Journal: Cells

Article Title: Machine Learning-Driven Multi-Omics Analysis Identifies CHP2 as a Key PANoptosis-Related Dual-Function Biomarker in Colorectal Cancer

doi: 10.3390/cells15050430

Figure Lengend Snippet: Experimental validation of CHP2 as a predictive biomarker for drug sensitivity and a driver of PANoptosis. ( A – C ) IC 50 growth inhibition curves of HCT116 and SW480 cells treated with first-line chemotherapeutic agents: ( A ) 5-fluorouracil (5-FU), ( B ) Oxaliplatin, and ( C ) Irinotecan. ( D , E ) IC50 curves for targeted agents: ( D ) Ribociclib and ( E ) Lapatinib. ( F ) Western blot analysis of key PANoptosis markers, including p-MLKL (necroptosis), N-GSDMD (pyroptosis), and Cleaved-Caspase 3 (apoptosis), in Control, CHP2-OV, and CHP2-OV-siRNA-3 groups. ( G – J ) Quantitative analysis of protein expression levels normalized to β-actin. Data are presented as mean ±SD from three independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: After the protein concentration was determined with a BCA protein assay kit (Beyotime, P0010), identical amounts of proteins were subjected to SDS-PAGE and transferred onto PVDF membranes, which were subsequently incubated with primary antibodies against CHP2 (Boster, Wuhan, China, A08478-2, 1:1000), p-MLKL (MCE, HY86069, 1:1000), GSDMD (N-terminal) (MCE, HY- P85810 , 1:1000), Cleaved Caspase-3 (HY- P86370 ) and β-actin (Servicebio, ZB15001-HRP, 1:2000).

Techniques: Biomarker Discovery, Inhibition, Western Blot, Control, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Targeting TANK-binding kinase 1 attenuates painful diabetic neuropathy via inhibiting microglia pyroptosis

doi: 10.1186/s12964-024-01723-6

Figure Lengend Snippet: Inhibition of TBK1 suppressed the expression of NF-κB, NLRP3 inflammasome and IL-1β. A-B Immunoblot analysis of NF-κB expression levels in the spinal cord. C-D Double immunofluorescence staining of p-NF-κB and Iba1 in different groups. E-F Immunoblot analysis of NLRP3 expression levels in the spinal cord. G-H Immunoblot analysis of caspase-1 expression levels in the spinal cord. I-J Immunoblot analysis of ASC and IL-1β expression levels in the spinal cord. K-L Double immunofluorescence staining of ASC and Iba1 in different groups. Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01

Article Snippet: The primary antibodies included anti-TBK1 (Cell Signaling Technology, Cat#38,066), anti-Phospo-TBK1 (Cell Signaling Technology, Cat#5483), anti-NF-κB p65 (Cell Signaling Technology, Cat#8242), anti-Phospho-NF-κB p65 (Cell Signaling Technology, Cat#3033), anti-NLRP3 (Immunoway, Cat#YT5382), anti-pro-Caspase-1 (Immunoway, Cat#YT5743), anti-cleaved Caspase-1 p20 (Cell Signaling Technology, Cat#89,332), anti-ASC (Abmart, Cat#PA7120), anti- IL-1β (Immunoway, Cat#YM4682), anti-GSDMD n-ternal (Immunoway, Cat#YT7991), anti-c-Fos (Cell Signaling Technology, Cat#2250), anti-Iba1 (Abcam, Cat#ab283319) and anti-TNF-α (Abcam, Cat#ab215188) (all diluted 1:1000).

Techniques: Inhibition, Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Frontiers in Pharmacology

Article Title: DL-3-n-Butylphthalide Attenuates Myocardial Hypertrophy by Targeting Gasdermin D and Inhibiting Gasdermin D Mediated Inflammation

doi: 10.3389/fphar.2021.688140

Figure Lengend Snippet: Involvement of GSDMD in NBP-mediated cardiomyocyte protection. (A) Primary cardiomyocytes were transfected with cDNA plasmids encoding GSDMD-N (plsGSDMD-N) or empty vector (negative control, NC). Western blotting was performed to assess the protein levels of GSDMD. Primary cardiomyocytes transfected with NC or plsGSDMD-N were pretreated with NBP (20 μm) for 1 h and then exposed to Ang II (1 μm) for the indicated times. (B) After exposure to Ang II for 24 h, the effects of NBP and GSDMD-N overexpression on MyHC were detected by western blotting. (C–E) After exposure to Ang II for 12 h, the mRNA levels of Myhc, Anp, and Bnp were detected by RT-qPCR. (F) Rhodamine phalloidin staining of GSDMD-N-overexpressing primary cardiomyocytes (DAPI: nucleus; × 400 magnification). (G–H) After exposure to Ang II for 24 h, LDH and IL-1 β release were detected by assay kits. (n = 4 in each group; *, vs. Ctrl group; #, vs. Ang II group; $, vs. Ang II + NBP group; # and $ p < 0.05, ** and ## p < 0.01, *** and ### p < 0.001).

Article Snippet: GSDMD-N overexpression in cells was achieved using GSDMD-N plasmids (GenePharma Co.,Ltd, Shanghai, China).

