Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 1. Smad protein phosphorylation by TGF-1 stimulation in LX2, PSC, and MEF cells. LX2, PSC, and MEF cells were serum-starved overnight, then treated with different concentrations of TGF-1 for 45 min. Antibodies against p-Smad1/5 (Ser-463/465), p-Smad2 (Ser-465/467), and p-Smad3 (Ser-423/425) were used. Both Smad1/5 and Smad2/3 were phosphorylated by TGF-1 stimulation in all of the cells. An asterisk (*) indicates the nonspecific band.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 2. The effects of eliminating or overexpressing NRP-1 on Smad protein phosphorylation and the Smad target gene expression. A, eliminating NRP-1 up-regulated Smad1/5 phosphorylation and down-regulated Smad2/3 phosphorylation. NRP-1 was knocked down by siRNA in LX2 and PSC. For MEF cells, wt MEF, and NRP-1/ MEF cells were used. All the cells were serum-starved overnight, and then treated with 10 ng/ml TGF-1 for the indicated time. An asterisk (*) indicates the nonspecific band. B, overexpress NRP-1 in NRP-1/ MEF cells down-regulated Smad1/5 phosphorylation and up-regulated Smad2/3 phosphorylation. NRP-1/ MEF cells were infected with NRP-1 encoding retrovirus. LacZ retrovirus was used as control. After 36 h, cells were serum-starved overnight, and then treated with 10 ng/ml TGF-1 for the indicated time. C–E, effect of knocking down NRP-1 on the Smad proteins transcriptional activity in the cells measured by the luciferase promoter assay. 5 103/well LX2 cells were plated into a 96-well plate transfected with NRP-1 siRNA for 24–30 h, then serum-starved overnight. The cells were then transfected with the corresponding luciferase promoter plasmids together with pRL-TKRenilla luciferase plasmid astheinternalcontrol.Onehourafterthetransfection,TGF-1wasaddedtothecorrespondingwelltothefinalconcentrationof10ng/ml.Fireflyluciferaseand Renilla luciferase activities were performed. C, Luc-Id-1 promoter. D, Luc-ARE, co-transfected with FAST. E, Luc-SBE. F, knocking down NRP-1 affected the Smad protein responding gene expression. Cells were transfected with NRP-1 siRNA for 24–30 h in the complete medium, then serum-starved overnight. 10 ng/ml TGF-1 was added to the cells for 24 h. In response to TGF-1 stimulation, Id-1 protein (the Smad1-responding gene) expression was more highly up-regulated in NRP-1 knocked down cells than in control cells stimulated with TGF-1. In response to TGF-1 stimulation, -SMA and PAI-1 protein (the Smad3-responding gene) expression was more highly down-regulated in NRP-1 knocked down cells than in control cells stimulated with TGF-1.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics, Targeted Gene Expression, Infection, Control, Activity Assay, Luciferase, Promoter Assay, Transfection, Plasmid Preparation, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 3. NRP-1 interacted with TGF- receptors. A, NRP-1 bound to TGF-RII. LX2 cells grown in the complete medium were made for cell lysate, and immunoprecipitation was performed using anti-NRP-1 antibody and West- ernblotforTGF-RII.ControlantibodywithcelllysateandNRP-1antibodywithoutcelllysatewereusedasnegative controls.B,TGF-1stimulationdidnotincreasetheassociationbetweenNRP-1andTGF-RII.LX2cellswereserum- starved overnight and stimulated with 10 ng/ml TGF-1 for 0, 15, and 45 min. The cell lysate was made, and immu- noprecipitation was performed using anti-NRP-1 antibody and Western blot for TGF-RII. C, siRNA knocked down TGF-RII eliminated the effect of NRP-1 on Smad protein phosphorylation. LX2 cells were transfected with control, NRP-1, and TGF-RII siRNA alone or combined, as indicated in the figure. 24–30 h later, cells were serum-starved overnight. 10 ng/ml TGF-1 was added to the indicated plates for 45 min, and the cell lysate was subjected to Western blot analysis. An asterisk (*) indicates the nonspecific band. D, TGF-RI (ALK5) inhibitors eliminated both Smad1/5 and Smad2/3 protein phosphorylation on TGF-1 stimulation. LX2 cells were serum-starved overnight. TGF-RI inhibitors SB431542 (10 M) and ALK5 Inhibitor (10 M) were added to the cells 30 min before TGF-1 stimulation. An asterisk (*) indicates the nonspecific band. E, NRP-1 associated with TGF-RI (ALK5) by TGF-1 stimulation. 293T cells were transfected with NRP-1 and Flag-ALK5-expressing plasmids. Cells were serum-starved overnight and stimulated with 10 ng/ml TGF-1 for 0, 5, and 15 min. The cell lysate was made, and immunoprecipi- tation was performed using anti-NRP-1 antibody and Western blot for Flag tag. F, knockdown TGF-RII eliminated theassociationbetweenNRP-1andTGF-RI(ALK5)inthepresentofTGF-stimulation.293Tcellsweretransfected with TGF-RII siRNA, and the following experiments were preformed as described in panel E.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Transfection, Control, Expressing, FLAG-tag, Knockdown

