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: Frontiers in Immunology

Article Title: Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction

doi: 10.3389/fimmu.2022.1078880

Figure Lengend Snippet: Polarization-related signaling pathway of macrophages was activated after T.cp -MIF treatment. The macrophages binded with TLR4, and T.cp -MIF activated NF-κB signaling pathway 24 hours after induction. At this time, macrophages showed M1 type polarization, while PI3K/AKT/mTOR signaling pathway is activated at 48 hours, making macrophages show M2 type polarization.

Article Snippet: After blocking, the corresponding primary antibodies such as TLR4 (Wuhan Sanying Biotechnology Co., Ltd.), NF-κB (Wuhan Sanying Biotechnology Co., Ltd.), p-NF-κB (Cell signaling technology, USA), PI3K (Wuhan Sanying Biotechnology Co., Ltd.), p-PI3K (Abcam, UK), Akt (Wuhan Sanying Biotechnology Co. Ltd.), and β-actin (Hangzhou Huaan Biotechnology Co., Ltd.) was separately added to the box (antibody concentrations were diluted according to the instructions), incubated at 4°C for over 12 h. After incubation, the membrane was washed 3 times with TBST buffer for 10 min each time.

Techniques:

Journal: Frontiers in Immunology

Article Title: Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction

doi: 10.3389/fimmu.2022.1078880

Figure Lengend Snippet: Effect of T.cp -MIF on the expression of macrophage polarization-related proteins in different time. (A–D) The protein expression levers of TLR4, NF-κBp65, PI3K and AKT were analyzed by western blot and normalized against β-actin expression. * indicated P <0.05. Results are showed as the mean of three times of independent experiments ( x̄ ±SD, n=3).

Article Snippet: After blocking, the corresponding primary antibodies such as TLR4 (Wuhan Sanying Biotechnology Co., Ltd.), NF-κB (Wuhan Sanying Biotechnology Co., Ltd.), p-NF-κB (Cell signaling technology, USA), PI3K (Wuhan Sanying Biotechnology Co., Ltd.), p-PI3K (Abcam, UK), Akt (Wuhan Sanying Biotechnology Co. Ltd.), and β-actin (Hangzhou Huaan Biotechnology Co., Ltd.) was separately added to the box (antibody concentrations were diluted according to the instructions), incubated at 4°C for over 12 h. After incubation, the membrane was washed 3 times with TBST buffer for 10 min each time.

Techniques: Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction

doi: 10.3389/fimmu.2022.1078880

Figure Lengend Snippet: The effect of TAK-242 on the expression of macrophage polarization-related proteins. (A–D) The cells were pretreated with TAK-242 (the TLR4 inhibitor) for 6 h, and the expression levels of TLR4, NF-κBp65, p-NF-κBp65, PI3K, p-PI3K, Akt and p-Akt were detected by western blot. * indicated P <0.05. Results are showed as the mean of three times of independent experiments ( x̄ ±SD, n=3).

Article Snippet: After blocking, the corresponding primary antibodies such as TLR4 (Wuhan Sanying Biotechnology Co., Ltd.), NF-κB (Wuhan Sanying Biotechnology Co., Ltd.), p-NF-κB (Cell signaling technology, USA), PI3K (Wuhan Sanying Biotechnology Co., Ltd.), p-PI3K (Abcam, UK), Akt (Wuhan Sanying Biotechnology Co. Ltd.), and β-actin (Hangzhou Huaan Biotechnology Co., Ltd.) was separately added to the box (antibody concentrations were diluted according to the instructions), incubated at 4°C for over 12 h. After incubation, the membrane was washed 3 times with TBST buffer for 10 min each time.

Techniques: Expressing, Western Blot