Journal: Acta pharmacologica Sinica

Article Title: Ginsenoside Rb1 alleviates colitis in mice via activation of endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 signaling pathway.

doi: 10.1038/s41401-020-00561-9

Figure Lengend Snippet: Fig. 2 Effects of the ginsenoside Rb1 (GRb1) on inflammation and apoptosis in a DSS-induced mouse model of colitis. Expression levels of the colonic cytokines a MPO, b IL-1β, c IL-6, and d TNF-α were determined by ELISA. Western blot analysis of the inflammation-related proteins e iNOS, f COX2, and g p-NF-κB-p65 (calculated as p-NF-κB-p65/NF-κB-p65) and the apoptosis-related protein h cleaved-caspase 3 (cl-caspase 3), calculated as cl-caspase 3/caspase 3. Data are expressed as the mean ± SD. Values in the sham group were set to 100%, and other values are given relative to those in the sham group, **P < 0.01 compared with the sham group; ##P < 0.01 compared with the DSS control group; n = 3 samples for Western blot experiments; n = 6 samples for other experiments.

Article Snippet: Antibodies against Hrd1(13473-1-AP), CHOP(15204-1-AP), Fas (13098-1-AP), caspase 8(13423-1-AP), caspase 3 (19677-1-AP), cleaved-caspase 3 (cl-caspase 3) (19677-1-AP), and iNOS (14142-1-AP) were purchased from Proteintech Co., Ltd. (Wuhan, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Acta pharmacologica Sinica

Article Title: Ginsenoside Rb1 alleviates colitis in mice via activation of endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 signaling pathway.

doi: 10.1038/s41401-020-00561-9

Figure Lengend Snippet: Fig. 3 Effects of the ginsenoside Rb1 (GRb1) on ER stress and Hrd1 in a DSS-induced mouse model of colitis. Western blot analysis of the ER stress marker proteins a GRP78, b PERK, c CHOP, and d caspase 12. e Western blot analysis of Hrd1. f Expression of Hrd1 determined by real- time PCR. g Immunohistochemical staining of Hrd1 in the colonic epithelium. Scale bars, 200, 100, and 50 μm. Data are expressed as the mean ± SD; **P < 0.01 compared with the sham group; ##P < 0.01 compared with the DSS control group; n = 3 samples for Western blot experiments; n = 6 samples for other experiments.

Article Snippet: Antibodies against Hrd1(13473-1-AP), CHOP(15204-1-AP), Fas (13098-1-AP), caspase 8(13423-1-AP), caspase 3 (19677-1-AP), cleaved-caspase 3 (cl-caspase 3) (19677-1-AP), and iNOS (14142-1-AP) were purchased from Proteintech Co., Ltd. (Wuhan, China).

Techniques: Western Blot, Marker, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Control

Journal: Acta pharmacologica Sinica

Article Title: Ginsenoside Rb1 alleviates colitis in mice via activation of endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 signaling pathway.

doi: 10.1038/s41401-020-00561-9

Figure Lengend Snippet: Fig. 5 Effect of the ginsenoside Rb1 (GRb1) on apoptosis. a Effect of GRb1 (200 μM) on IEC-6 cell apoptosis, as determined by flow cytometry. Effect of GRb1 on the expression of b Fas and c caspase 8 in vivo. Effect of GRb1 (200 μM) on the expression of Fas in LPS-treated IEC-6 cells in the absence (d) and presence (e) of siRNA targeting Hrd1. f Effects of GRb1 (200 μM) on IEC-6 cell apoptosis in the presence of siRNA targeting Hrd1. Data are expressed as the mean ± SD. **P < 0.01 compared with the corresponding control group; ##P < 0.01 compared with the DSS control group. n = 3 samples for Western blot experiments, n = 6 samples for other experiments.

Article Snippet: Antibodies against Hrd1(13473-1-AP), CHOP(15204-1-AP), Fas (13098-1-AP), caspase 8(13423-1-AP), caspase 3 (19677-1-AP), cleaved-caspase 3 (cl-caspase 3) (19677-1-AP), and iNOS (14142-1-AP) were purchased from Proteintech Co., Ltd. (Wuhan, China).

Techniques: Cytometry, Expressing, In Vivo, Control, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: Ginsenoside Rb1 alleviates colitis in mice via activation of endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 signaling pathway.

doi: 10.1038/s41401-020-00561-9

Figure Lengend Snippet: Fig. 7 Effects of the ginsenoside Rb1 (GRb1) on Hrd1, ER stress, apoptosis, and inflammation in a TNBS-induced colitis model. Western blot analysis of a Hrd1, b CHOP, c GRP78, d cleaved-caspase 3 (cl-caspase 3) (calculated as cl-caspase 3/caspase 3), and e p-NF-κB-p65 (calculated as p-NF-κB-p65/NF-κB-p65). Data are expressed as the mean ± SD. **P < 0.01 compared with the sham group (n = 3 mice); ##P < 0.01 compared with the TNBS-induced colitis group; n = 3 mice.

Article Snippet: Antibodies against Hrd1(13473-1-AP), CHOP(15204-1-AP), Fas (13098-1-AP), caspase 8(13423-1-AP), caspase 3 (19677-1-AP), cleaved-caspase 3 (cl-caspase 3) (19677-1-AP), and iNOS (14142-1-AP) were purchased from Proteintech Co., Ltd. (Wuhan, China).

