Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: A qPCR showed increased TLR4 expression in the 216911KO cell line. ** p < 0.01 by student T tests. B The diagram presented two pir-hsa-216911 binding sites in 3ʹUTR of TLR4. C The dual luciferase assay revealed that pir-has-216911 could bind both TLR4 binding sites TLR4S1 and TLR4S2. Plasmids carrying TLR4S1 and TLR4S2 generate a higher Firefly/Renilla Luciferase ratio in the 216911KO cell line, which pir-hsa-216911 was absent. **p < 0.01; ***p < 0.001 by student T tests. D TCGA-LIHC survival analysis found low expression of TLR4 to be unfavorable to the survival of HCC patients. E Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway. TLR4, p-p65, and NLRP3 levels were elevated in the 216911KO cell line, which pir-hsa-216911 was absent. F Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway through silencing TLR4. After silencing TLR4 with siRNA, pir-hsa-216911 absent could not elevate p-p65, and NLRP3 levels were elevated in the 216911KO cell line.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Expressing, Binding Assay, Luciferase, Western Blot

Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: A The diagram presented the xenograft modeling process. B Euthanized mouse to display the subcutaneous xenografted tumors. C 216911KO group resulted in much smaller tumors in size compared to the control group. D 216911KO group resulted in much smaller tumors in weight compared to the control group. The histogram represents the mean tumor weight of each group. ***p < 0.001 by student T tests. E Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway in vivo. Both TLR4 and p-p65 levels were elevated in tumors formed in the 216911KO group compared to the control group.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Control, Western Blot, In Vivo

Journal: bioRxiv

Article Title: Identification of A Disintegrin and Metalloproteinase 9 domain (ADAM9) required in the early stages of encephalomyocarditis virus infection

doi: 10.1101/491068

Figure Lengend Snippet: WT and ADAM9 KO HeLa cells were transduced with retroviral vectors with wild-type (WT) murine ADAM9 (mADAM9), catalytically inactive mutant ADAM9 (E>A), cytoplasmic tail deleted (ΔCT) ADAM9 constructs, or GFP control vectors. Transduced cells were selected and cloned by limiting dilution, and expression of mADAM9 was confirmed by western blot analysis. (A) WT, KO and rescue cell lysates were prepared in RIPA buffer and run on 12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 3% BSA and incubated with ADAM9 antibodies. Upper panel, rabbit anti-human ADAM9 (Cell signaling #2099) that detects an epitope in the intracellular domain of human ADAM9 and cross-reacts with mouse ADAM9. Middle panel, goat anti-mouse ADAM9 (R&D systems AF949) that detects the extracellular domain of murine ADAM9, but not human ADAM9. Lower panel, anti-Actin (Santa Cruz sc-1616) which detects both human and murine ϟ-Actin. Bands were visualized using HRP and ECL reagent. Upper panel: WT but not KO cells expressed human ADAM9. Middle panel: Rescue but not KO cells expressed murine ADAM9. Lower panel: Actin loading control. (B) WT clones, ADAM9 KO clones and rescued ADAM9-expressing clones were infected with EMCV or CVB3 at varying MOI and incubated at 37 °C for 24 h. Viability of EMCV-infected and CVB3-infected clones was measured by CellGlo ATP luminescence. (C) EMCV replication was quantified in infected culture supernatants by plaque assay using BHK-21 cells. Neither the functional sequence of the ADAM9 metalloproteinase domain nor the cytoplasmic tail are required for EMCV infection. ***, P <0.0001, KO vs. WT; KO vs. rescue.

Article Snippet: 3% BSA blocking buffer), anti-goat IgG HRP (1:2000 dilution, R&D Systems HAF109), and anti-rabbit IgG HRP (1:5000 dilution Vector Labs PI1000).

Techniques: Transduction, Mutagenesis, Construct, Clone Assay, Expressing, Western Blot, SDS Page, Incubation, Infection, Plaque Assay, Functional Assay, Sequencing

Journal: Circulation Research

Article Title: Hemoglobin Scavenger Receptor CD163 Mediates Interleukin-10 Release and Heme Oxygenase-1 Synthesis

doi: 10.1161/01.res.0000109414.78907.f9

Figure Lengend Snippet: Figure 3. Induction of HO-1 by Hb:Hp via a CD163 receptor IL-10 autocrine mechanism. A, Representative HO-1 and HO-2 immunoblots are shown from in vitro differentiated day 7 macrophages stimulated with Hb, Hp, Hb:Hp, or Hb:Hp plus blocking anti–IL-10, IgG control, or RM3/1 antibody. Induction of the HO-1 isomer was established relative to con- stitutive expression of the HO-2 isomer by probing blots with polyclonal antibody against HO-1, followed by stripping and reprobing with anti–HO-2. B, Lysates in A were subjected to quantification of intracellular HO-1 levels by HO-1–spe- cific ELISA and normalized with respect to total cell protein. Values shown repre- sent meanSEM. Results are represen- tative of 3 similar experiments.

Article Snippet: Neutralizing anti- human IL-10 polyclonal antibody and control goat IgG antibody were purchased from R&D Systems (Abingdon, UK).

Techniques: Western Blot, In Vitro, Blocking Assay, Control, Expressing, Stripping Membranes, Enzyme-linked Immunosorbent Assay