Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis.

doi: 10.4049/jimmunol.1701643

Figure Lengend Snippet: FIGURE 3. REDD1 overexpression by neutrophils in colonic tissue of active UC or by control neutrophils stimulated in vitro with colonic tissue culture CM from active UC is paralleled with autophagy induction. (A) Colonic biopsy sections derived from patients with active UC stained with (A) REDD1 and NE (confocal microscopy; NE [green], REDD1 [red]) or (B) Beclin-1 and NE (confocal microscopy; Beclin-1 [green], NE [red]) compared with controls or active CD. (C) REDD1 mRNA levels and (D) REDD1, mTOR p2481, or p62/SQSTM1 immunoblotting in control neutrophils stimulated with CM from active UC patients in comparison with CM from healthy subjects or active CD. ***p , 0.001. (E) Results after measurement of the relative IOD of bands presented in (D), expressed as mTOR p2481/mTOR or p62/GAPDH. *,#p , 0.001. For (C) and (E), data presented as mean 6 SD. For (A) and (B), n = 4, (C)–(E) n = 6.

Article Snippet: Moreover, to detect REDD1 production, an anti-REDD1 polyclonal Ab (1/1000 dilution; Proteintech Group) was used.

Techniques: Over Expression, Control, In Vitro, Derivative Assay, Staining, Confocal Microscopy, Western Blot, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis.

doi: 10.4049/jimmunol.1701643

Figure Lengend Snippet: FIGURE 4. In mucosal tissue of patients with active UC, neutrophil REDD1, Beclin-1 expression, and NETosis are diminished according to the distance from the inflamed area. Colonic biopsy sections derived from distinct mucosa areas of patients with active UC stained with (A) REDD1 and NE (confocal microscopy; NE [green], REDD1 [red]) or (B) Beclin-1 and NE (confocal microscopy; Beclin-1 [green], NE [red]). (C) REDD1 mRNA, (D) REDD1 immunoblotting, (E) percentage of NET-releasing neutrophils, (F) MPO–DNA complex, and (G) IL-1b expression in isolated NET structures (NET ELISA) of control neutrophils stimulated with colonic tissue culture CM from inflamed, marginal, or noninflamed mucosal area of patients with active UC. For (A) and (B), n = 4. For (C)–(G), data presented as mean 6 SD, n = 6. ***p , 0.001.

Article Snippet: Moreover, to detect REDD1 production, an anti-REDD1 polyclonal Ab (1/1000 dilution; Proteintech Group) was used.

Techniques: Expressing, Derivative Assay, Staining, Confocal Microscopy, Western Blot, Isolation, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis.

doi: 10.4049/jimmunol.1701643

Figure Lengend Snippet: FIGURE 5. Colonic tissue culture CM from inflamed area of patients with active UC constitute a more potent inducer of REDD1 expression than the respective sera. (A) REDD1 mRNA and (B) REDD1 immunoblotting in control neutrophils stimulated either with CM from the inflamed mucosal area of patients with active UC or with the respective sera. For (A), data presented as mean 6 SD. For (A) and (B), n = 6. ***p , 0.001.

Article Snippet: Moreover, to detect REDD1 production, an anti-REDD1 polyclonal Ab (1/1000 dilution; Proteintech Group) was used.

Techniques: Expressing, Western Blot, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis.

doi: 10.4049/jimmunol.1701643

Figure Lengend Snippet: FIGURE 6. REDD1/neutrophils/IL-1b axis offers candidate targets for differential diagnosis and treatment of active UC. (A and B) Representative histograms and (C and D) data from flow cytometry analysis of CitH3+ or IL-1b–positive ex vivo isolated neutrophils from patients with active UC (n = 15) compared with controls (n = 25), active CD (n = 11), or IC (n = 15). (E) Heat map analysis of colonic tissue neutrophils positive for REDD1 expression (n = 6). (F) Visual analog scale (VAS) pain during sacroiliitis attacks of two active UC patients treated with anakinra (100 mg/daily s.c.). (G) Intracellular ex- pression of IL-1b in neutrophils or (H) CitH3-positive neutrophils ex vivo isolated from patients with active UC under mesalazine/5-ASA monotherapy (n = 8) compared with naive ones (n = 15), as assessed by flow cytometry analysis. For (C), (D), (G), and (H), data presented as mean 6 SD. ***p , 0.001.

