Journal: Pharmacognosy magazine

Article Title: Poncirus trifoliata Rafin. induces the apoptosis of triple-negative breast cancer cells via activation of the c-Jun NH(2)-terminal kinase and extracellular signal-regulated kinase pathways.

doi: 10.4103/0973-1296.166056

Figure Lengend Snippet: Figure 4: The expressions and activations of death receptor proteins in P. trifoliata methanol extract (MEPT)‑treated MDA‑MB‑231 cells. Western blotting was performed for tumor necrosis factor receptor, TNFR type 1‑associated death domain, Fas, and FADD after administering MEPT at 0, 25, 50, or 100 μg/mL for 24 h, or 30 nM paclitaxel for 24 h (a), and expression levels were normalized using loading control (b)

Article Snippet: Primary antibodies for cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, TNFR, TRADD, Fas, FADD, p38, phospho‐p38, JNK, and phospho‐JNK were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Western Blot, Expressing, 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. 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: Clinical and Translational Medicine

Article Title: NLK facilitates Caspase‐8 activation to drive macrophage PANoptosis in sepsis

doi: 10.1002/ctm2.70616

Figure Lengend Snippet: NLK deficiency disrupts PANoptosome assembly and augments RIPK1/3‐ dependent necrosome formation in vivo. (A, B) Representative immunoblots of FADD‐ and RIPK1‐associated complexes, together with corresponding input samples, in macrophages isolated from WT and NKO mice at 8 h post‐CLP. Co‐immunoprecipitates were probed for NLK, Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3. (C–I) Quantification analysis of Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3 in FADD‐associated complexes ( n = 3 independent biological replicates). (J–L) Quantification analysis of p‐RIPK1, RIPK3, and p‐RIPK3 in RIPK1‐associated complexes ( n = 3 independent biological replicates). (M) Representative confocal immunofluorescence images of lung macrophages stained for CD68 (green), Caspase‑8 (red), and ASC (cyan). Merged images indicate ASC–Caspase‐8 colocalisation; boxed regions show magnified views. Scale bars: 50 µm. Statistical significance was determined by using one‑way ANOVA with Bonferroni's post hoc test; * p < .05, ** p < .01 and ns indicates p > .05.

Article Snippet: Antibodies against CD68 (28058‐1‐AP), Caspase‐1 (22915‐1‐AP), p‐RIPK1 (66854‐1‐Ig), FADD ( P14906 ‐1‐AP), HA‐Tag (51064‐2‐AP, 66006‐2‐Ig), Flag‐Tag (66008‐4‐Ig, 20543‐1‐AP), and IgG (B900620) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: In Vivo, Western Blot, Isolation, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: NLK facilitates Caspase‐8 activation to drive macrophage PANoptosis in sepsis

doi: 10.1002/ctm2.70616

Figure Lengend Snippet: NLK deficiency impairs PANoptosome assembly and enhances RIPK1/3‐dependent necrosome formation in macrophages. (A, B) Representative immunoblots of FADD‐ and RIPK1‐associated complexes, together with corresponding input samples, in macrophages isolated from WT and NKO mice at 3 h post‐LPS. Co‐IP were probed for NLK, Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3. (C–I) Quantification analysis of Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3 in FADD‐associated complexes ( n = 3 independent biological replicates). (J–L) Quantification analysis of p‐RIPK1, RIPK3, and p‐RIPK3 in RIPK1‐associated complexes ( n = 3 independent biological replicates). (M) Representative confocal immunofluorescence images showing the co‑localisation of RIPK3 (cyan), ASC (green), and Caspase‑8 (red) in PBS‐ or LPS‐treated BMDMs. Merged images indicate RIPK3–ASC–Caspase‐8 colocalisation; boxed regions show magnified views. Scale bars: 25 µm (merged), 10 µm (zoomed). Statistical differences were analysed by one‑way ANOVA with Bonferroni's post hoc test, * p < .05 and ** p < .01.