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 8. The schematic illustration of NRP-1 function in regulating TGF- signaling. TGF- induced both Smad1/5/8 and Smad2/3 phosphorylation in the fibroblast cells. Without NRP-1 (left panel), Smad1/5/8 is more phosphorylated and Smad2/3 less phosphorylated, and the corresponding gene expression controlled by the Smad proteins (e.g. Smad1/Id-1, Smad3/a-SMA, PAI-1) caused the cell to enter a less activated state (more quiescent). With NRP-1 (right panel), Smad1/5/8 is less phosphorylated, and Smad2/3 is more phosphorylated, and the corresponding gene expression controlled by the Smad proteins caused the cell to enter a more activated state (less quiescent).

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics, Gene Expression

Journal: Molecular Cancer

Article Title: Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia

doi: 10.1186/1476-4598-13-116

Figure Lengend Snippet: Validation of nutlin-induced modulation of acetylated proteins in the AML cell line MOLM-13. (A) Western blots of total lysates from SILAC (stable labeling with amino acids in cell culture) labeled MOLM-13 cells treated with DMSO or 6 μM nutlin-3 for 6 hours using antibodies against p53, MDM2, Histone H2B, acetylated Histone H2B (Lys120), Hsp27, Hsp90, acetylated Hsp90 (Lys294) and actin. Bands were quantified using region of interest imaging analysis, and values are given as fold induction of control relative to actin. (B) MOLM-13 cells were treated nutlin-3 as described above, and immunoprecipitations using anti-acetyl lysine antibody or an unspecific antibody (rabbit IgG) were performed. Western blots of Hsp27 of the immunoprecipitated proteins and of the total lysates used in immunoprecipitations are shown. (C) Total levels of Hsp90 and Hsp27 in MOLM-13 cells treated with nutlin-3 as described above analyzed by flow cytometry. Results are given as representative flow diagrams and median fluorescence intensity (MFI) relative to control.

Article Snippet: The following antibodies were used; p53 (Bp53-12), Mdm2 (SMP-14) (Santa Cruz Biotechnology, CA, USA), Mdm2 (2A10), Mdm2 (IF2), anti-Hsp27 (G3.1) (Calbiochem, San Diego, CA, USA), p21 (SX118) (BD Biosciences, San Jose, CA, USA), phospho-p53 (Ser15), phospho-p53 (Ser20), ac-p53 (Lys382) (Cell Signaling Technologies, Beverly, MA, USA), anti-Histone H2B, anti-Hsp90 (Millipore, Temecula, CA, USA), anti-acetyl-Histone H2B (Lys120) (Upstate cell signaling solutions, Lake Placid, NY, USA), anti-acetyl-Hsp90 (Lys294) (Rockland Immunochemicals, Inc., Gilbertsville, PA, USA), secondary horse radish peroxidase conjugated mouse and rabbit antibody (Jackson ImmunoResearch, West Grove, PA, USA), actin (AC-15) (Abcam plc, Cambridge, UK).