Techniques: Western Blot

Journal: Molecular Systems Biology

Article Title: An incoherent feedforward loop interprets NFκB/RelA dynamics to determine TNF‐induced necroptosis decisions

doi: 10.15252/msb.20209677

Figure Lengend Snippet:

Article Snippet: Rat FLIP Antibody [Dave‐2] , ProSci , Cat # XA‐1008.

Techniques: Recombinant, Sequencing, CRISPR, Control, cDNA Synthesis, SYBR Green Assay, Software, Microscopy, Imaging

Journal: Journal of Clinical Investigation

Article Title: ZBP1-mediated apoptosis and inflammation exacerbate steatotic liver ischemia/reperfusion injury

doi: 10.1172/jci180451

Figure Lengend Snippet: Figure 5. Palmitic acid upregulates hepatocyte ZBP1 expression level through JNK pathway to exacerbate I/R injury of steatotic livers. (A and B) ZBP1 mRNA levels in donor livers (n = 6) or mouse I/R model (n = 4 for sham and n = 8 for I/R) were quantified. (C and D) mRNA and protein levels of ZBP1 after different concentration of PA in normal PMH for 48 hours (n = 5). (E and F) mRNA and protein levels of ZBP1 after stimulation of 0.4 mM PA and 10μM SP600125 in normal PHH for 24 hours (n = 5). (G and H) mRNA and protein levels of ZBP1 after stimulation of 0.4 mM PA for 24 hours in c-Jun knockdown normal PHH (n = 5). (I) ZBP1 promoter mutation schema and luciferase activity after c-Jun overexpression (n = 6). (J) Binding of c-Jun to ZBP1 promoter after stimulation of PA and SP600125 treatment or c-Jun knockdown in normal PHH (n = 6). (K–M) Zbp1+/+ or Zbp1–/– PMH (n = 3) were pretreated with 0.2 mM PA for 24 hours and subjected to 10 h hypoxia. Cell death (K) was analyzed after 24 hours reoxygenation and apoptosis (L) and mRNA levels of cyto- kines (M) was analyzed after 3 hours reoxygenation. All data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. 2-way ANOVA, post hoc Bonferroni’s test (A, B, I, J, and M). 1-way ANOVA, post hoc Dunnett’s test (C, E, and G). Unpaired, 2-tailed Student’s t test (K).

Article Snippet: Casp8fl/fl (S-CKO-01552), Mlkl–/– (S-KO-14468), Ripk3–/– (S-KO-10874), Zbp1–/– (S-KO-11088) mice were obtained from Cyagen, China.

Techniques: Expressing, Concentration Assay, Knockdown, Mutagenesis, Luciferase, Activity Assay, Over Expression, Binding Assay

Journal: Journal of Clinical Investigation

Article Title: ZBP1-mediated apoptosis and inflammation exacerbate steatotic liver ischemia/reperfusion injury

doi: 10.1172/jci180451

Figure Lengend Snippet: Figure 6. Z-NA sensing is not required for ZBP1-induced liver I/R injury. (A–F) Different truncation mutants of Zbp1 (A) were packaged into AAV8 and injected to ND-fed mice and I/R operation was conducted 1 month later (n = 8). Cell death and ZBP1-RIPK1 interaction analysis (B), serum ALT/AST detec- tion (C), H&E staining (D), TUNEL staining (E), and detection of serum levels of the proinflammatory cytokines (F) were performed. All data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. 1-way ANOVA, post hoc Dunnett’s test. Scale bars: 200 μm.

Article Snippet: Casp8fl/fl (S-CKO-01552), Mlkl–/– (S-KO-14468), Ripk3–/– (S-KO-10874), Zbp1–/– (S-KO-11088) mice were obtained from Cyagen, China.

Techniques: Injection, Staining, TUNEL Assay

Journal: Journal of Clinical Investigation

Article Title: ZBP1-mediated apoptosis and inflammation exacerbate steatotic liver ischemia/reperfusion injury

doi: 10.1172/jci180451

Figure Lengend Snippet: Figure 7. ROS triggers ZBP1 aggregation and activation in liver I/R injury. (A) Flag immunostaining was performed in I/R-challenged ND-fed mouse livers overexpressing Zbp1-Flag (n = 8). (B) ZBP1 aggregation was detected with immunoblot under nonreducing or reducing conditions in I/R-challenged mouse livers or transplantation-challenged donor livers (n = 3). (C) hZBP1-GFP plasmids were transfected into HEK293T cells. After 1 mM H2O2 treatment for 4 hours or H/R challenge after pretreatment with 10 mM NAC, ZBP1 oligomers were detected from green fluorescence (n = 8). (D–H) After AAV8-mediated Zbp1-Flag overexpression in ND-fed mice and pretreatment with NAC, I/R operation was performed (n = 8). Flag immunostaining (D), ZBP1 aggregation and ZBP1-RIPK1 interaction (E), serum ALT/AST levels (F), H&E and TUNEL staining (G), and detection of serum levels of the proinflammatory cytokines (H) were detected. All data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired, 2-tailed Student’s t test (A and D). 2-way ANOVA, post hoc Bonferroni’s test (C, G, and H). Scale bars (A, C, and D): 10 μm, (G) 200 μm.

Article Snippet: Casp8fl/fl (S-CKO-01552), Mlkl–/– (S-KO-14468), Ripk3–/– (S-KO-10874), Zbp1–/– (S-KO-11088) mice were obtained from Cyagen, China.

Techniques: Activation Assay, Immunostaining, Western Blot, Transplantation Assay, Transfection, Fluorescence, Over Expression, TUNEL Assay, Staining