Journal: Cancer Research

Article Title: Heat Shock Protein 90α Recruits FLIPSto the Death-Inducing Signaling Complex and Contributes to TRAIL Resistance in Human Glioma

doi: 10.1158/0008-5472.can-07-0569

Figure Lengend Snippet: Figure 3. siRNA targeting HSP90a disrupts the recruitment of FLIPS to the DISC and sensitizes cells to TRAIL-induced apoptosis. TRAIL-sensitive E6/E7/ hTERT/Ras and G37 cells were either left untransfected/uninfected, or were subjected to retroviral infection/selection with an empty or cdc42-encoding construct, and transfected with a scrambled siRNA or a siRNA targeting HSP90a. A, all cells were then exposed to vehicle (CTRL) or TRAIL (800 ng/mL, 24 h) 48 h after initiation of siRNA exposure (where siRNA was used). Cells were then stained with propidium iodide, and analyzed by flow cytometry for the percentage of cells having <2N DNA content (apoptotic cells). B, recruitment of HSP90a and FLIPS to the DISC was assessed from anti-FADD antibody DISC immunoprecipitates of groups described in (A). C, total levels of HSP90a and FLIPS proteins in the lysates of groups described in (A) was determined by Western blotting using an aliquot of lysate taken prior to immunoprecipitation. a-Tubulin was used as a loading control. A, columns, means; bars, SE (n = 3). B and C, Western blots representative of one of three independent experiments for each group.

Article Snippet: Following incubation with TRAIL (0 or 800 ng/mL) for 24 h, the cells were washed once with ice-cold PBS and harvested using 1 ice-cold cell lysis buffer supplemented with 1 mmol/L of phenylmethylsulfonyl fluoride and incubated on ice for 10 min. HSP90a, FLIPS, FLIPL, or RIP was selectively immunoprecipitated from 200 Ag of protein (whole cell lysates) by combining the cell lysate with 20 AL of FADD antibody (Cell Signaling Technology) conjugated to agarose A/G beads (Santa Cruz Biotechnology) followed by gentle rotation for 4 h at 4jC.

Techniques: Retroviral, Infection, Selection, Construct, Transfection, Staining, Flow Cytometry, Western Blot, Immunoprecipitation, Control

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