Techniques: Biomarker Discovery, Western Blot, Multiplex sample analysis, Labeling, Cell Culture, Imaging, Control, Immunoprecipitation, Flow Cytometry, Fluorescence

Journal: Molecular Cancer

Article Title: Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia

doi: 10.1186/1476-4598-13-116

Figure Lengend Snippet: Functional role of p53 acetylation in nutlin-sensitivity. (A) SAOS-2 and H1299 cells were transiently transfected with empty vector, p53 full length (FL) or the actylation defective mutant p53 6KR and treated with 20 μM nutlin-3 for 24 hours. Cell viability was determined using the WST-1 viability/proliferation assay (*** p < 0.001, ** p < 0.01). Results were analyzed in triplicates in tree independent experiments and error bars represent standard error of mean. Transfections were verified in Western blots with antibodies against p53 and actin. (B) H1299 cells were transiently transfected with empty vector (EV), p53 full length (FL), or p53 6KR and treated with DMSO or 20 μM nutlin-3 for 6 hours. Western blotting was performed using antibodies against p53, MDM2, acetylated p53 (Lys382), Hsp27, Hsp90 and acetylated Hsp90 (Lys294) and actin. Bands were quantified using region of interest imaging analysis, and values are given as fold induction of control (DMSO treated sample for each transfection for EV/p53 FL/p53 6KR) relative to actin.

Article Snippet: The following antibodies were used; p53 (Bp53-12), Mdm2 (SMP-14) (Santa Cruz Biotechnology, CA, USA), Mdm2 (2A10), Mdm2 (IF2), anti-Hsp27 (G3.1) (Calbiochem, San Diego, CA, USA), p21 (SX118) (BD Biosciences, San Jose, CA, USA), phospho-p53 (Ser15), phospho-p53 (Ser20), ac-p53 (Lys382) (Cell Signaling Technologies, Beverly, MA, USA), anti-Histone H2B, anti-Hsp90 (Millipore, Temecula, CA, USA), anti-acetyl-Histone H2B (Lys120) (Upstate cell signaling solutions, Lake Placid, NY, USA), anti-acetyl-Hsp90 (Lys294) (Rockland Immunochemicals, Inc., Gilbertsville, PA, USA), secondary horse radish peroxidase conjugated mouse and rabbit antibody (Jackson ImmunoResearch, West Grove, PA, USA), actin (AC-15) (Abcam plc, Cambridge, UK).

Techniques: Functional Assay, Transfection, Plasmid Preparation, Mutagenesis, Proliferation Assay, Western Blot, Imaging, Control

Journal: Cancer cell

Article Title: The osteogenic niche is a calcium reservoir of bone micrometastases and confers unexpected therapeutic vulnerability

doi: 10.1016/j.ccell.2018.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Western blots were performed using antibodies against NFAT1(Cell Signalling, 5861S), pS6K(T389) (Cell Signaling, 9234S), pCamKII(T286) (Cell Signalling, 12716S), MeCP2 (Cell Signalling, 3456S), pMeCP2(S421)(Rockland, 600-401-X14), Firefly luciferase (Thermo, PA5-32209).

Techniques: Luciferase, Recombinant, Expressing, In Vivo, Software

Journal: Cell Death & Disease

Article Title: ANGPTL2 inhibits macrophage pyroptosis and alleviates rheumatoid arthritis progression by regulating mitophagy via IGFBP5

doi: 10.1038/s41419-026-08537-z

Figure Lengend Snippet: A Representative macroscopic images showing paw swelling in WT and Angptl2 –/– CIA mice. B Assessment of arthritis progression, including body weight changes, paw thickness, and clinical arthritis scores over time. C Representative 3D μCT reconstruction images of ankle joints. Quantification of talus bone volume (mm³) ( D ) and the ratio of bone volume to tissue volume (BV/TV, %) ( E ) in ankle joints. F Representative H&E-stained sections of ankle joints showing inflammation and structural damage. G Representative TRAP-stained sections of ankle joints showing osteoclast distribution and activity. H Representative ALP-stained sections of ankle joints indicating osteoblast activity and bone formation in periarticular regions. I Dual immunofluorescence (IF) staining of ankle joint sections using anti-F4/80 and antibodies against NLRP3, Caspase-1, GSDMD, or IL-1β in CIA mice. The average IF signals for total NLRP3, Caspase-1, GSDMD, and IL-1β expression, and co-localization with F4/80 + macrophages were determined. T: tibia; TAL: talus; ST: synovial tissue. J Western blot analysis showing protein levels of ANGPTL2, NLRP3, Caspase-1, GSDMD, and IL-1β in the joints of CIA mice. * p < 0.05, ** p < 0.01, *** p <0.001, **** p <0.0001 in the indicated groups.

Article Snippet: The membranes were treated with 5% non-fat milk in TBST for an hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies: ANGPTL2 (Proteintech, Wuhan.