Techniques: Transfection, Plasmid Preparation, Negative Control, Western Blot, Over Expression, Quantitative RT-PCR, Staining

Journal: Frontiers in Pharmacology

Article Title: DL-3-n-Butylphthalide Attenuates Myocardial Hypertrophy by Targeting Gasdermin D and Inhibiting Gasdermin D Mediated Inflammation

doi: 10.3389/fphar.2021.688140

Figure Lengend Snippet: Structural and energetic analysis of NBP to the binding site of GSDMD-N. (A) RMSD curves for the 200 ns MD simulation. (B) The 10 top contributing residues to NBP bound to GSDMD-N. (C) Overview of the predicted binding mode between GSDMD-N and NBP. (D) Detailed view of the 10 top contributing residues of NBP bound to GSDMD-N and the hydrogen bonds (red). (E) Schematic illustration of the role of NBP in GSDMD-related inflammation in TAC- or Ang II-induced myocardial hypertrophy.

Article Snippet: GSDMD-N overexpression in cells was achieved using GSDMD-N plasmids (GenePharma Co.,Ltd, Shanghai, China).

Techniques: Binding Assay

Journal: Journal of physiological investigation.

Article Title: Itaconate Attenuates Homocysteine-induced Nod-like Receptor Family Protein 3 Inflammasome-mediated Pyroptosis in Hippocampal Neurons: Involvement of Inhibiting Succinate Dehydrogenase Complex Subunit A Level

doi: 10.4103/ejpi.ejpi-d-25-00018

Figure Lengend Snippet: Figure 2: Effects of homocysteine (Hcy) on the expression and the membrane localization of GSDMD in HT22 cells. After treatment of HT22 cells with Hcy (1.25, 2.5, and 5 mM) for 24 h, the expression levels of GSDMD and GSDMD‑N were detected by Western blot (a), and the colocalization of GSDMD in cell membrane was observed by immunofluorescence (b). Data were shown as the mean ± standard error of the mean. Statistical significance was determined by one‑way analysis of variance with least‑significant difference post hoc test (n = 4).

Article Snippet: Apoptosis‐associated speck‐like protein (ASC) Rabbit pAb (DF6304) and Gasdermin D (GSDMD) Rabbit pAb (AF4012) were obtained from Affinity Biosciences (OH, USA).

Techniques: Expressing, Membrane, Western Blot, Immunofluorescence

Journal: Journal of physiological investigation.

Article Title: Itaconate Attenuates Homocysteine-induced Nod-like Receptor Family Protein 3 Inflammasome-mediated Pyroptosis in Hippocampal Neurons: Involvement of Inhibiting Succinate Dehydrogenase Complex Subunit A Level

doi: 10.4103/ejpi.ejpi-d-25-00018

Figure Lengend Snippet: Figure 6: Effects of itaconate on the expression and membrane localization of GSDMD in homocysteine (Hcy)‑exposed HT22 cell. After pretreatment with itaconate (25, 50, and 100 μM) for 10 h, HT22 cells were exposed to Hcy (2.5 mM) for 24 h. The expression levels of GSDMD and GSDMD‑N were detected by Western blot (a). The colocalization of GSDMD and cell membrane was observed by immunofluorescence (b). Data was shown as the mean ± standard error of the mean. OI, 4‑Octyl itaconate. Statistical significance was determined by one‑way analysis of variance with least‑significant difference post hoc test. (n = 5).

Article Snippet: Apoptosis‐associated speck‐like protein (ASC) Rabbit pAb (DF6304) and Gasdermin D (GSDMD) Rabbit pAb (AF4012) were obtained from Affinity Biosciences (OH, USA).

Techniques: Expressing, Membrane, Western Blot, Immunofluorescence

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Vericiguat reduces pyroptosis in rats with coronary microembolization by inhibiting the AMPK/Nrf2/NLRP3 signaling pathway

doi: 10.4196/kjpp.25.008

Figure Lengend Snippet: (A) The change of mRNA expression levels in NLRP3, IL-1β and ASC in cardiac tissue (n = 6). (B) The change of protein expression levels in NLRP3, ASC, GSDMD-N, IL-1β, and caspase-1 p20 in cardiac tissue (n = 4), for each group. The obtained data were presented as the mean ± SD. CME, coronary microembolization; CME + VER, CME + Vericiguat; CME + VER + CC, CME + Vericiguat + AMPK inhibitor; AMPK, adenosine monophosphate-activated protein kinase; NLRP3, NOD-like receptor pyrin containing 3; IL-1β, interleukin-1 beta; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *p < 0.05 vs . Sham group; **p < 0.05 vs . CME group; ***p < 0.05 vs. CME + VER group.

Article Snippet: Following the blocking step with 5% skim milk for one hour, the membranes were then left to incubate overnight at 4°C with primary antibodies specific to NLRP3, gasdermin D-N-terminal domain (GSDMD-N), cleaved caspase-1 p20, ASC, IL-1β, GAPDH, p-AMPK, AMPK and Nrf2 (all antibodies sourced from Wanleibio, 1:1,000).

Techniques: Expressing