caspase 9  (Proteintech)

Journal: Frontiers in Pharmacology

Article Title: Hyperoside ameliorates lupus nephritis by suppressing AKT1-mediated PANoptosis in podocytes: integrating network pharmacology and experimental validation

doi: 10.3389/fphar.2025.1726254

Figure Lengend Snippet: PANoptosis contributes to renal pathology in lupus nephritis. (A) Comparison of 24-h urinary protein levels between MRL/MpJ and MRL/lpr mice (n = 6). (B) Representative renal histology evaluated by H&E staining (n = 6). (C) TUNEL staining showing cell death in glomeruli (n = 6). (D) IHC analysis of podocyte markers (Podocalyxin, Nephrin, Synaptopodin, Podocin) in renal tissues. (E–G) Immunofluorescence staining of PANoptosis-related markers (NLRP3, RIPK3, Caspase-9) in kidney sections. (H) WB analysis of PANoptosis executor proteins (caspase-3, caspase-1, p-MLKL) in renal tissues, with bar graphs showing quantitative results relative to GAPDH (n = 3). (I) IF triple-staining of caspase-3, caspase-1, and p-MLKL in mouse kidney tissues. (J) Viability of MPC-5 cells treated with ICs at different concentrations and time points, measured by CCK-8 assay. (K) Flow cytometric analysis of cell death in MPC5 cells pretreated with VX-765 (10 μM), Nec-1 (20 μM), or Z-VAD (5 μM), followed by IC stimulation for 24 h. Bar graph shows the percentage of dead cells (n = 3). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, ns: not significant.

Article Snippet: Sequential sections (4 μm) were used for the following staining and analyses: Hematoxylin and eosin (H&E) staining was performed to evaluate pathological changes in renal tissues; immunohistochemistry (IHC) was conducted to examine the expression of synaptopodin (21064-1-AP, Proteintech, USA), nephrin (bs-10233R, Bioss, China), podocalyxin (bs-1345R, Bioss, China), and podocin (bs-6597R, Bioss, China); immunofluorescence (IF) staining was used to detect NLRP3 (DF7438, Affinity, UK), RIPK3 (bs-3551R, Bioss, China), and Caspase-9 (bs-0049R, Bioss, China) expression; and a TUNEL assay kit (E-CK-A320, Elabscience, China) was employed to assess cell death.

Techniques: Comparison, Staining, TUNEL Assay, Immunofluorescence, CCK-8 Assay

Journal: Frontiers in Pharmacology

Article Title: Hyperoside ameliorates lupus nephritis by suppressing AKT1-mediated PANoptosis in podocytes: integrating network pharmacology and experimental validation

doi: 10.3389/fphar.2025.1726254

Figure Lengend Snippet: Hyperoside protects podocytes from IC-induced PANoptosis. MPC5 cells were treated with different concentrations of Hyperoside followed by IC stimulation for 24 h (A) TUNEL staining showing cell death in MPC5 cells. (B) Flow cytometric analysis of apoptosis in MPC5 cells. (C) Quantitative analysis of the percentage of dead cells by flow cytometry (n = 3). (D–F) mRNA expression levels of PANoptosis-related genes (NLRP3, RIPK3, Caspase-9) measured by qPCR (n = 6). (G) IL-18 secretion detected by ELISA (n = 6). (H–M) Western blot analysis showing relative protein expression of PANoptosis executors (p-MLKL, Caspase-1, Caspase-3, GSDMD-N, Caspase-8) (n = 3). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, ns: not significant.

Article Snippet: Sequential sections (4 μm) were used for the following staining and analyses: Hematoxylin and eosin (H&E) staining was performed to evaluate pathological changes in renal tissues; immunohistochemistry (IHC) was conducted to examine the expression of synaptopodin (21064-1-AP, Proteintech, USA), nephrin (bs-10233R, Bioss, China), podocalyxin (bs-1345R, Bioss, China), and podocin (bs-6597R, Bioss, China); immunofluorescence (IF) staining was used to detect NLRP3 (DF7438, Affinity, UK), RIPK3 (bs-3551R, Bioss, China), and Caspase-9 (bs-0049R, Bioss, China) expression; and a TUNEL assay kit (E-CK-A320, Elabscience, China) was employed to assess cell death.

Techniques: TUNEL Assay, Staining, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Hyperoside ameliorates lupus nephritis by suppressing AKT1-mediated PANoptosis in podocytes: integrating network pharmacology and experimental validation

doi: 10.3389/fphar.2025.1726254

Figure Lengend Snippet: AKT1 mediates podocyte PANoptosis and LN progression. (A–E) MRL/lpr mice were injected via tail vein with KD-NC or KD-AKT1 AAV and maintained for 6 weeks (n = 6). (A,B) Western blot analysis of AKT1 and PANoptosis markers (p-MLKL, Caspase-1, Caspase-3) in renal tissues (n = 3). (C) Renal pathology assessed by H&E staining. (D) Immunohistochemical staining of podocyte-associated proteins (Podocalyxin, Nephrin, Synaptopodin, Podocin). (E) Comparison of 24 h urinary protein levels between the two groups. (F–P) MPC-5 cells were transfected with Si-NC, Si-AKT1, OE-NC, or OE-AKT1, followed by IC treatment for 24 h. (F) Percentage of dead cells measured by flow cytometry (n = 6). (G) Representative flow cytometry plots showing apoptosis. (H–J) mRNA expression levels of PANoptosis-related genes (NLRP3, RIPK3, Caspase-9) detected by qPCR (n = 6). (K–P) Western blot analysis of PANoptosis executor proteins (p-MLKL, Caspase-1, Caspase-3, GSDMD-N, Caspase-8) in MPC5 cells (n = 3). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, ns: not significant.

Article Snippet: Sequential sections (4 μm) were used for the following staining and analyses: Hematoxylin and eosin (H&E) staining was performed to evaluate pathological changes in renal tissues; immunohistochemistry (IHC) was conducted to examine the expression of synaptopodin (21064-1-AP, Proteintech, USA), nephrin (bs-10233R, Bioss, China), podocalyxin (bs-1345R, Bioss, China), and podocin (bs-6597R, Bioss, China); immunofluorescence (IF) staining was used to detect NLRP3 (DF7438, Affinity, UK), RIPK3 (bs-3551R, Bioss, China), and Caspase-9 (bs-0049R, Bioss, China) expression; and a TUNEL assay kit (E-CK-A320, Elabscience, China) was employed to assess cell death.

Techniques: Injection, Western Blot, Staining, Immunohistochemical staining, Comparison, Transfection, Flow Cytometry, Expressing