Techniques: Staining, Activity Assay, Immunofluorescence, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: ANGPTL2 inhibits macrophage pyroptosis and alleviates rheumatoid arthritis progression by regulating mitophagy via IGFBP5

doi: 10.1038/s41419-026-08537-z

Figure Lengend Snippet: A qPCR analysis of Angptl2 , Nlrp3 , Caspase-1 , Gsdmd , and Il-1β expression in RAW264.7 cells stimulated with LPS alone or LPS + ATP. B ELISA quantification of IL-1β secretion in culture supernatants from RAW264.7 cells stimulated with LPS alone or LPS + ATP. C Time-course qPCR analysis of Angptl2 , Nlrp3 , Caspase-1 , Gsdmd , and Il-1β mRNA expression in RAW264.7 cells primed with LPS for 0, 3, 6, 12, and 24 h, followed by 1-h ATP stimulation prior to RNA extraction. D Corresponding time-course Western blot analysis of ANGPTL2, NLRP3, GSDMD, Caspase-1, and IL-1β expression in RAW264.7 cells under identical conditions. E qPCR analysis of Angptl2 , Nlrp3 , Caspase-1 , Gsdmd , and Il-1β expression in BMDMs stimulated with LPS alone or LPS + ATP. F ELISA quantification of IL-1β secretion in culture supernatants from BMDMs stimulated with LPS alone or LPS + ATP. G Time-course qPCR analysis of Angptl2 , Nlrp3 , Caspase-1 , Gsdmd , and Il-1β mRNA expression in BMDMs primed with LPS for 0, 3, 6, 12, and 24 h, followed by 1-h ATP stimulation prior to RNA extraction. H Corresponding time-course Western blot analysis of ANGPTL2, NLRP3, GSDMD, Caspase-1, and IL-1β expression in BMDMs under identical conditions. * p < 0.05, ** p < 0.01, *** p <0.001, **** p <0.0001 in the indicated groups.

Article Snippet: The membranes were treated with 5% non-fat milk in TBST for an hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies: ANGPTL2 (Proteintech, Wuhan.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, RNA Extraction, Western Blot

Journal: Cell Death & Disease

Article Title: ANGPTL2 inhibits macrophage pyroptosis and alleviates rheumatoid arthritis progression by regulating mitophagy via IGFBP5

doi: 10.1038/s41419-026-08537-z

Figure Lengend Snippet: qPCR analysis of Angptl2 , Nlrp3 , Caspase-1 , Gsdmd , and Il-1β mRNA expression in RAW264.7 cells ( A ) and BMDMs ( B ) following si Angptl2 or siNC transfection, with or without LPS + ATP stimulation. Western blot analysis of ANGPTL2, NLRP3, GSDMD, Caspase-1, and IL-1β protein expression in RAW264.7 cells ( C ) and BMDMs ( D ) under identical conditions. ELISA measurement of IL-1β levels in culture supernatants of RAW264.7 cells ( E ) and BMDMs ( F ). Cell death assessment using LDH release assay in RAW264.7 cells ( G ) and BMDMs ( H ) under identical conditions. Representative TEM images showing morphological features of pyroptotic cell death in RAW264.7 cells ( I ) and BMDMs ( J ). Representative flow cytometry plots showing Annexin V and PI staining of RAW264.7 cells ( K ) and BMDMs ( L ) under identical conditions. Quantification of Annexin V + PI + populations in RAW264.7 cells ( M ) and BMDMs ( N ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 in the indicated groups.

Article Snippet: The membranes were treated with 5% non-fat milk in TBST for an hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies: ANGPTL2 (Proteintech, Wuhan.

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Flow Cytometry, Staining

Journal: Cell Death & Disease

Article Title: ANGPTL2 inhibits macrophage pyroptosis and alleviates rheumatoid arthritis progression by regulating mitophagy via IGFBP5