Article Snippet: Moreover, to detect REDD1 production, an anti-REDD1 polyclonal Ab (1/1000 dilution; Proteintech Group) was used.

Techniques: Biomarker Discovery, Cytometry, Ex Vivo, Isolation, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis.

doi: 10.4049/jimmunol.1701643

Figure Lengend Snippet: FIGURE 8. Schematic illustration of the proposed mechanism for active UC. Intestinal inflammatory environment activates colonic mucosa neutrophils to release inflammatory NETs through local upregulation of REDD1, leading to autophagy induction. Targeting the REDD1/autophagy/NETs/IL-1b pathway promises novel therapeutic approaches for intestinal and systemic inflammation in active UC.

Article Snippet: Moreover, to detect REDD1 production, an anti-REDD1 polyclonal Ab (1/1000 dilution; Proteintech Group) was used.

Techniques:

Journal: Ecotoxicology and environmental safety

Article Title: DDIT4 is essential for DINP-induced autophagy of ovarian granulosa cells.

doi: 10.1016/j.ecoenv.2023.115686

Figure Lengend Snippet: Fig. 2. DINP upregulates DDIT4 expression in ovarian granulosa cells. (A) DDIT4 expression in the ovary tissue was detected by IHC after adult female mice were administered with 0–200 mg/kg DINP for 14 days. The scale bar was 100 µm. (B) The mRNA level of DDIT4 was determined by qPCR after KGN cells were exposed to 0 or 800 μM DINP for 24 h. (C, D) The protein expression of DDIT4 was detected by Western blot after KGN cells were exposed to 0–800 μM DINP for 24 h. *P < 0.05.

Article Snippet: Rabbit anti-DDIT4 polyclonal antibody (10638–1-AP) and mouse IgG (B900620) were purchased from Proteintech Group, Inc (Wuhan, China).

Techniques: Expressing, Western Blot

Journal: Ecotoxicology and environmental safety

Article Title: DDIT4 is essential for DINP-induced autophagy of ovarian granulosa cells.

doi: 10.1016/j.ecoenv.2023.115686

Figure Lengend Snippet: Fig. 3. DDIT4 is essential for DINP-induced autophagy of ovarian granulosa cells. (A, B) The protein levels of LC3, Beclin 1, Atg 5, p62 and DDIT4 were determined by Western blot after KGN cells were transfected with 0, 1, 2 μg pcDNA3.1-DDIT4 plasmid for 48 h. (C) Autophagic vesicles were observed by TEM after KGN cells were transfected with 0 or 2 μg pcDNA3.1-DDIT4 for 48 h. (D, E) The expression of LC3, Beclin 1, Atg 5, p62 and DDIT4 in KGN cells was detected by Western blot after the cells were transfected with si-NC or si-DDIT4 for 24 h. The protein levels of LC3, Beclin 1, Atg 5, p62 and DDIT4 (F, G) and the DDIT4 mRNA level (H) in KGN cells were detected after the cells were exposed to 0 or 800 μM DINP in the presence or absence of si-DDIT4 for 24 h.*P < 0.05.