Article Snippet: Antibodies against CD68 (28058‐1‐AP), Caspase‐1 (22915‐1‐AP), p‐RIPK1 (66854‐1‐Ig), FADD ( P14906 ‐1‐AP), HA‐Tag (51064‐2‐AP, 66006‐2‐Ig), Flag‐Tag (66008‐4‐Ig, 20543‐1‐AP), and IgG (B900620) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Western Blot, Isolation, Co-Immunoprecipitation Assay, Immunofluorescence

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: Cell death & disease

Article Title: c-FLIP and CD95 signaling are essential for survival of renal cell carcinoma.

doi: 10.1038/s41419-019-1609-y

Figure Lengend Snippet: Fig. 1 Cycloheximide sensitizes clearCa cells towards CD95L-induced apoptosis. a Surface expression of death receptors CD95, TRAIL-R1, TRAIL- R2, or TNF-R1 (black line) on clearCa-2, -3, -4, and -6 cells was detected by flow cytometry with specific antibodies. Unstained samples are shown in gray. b Expression levels of the DISC proteins c-FLIP, FADD, and caspase-8 as well as caspase-3 in clearCa-2, -3, -4, and -6 cells were analyzed via immunoblotting. Tubulin served as loading control. c Analysis of DNA fragmentation after stimulation of clearCa cells with 0, 2, 4, or 10 ng/mL CD95L in the presence or absence of 10 µg/mL CHX for 16 h. Bars display the mean of at least three experiments, error bars represent SD. Statistical significances were calculated by one-tailed Mann–Whitney U test; * p ≤0.05

Article Snippet: Primary antibodies for specific detection of proteins were: β-Actin (Ac-74, Sigma Aldrich), Bcl-x (Polyclonal, Transduction Laboratories), Caspase-8 (12F5, Dr. Klaus Schulze-Osthoff, Tübingen), Caspase-3 (Polyclonal, R&D Systems), CD95 (C-20, Santa Cruz), c-FLIP (NF6, Adipogen), Cleaved Caspase-3 (Asp175, 9661, Cell Signaling Technology), Cleaved Caspase-8 (18C8, Cell Signaling Technology), FADD (1F7, Upstate), FADD (TA332936, Origene), PARP (4C10–5, BD Biosciences), phospho-Erk1/2 (4370, Cell Signaling Technology), phospho-p65 (3033, Cell Signaling Technology), Tubulin (DM-1A, Sigma Aldrich), XIAP (48, BD Bioscience).

Techniques: Expressing, Cytometry, Western Blot, Control, One-tailed Test, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: RIPK1 can mediate apoptosis in addition to necroptosis during embryonic development

doi: 10.1038/s41419-019-1490-8

Figure Lengend Snippet: Analysis was performed to determine the effect of disrupting FADD-mediated apoptosis. Defects in the three mutant E12.5 embryos of the indicated genotypes are similar, contrasting normal embryos in the wild type control ( a ). The ruler division in mm is shown to the left of embryos. At two-month age, RIPK1 D324A/D324A RIPK3 −/− FADD −/− (TM) mice are indistinguishable in appearance ( b ) and in body weights ( c ) from the wild type (WT) and RIPK3 −/− FADD −/− (DKO) control mice. Western blot analysis of total splenocytes confirming presence or absence of RIPK1, RIPK3, and FADD proteins ( d ). Ponceau S staining (pink) was performed as protein loading and transfer control

Article Snippet: Antibodies specific for FADD (generated in house), RIPK3 (ProSci, #2283), RIPK1 (BD Biosciences, #610459), p65 (Cell Signaling, #3034), p44/42 MAPK (Erk1/2) (Cell signaling, #9102) were incubated with the membrane overnight at 4 °C followed by streptavidin-horseradish peroxidase (HRP)-conjugated anti-rabbit (1/10,000,Vector laboratories #PI-1000) or anti-mouse antibodies (1/10,000, Vector laboratories #PI-2000).

Techniques: Mutagenesis, Control, Western Blot, Staining