region of mice. Scale bar, 20 μm. n = 3. (F) Expression levels of IL-6 and TNF-α in mouse serum. n = 3. (G and H) Dendritic spine density in the hippocampus of mice. Scale bar, 20 or 10 μm. n = 3. (I and J) Expression levels of MAP2, PSD95, and BDNF proteins in the hippocam- pus of mice. n = 4. (K and L) Western blot analysis of the expression levels of HMGB1, NF-κB, NLRP3, and IL-1β in the hippocampus of mice. n = 4.

Article Snippet: For western blot analysis and immunofluorescence, we used the following antibodies: anti- Nrf2 antibody (1: 3000, Proteintech, 66,009- 1- IG), anti- HMGB1 antibody(1: 1000, Abcam, ab18256), anti- P- NF- κB antibody(1: 1000, Cell Signaling Technology, 3033S), anti- NF- κB p65 (D14E12) antibody (1: 1000, Cell Signaling Technology, 8242), anti- NLRP3 antibody (1: 1000, Cell Signaling Technology, 15,101), anti- IL- 1β antibody (1: 1000, Affinity, AF5103), anti- BDNF antibody (1: 1000, Cell Signaling Technology, 47,808), anti- Nrf2 antibody (1: 3000, Proteintech, 16,396- 1- AP), anti- IBA- 1 antibody (1: 1000, Abcam, ab178846), anti- MAP2 antibody (1: 1000, Abcam, ab11267), anti- PSD95 antibody (1: 1000, Abcam, ab18258), goat anti- rabbit IgG H&L (HRP) (1: 5000, Abcam, ab6721), goat anti- mouse IgG H&L (HRP) (1: 5000, Abcam, ab6789), goat anti- mouse IgG (H+L) cross- adsorbed secondary antibody, Alexa Fluor 488 (1: 500, nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cns.70406 by IN A SP - N E PA L , W iley O nline L ibrary on [25/05/2025].

Techniques: Activation Assay, Expressing, Western Blot

Journal: CNS neuroscience & therapeutics

Article Title: 11,12-Diacetyl-Carnosol Ameliorates Depression-Like Behaviors and Memory Dysfunction in CUMS Mouse Model via Inhibiting HMGB1-Mediated Neuroinflammation.

doi: 10.1111/cns.70406

Figure Lengend Snippet: FIGURE 6 | The HMGB1/NF-κB/NLRP3 pathway is involved in the regulation of DACA on LPS+ATP-induced BV2 cell injury, inflammation, and HMGB1 nuclear translocation. (A) Schematic timeline of the BV2 cell experimental procedure. (B) CCK-8 assay to assess BV2 cell viability. n = 5. (C) NO kit to measure the NO levels in BV2 cell supernatant. n = 5. (D and E) ELISA to measure IL-6 and TNF-α levels in BV2 cell super- natant. n = 3. (F and G) Western blot analysis of the expression of HMGB1, P-P65/NF-κB, NLRP3, IL-1β, and BDNF in BV2 cells. n = 4. (H and I) Immunofluorescence staining for IBA1 expression and HMGB1 nuclear translocation in BV2 cells. Scale bar, 20 μm. n = 3. ##p < 0.1, ###p < 0.01,

Article Snippet: For western blot analysis and immunofluorescence, we used the following antibodies: anti- Nrf2 antibody (1: 3000, Proteintech, 66,009- 1- IG), anti- HMGB1 antibody(1: 1000, Abcam, ab18256), anti- P- NF- κB antibody(1: 1000, Cell Signaling Technology, 3033S), anti- NF- κB p65 (D14E12) antibody (1: 1000, Cell Signaling Technology, 8242), anti- NLRP3 antibody (1: 1000, Cell Signaling Technology, 15,101), anti- IL- 1β antibody (1: 1000, Affinity, AF5103), anti- BDNF antibody (1: 1000, Cell Signaling Technology, 47,808), anti- Nrf2 antibody (1: 3000, Proteintech, 16,396- 1- AP), anti- IBA- 1 antibody (1: 1000, Abcam, ab178846), anti- MAP2 antibody (1: 1000, Abcam, ab11267), anti- PSD95 antibody (1: 1000, Abcam, ab18258), goat anti- rabbit IgG H&L (HRP) (1: 5000, Abcam, ab6721), goat anti- mouse IgG H&L (HRP) (1: 5000, Abcam, ab6789), goat anti- mouse IgG (H+L) cross- adsorbed secondary antibody, Alexa Fluor 488 (1: 500, nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cns.70406 by IN A SP - N E PA L , W iley O nline L ibrary on [25/05/2025].

Techniques: Translocation Assay, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence, Staining