doi: 10.1038/s41419-026-08537-z

Figure Lengend Snippet: A Volcano plot of DEGs comparing Angptl2 –/– and NC BMDMs following LPS + ATP stimulation. Genes with significant upregulation (yellow) or downregulation (blue) are highlighted based on thresholds of adjusted p < 0.05 and |log2(fold change)| ≥2. Non-significant genes are shown in gray. B Heatmap showing hierarchical clustering of DEGs between Angptl2 –/– and NC BMDMs following LPS + ATP stimulation. Each column represents an individual sample, and each row represents a gene. Color gradient indicates Z-score normalized expression levels (red: upregulated, green: downregulated). C GSEA plot showing enrichment of the gene set “negative regulation of autophagy of mitochondrion” between groups. D GO enrichment dot plot for DEGs. Enriched terms are classified by Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). Dot size reflects gene count, color indicates adjusted p-value, and X-axis denotes gene ratio. E KEGG pathway enrichment map for DEGs in KO-LPS vs. NC-LPS. Dot size reflects gene count, color indicates adjusted p-value, and X-axis denotes gene ratio.

Article Snippet: The membranes were treated with 5% non-fat milk in TBST for an hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies: ANGPTL2 (Proteintech, Wuhan.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: ANGPTL2 inhibits macrophage pyroptosis and alleviates rheumatoid arthritis progression by regulating mitophagy via IGFBP5

doi: 10.1038/s41419-026-08537-z

Figure Lengend Snippet: A Western blot analysis of mitophagy-related proteins (PINK1, PARKIN, LC3B, and p62) in joint tissues from WT and Angptl2 –/– mice under basal and CIA-induced conditions. B Western blot analysis of mitophagy markers in RAW264.7 cells and BMDMs transfected with si Angptl2 or siNC, with or without LPS + ATP stimulation. Flow cytometry analysis of mitochondrial membrane potential using JC-1 staining in RAW264.7 cells ( C ) and BMDMs ( D ). The ratio of red (aggregates) to green (monomers) fluorescence (R/G) was calculated to assess mitochondrial integrity. Intracellular ROS levels measured by flow cytometry in RAW264.7 cells ( E ) and BMDMs ( F ) under the indicated conditions. G Flow cytometry analysis of mito-Keima-loaded RAW264.7 cells to assess mitophagic flux under different conditions. H Representative confocal microscopy images showing colocalization of LC3B (red) and mitochondria (MitoTracker Green) in BMDMs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 in the indicated groups.

Article Snippet: The membranes were treated with 5% non-fat milk in TBST for an hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies: ANGPTL2 (Proteintech, Wuhan.

Techniques: Western Blot, Transfection, Flow Cytometry, Membrane, Staining, Fluorescence, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: ANGPTL2 inhibits macrophage pyroptosis and alleviates rheumatoid arthritis progression by regulating mitophagy via IGFBP5

doi: 10.1038/s41419-026-08537-z

Figure Lengend Snippet: A Western blot analysis of ANGPTL2 expression in BMDMs following treatment with recombinant mouse ANGPTL2 protein (rMmANGPTL2). B qPCR analysis of Nlrp3 , Caspase-1 , Gsdmd , and Il-1β mRNA expression in BMDMs stimulated with LPS + ATP and treated with increasing concentrations of rMmANGPTL2 (0, 250, 500, 750, 1000 ng/mL). C Flow cytometry analysis of mitochondrial membrane potential using JC-1 staining in BMDMs under NC, LPS + ATP, or LPS + ATP + rMmANGPTL2 treatment conditions. D Flow cytometry analysis of intracellular ROS levels in BMDMs under NC, LPS + ATP, or LPS + ATP + rMmANGPTL2 treatment conditions. qPCR ( E ) and Western blot ( F ) analysis of mitophagy-related markers in BMDMs following LPS + ATP stimulation with or without rMmANGPTL2 and the autophagy inhibitor 3-MA. qPCR ( G ) and Western blot ( H ) analysis of pyroptosis-associated markers in BMDMs under the same treatment conditions. I Flow cytometry analysis of mito-Keima-loaded RAW264.7 cells to assess mitophagic flux under the same treatment conditions. J Representative confocal microscopy images showing LC3B and mitochondria colocalization in BMDMs under the same treatment conditions. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 in the indicated groups.

Article Snippet: The membranes were treated with 5% non-fat milk in TBST for an hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies: ANGPTL2 (Proteintech, Wuhan.