Article Snippet: Rabbit anti-DDIT4 polyclonal antibody (10638–1-AP) and mouse IgG (B900620) were purchased from Proteintech Group, Inc (Wuhan, China).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Ecotoxicology and environmental safety

Article Title: DDIT4 is essential for DINP-induced autophagy of ovarian granulosa cells.

doi: 10.1016/j.ecoenv.2023.115686

Figure Lengend Snippet: Fig. 4. ATF4 promotes gene transcription of DDIT4. (A) Three potential ATF4 responsive elements (RE) in the DDIT4 promoter region were predicted by JASPAR. (B- D) The mRNA and protein levels of ATF4 and DDIT4 were detected by qPCR and Western blot, respectively, after KGN cells were transfected with 0, 1, 2 μg pcDNA3.1-ATF4 for 24 or 48 h. (E-G) The mRNA and protein levels of ATF4 and DDIT4 were determined by qPCR and Western blot, respectively, after KGN cells were transfected with si-NC or si-ATF4 for 24 h. (H) Luciferase activity was detected after KGN cells were transfected with pGL4.2 or pGL4.2-DDIT4 prom together with pcDNA3.1 or pcDNA3.1-ATF4 for 48 h. (I) Luciferase activity was determined after KGN cells were transfected with pGL4.2-RE1, pGL4.2-RE2 or pGL4.2-RE3, respectively, together with pcDNA3.1 or pcDNA3.1-ATF4 for 48 h. (J) Luciferase activity was determined after KGN cells were transfected with pGL4.2-RE2 or pGL4.2-mutRE2 for 48 h in the presence or absence of pcDNA3.1-ATF4. (K) ChIP-qPCR analysis was utilized to identify the binding of ATF4 to the RE2 site. *P < 0.05.

Article Snippet: Rabbit anti-DDIT4 polyclonal antibody (10638–1-AP) and mouse IgG (B900620) were purchased from Proteintech Group, Inc (Wuhan, China).

Techniques: Western Blot, Transfection, Luciferase, Activity Assay, ChIP-qPCR, Binding Assay

Journal: Ecotoxicology and environmental safety

Article Title: DDIT4 is essential for DINP-induced autophagy of ovarian granulosa cells.

doi: 10.1016/j.ecoenv.2023.115686

Figure Lengend Snippet: Fig. 5. ATF4 is involved in DINP-induced upregulation of DDIT4 in ovarian granulosa cells. (A) The ATF4 expression in the ovary tissue was observed by IHC after adult female mice were administered with 0–200 mg/kg DINP for 14 d. The scale bar was 20 µm. (B-D) The ATF4 mRNA and protein level were detected by qPCR and Western blot, respectively, after KGN cells were treated with the indicated concentration of DINP for 24 h. (E-G) The expression of DDIT4 and ATF4 was detected by qPCR and Western blot, respectively, after KGN cells were exposed to 0 or 800 μM DINP for 24 h in the presence or absence of si-ATF4. *P < 0.05.

Article Snippet: Rabbit anti-DDIT4 polyclonal antibody (10638–1-AP) and mouse IgG (B900620) were purchased from Proteintech Group, Inc (Wuhan, China).

Techniques: Expressing, Western Blot, Concentration Assay

Journal: Ecotoxicology and environmental safety

Article Title: DDIT4 is essential for DINP-induced autophagy of ovarian granulosa cells.

doi: 10.1016/j.ecoenv.2023.115686

Figure Lengend Snippet: Fig. 6. DINP induces autophagy of ovarian granulosa cells via ATF4/DDIT4 signals. (A, B) The protein levels of LC3, Atg 5, Beclin 1, p62 and ATF4 were determined after KGN cells were transfected with 0, 1, 2 μg pcDNA3.1-ATF4 for 48 h. (C) Autophagic vesicles were detected by TEM after KGN cells were transfected with 0 or 2 μg pcDNA3.1-ATF4 for 48 h. (D, E) The protein levels of LC3, Beclin 1, Atg 5, p62 and ATF4 were detected after KGN cells were transfected with si-NC or si-ATF4 for 24 h. (F, G) The protein levels of LC3, Beclin 1, Atg 5, p62 and ATF4 were determined after KGN cells were exposed to 0 or 800 μM DINP for 24 h in the presence or absence of si-ATF. *P < 0.05.

Article Snippet: Rabbit anti-DDIT4 polyclonal antibody (10638–1-AP) and mouse IgG (B900620) were purchased from Proteintech Group, Inc (Wuhan, China).

Techniques: Transfection