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: Cancer Chemotherapy and Pharmacology

Article Title: The newly synthesized anticancer drug HUHS1015 is useful for treatment of human gastric cancer

doi: 10.1007/s00280-014-2661-z

Figure Lengend Snippet: Primers used for real-time RT-PCR

Article Snippet: After blocking with TBST (20 mM Tris, 150 mM NaCl, 0.1 % (v/v) Tween-20, pH 7.5) containing 5 % (w/v) of bovine serum albumin, blotting membrane was reacted with antibodies against FasL (Cell Signaling Technology, Inc., Danvers, MA, USA), Fas (Cell Signaling Technology), FADD (Cell Signaling Technology), tumor necrosis factor α (TNFα) (Cell Signaling Technology), TNFR1 (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), or TRADD (Santa Cruz Biotechnology) followed by a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-goat IgG antibody.

Techniques:

Journal: Cancer Chemotherapy and Pharmacology

Article Title: The newly synthesized anticancer drug HUHS1015 is useful for treatment of human gastric cancer

doi: 10.1007/s00280-014-2661-z

Figure Lengend Snippet: Real-time RT-PCR analysis. MKN45 cells were treated with HUHS1015 (100 μM) for periods of time as indicated, and then, real-time RT-PCR was carried out. The mRNA quantity for FasL ( a ), Fas ( b ), FADD ( c ), TNFα ( d ), TNFR1 ( e ), and TRADD ( f ) was calculated from the standard curve made by amplifying different amount of the GAPDH mRNA and normalized by regarding the average of independent basal mRNA quantity at 0 h as 1. In the graphs, each point represents the mean (±SEM) ratio relative to basal mRNA levels ( n = 4 independent experiments)

Article Snippet: After blocking with TBST (20 mM Tris, 150 mM NaCl, 0.1 % (v/v) Tween-20, pH 7.5) containing 5 % (w/v) of bovine serum albumin, blotting membrane was reacted with antibodies against FasL (Cell Signaling Technology, Inc., Danvers, MA, USA), Fas (Cell Signaling Technology), FADD (Cell Signaling Technology), tumor necrosis factor α (TNFα) (Cell Signaling Technology), TNFR1 (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), or TRADD (Santa Cruz Biotechnology) followed by a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-goat IgG antibody.

Techniques: Quantitative RT-PCR

Journal: Cancer Chemotherapy and Pharmacology

Article Title: The newly synthesized anticancer drug HUHS1015 is useful for treatment of human gastric cancer

doi: 10.1007/s00280-014-2661-z

Figure Lengend Snippet: Western blot analysis. MKN45 cells were treated with HUHS1015 (100 μM) for periods of time as indicated, and Western blotting was carried out. The signal intensity for FasL ( a ), Fas ( b ), FADD ( c ), TNFα ( d ), TNFR1 ( e ), or TRADD protein ( f ) was normalized by that for β-actin. In the graphs, each column represents the mean (±SEM) normalized intensity for each protein ( n = 4 independent experiments). P value, Dunnett’s test

Article Snippet: After blocking with TBST (20 mM Tris, 150 mM NaCl, 0.1 % (v/v) Tween-20, pH 7.5) containing 5 % (w/v) of bovine serum albumin, blotting membrane was reacted with antibodies against FasL (Cell Signaling Technology, Inc., Danvers, MA, USA), Fas (Cell Signaling Technology), FADD (Cell Signaling Technology), tumor necrosis factor α (TNFα) (Cell Signaling Technology), TNFR1 (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), or TRADD (Santa Cruz Biotechnology) followed by a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-goat IgG antibody.

Techniques: Western Blot