Techniques: Western Blot, Expressing, Recombinant, Flow Cytometry, Membrane, Staining, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: ANGPTL2 inhibits macrophage pyroptosis and alleviates rheumatoid arthritis progression by regulating mitophagy via IGFBP5

doi: 10.1038/s41419-026-08537-z

Figure Lengend Snippet: A Heatmap showing the expression correlation between ANGPTL2 and insulin-like growth factor (IGF) family members, including IGF1, IGF2, and IGFBP proteins. qPCR ( B ) and Western blot ( C ) analysis of IGFBP5 expression in BMDMs following Igfbp5 knockdown and LPS + ATP stimulation. D Western blot analysis of mitophagy-related proteins (PINK1, PARKIN, LC3B, p62) and NLRP3 in BMDMs after Igfbp5 knockdown with LPS + ATP treatment. qPCR ( E ) and Western blot ( F ) analysis of IGFBP5 expression in BMDMs following Angptl2 knockdown and LPS + ATP stimulation. qPCR ( G ) and Western blot ( H ) analysis of IGFBP5 expression in BMDMs treated with rMmANGPTL2 under LPS + ATP stimulation. I – J Co-immunoprecipitation (Co-IP) assay to evaluate protein-protein interaction between ANGPTL2 and IGFBP5 in RAW264.7 cells. K 3D model of structured ANGPTL2 interfaces with IGFBP5 predicted by HDOCK. L Western blot analysis of mitophagy-related proteins in BMDMs treated with LPS + ATP and rMmANGPTL2, with or without Igfbp5 knockdown. * p < 0.05, ** p < 0.01, *** p <0.001, **** p <0.0001 in the indicated groups.

Article Snippet: The membranes were treated with 5% non-fat milk in TBST for an hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies: ANGPTL2 (Proteintech, Wuhan.

Techniques: Expressing, Western Blot, Knockdown, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: ANGPTL2 inhibits macrophage pyroptosis and alleviates rheumatoid arthritis progression by regulating mitophagy via IGFBP5

doi: 10.1038/s41419-026-08537-z

Figure Lengend Snippet: A Representative images of mouse paws from control, CIA, and AAV- Angptl2 -treated groups showing macroscopic joint swelling. B Assessment of arthritis progression, including body weight changes, paw thickness, and clinical arthritis scores over time. C Representative 3D μCT reconstruction images of ankle joints. D Representative H&E-stained sections of ankle joints illustrating synovial inflammation and tissue architecture. E Representative TRAP-stained sections showing osteoclast distribution and activity. F Representative ALP-stained sections indicating osteoblast activity and bone formation. G Representative dual immunofluorescence images of ankle joint sections stained for F4/80 and either NLRP3, GSDMD, Caspase-1, or IL-1β. The average IF signals for total NLRP3, GSDMD, Caspase-1, and IL-1β expression, and co-localization with F4/80 + macrophages were determined. T: tibia; TAL: talus; ST: Synovial Tissue. H Western blot analysis of ANGPTL2 protein levels in joint tissues from each group. I Western blot analysis of pyroptosis and inflammasome-related proteins (NLRP3, GSDMD, Caspase-1, IL-1β) in joint tissues. J Western blot analysis of mitophagy-related proteins (PINK1, PARKIN, LC3B, p62) in joint tissues. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 in the indicated groups.

Article Snippet: The membranes were treated with 5% non-fat milk in TBST for an hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies: ANGPTL2 (Proteintech, Wuhan.

Techniques: Control, Staining, Activity Assay, Immunofluorescence, Expressing, Western Blot

Journal: Foods

Article Title: Sea Buckthorn Pericarp Flavonoids Improve Diet-Induced Hyperlipidemia via Coordinated Modulation of Hepatic Lipid Metabolism and Gut Microbiota

doi: 10.3390/foods15061049

Figure Lengend Snippet: TFSP modulates the expression of ACC, FAS, CPT-1α, PPARα and ATGL proteins in the livers of HFD mice. n = 6 per group. Data are presented as mean ± SD. Different letters above bars indicate statistically significant differences ( p < 0.05) by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Peroxisome proliferator-activated receptor alpha (PPARα) primary antibody (Cat. No. A00600-2), carnitine palmitoyltransferase-1 alpha (CPT-1α) primary antibody (Cat. No. A00917-3), acetyl-CoA carboxylase (ACC) primary antibody (Cat. No. M01802-2), fatty acid synthase (FAS) primary antibody (Cat. No. BA0484), adipose triglyceride lipase (ATGL) primary antibody (Cat. No. A01800-1), and GAPDH primary antibody (Cat. No. BM1623) were obtained from BOSTER Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing