lscm  (Bioss)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neutrophilic granule protein promotes lipopolysaccharide-induced iNOS/NO expression via JAK2/STAT1 signaling pathway and augments bacteria clearance of macrophages

doi: 10.1007/s00018-025-05865-9

Figure Lengend Snippet: NGP controls the expression of iNOS/NO by regulating the JAK2/STAT1 signaling pathway. ( A ) Ngp NC /RAW and Ngp HI /RAW cells were treated with LPS (10 μg/mL) for 6 h and lysed for RNA sequencing. The KEGG database was utilized to enrich signal pathways exhibiting significant differences. ( B ) Ngp NC /RAW and Ngp HI /RAW cells were stimulated with LPS for 6 h. The cells were harvested and subjected to CSP100 PLUS phosphorylation microarray analysis. ( C ) The Ngp NC /RAW and Ngp HI /RAW cells were treated without or with LPS for 6 and 12 h respectively. Western blotting analysis was performed to assess the phosphorylation levels of JAK2 and STAT1. ( D ) The Ngp NC /RAW and Ngp HI /RAW cells were treated with LPS for 6 h and subjected for IP-MS to screen for the binding protein of NGP and verify the interaction. ( E ) The Ngp WT /RAW cells were treated without or with LPS for 6 h, harvested for Co-IP, and STAT1 expression levels were quantified via grayscale intensity analysis. ( F – H ) The predicted structures of NGP and STAT1 were generated by Alphafold and subjected to GRAMM Web Server for molecular docking. ( F ) Molecular docking generic matching information. ( G ) Predicted binding sites between NGP and STAT1. ( H ) The protein–protein interaction cartoon model generated by PyMOL. (I-J) The phosphorylation levels in PBS- or LPS-treated Ngp NC /RAW and Ngp HI /RAW cells were examined by LSCM (Scale bar = 20 μm). (K-M) Ngp NC /RAW and Ngp HI /RAW cells were pre-treated without or with JAK2 inhibitor (10 μM), STAT1 inhibitor (50 μM), or control solution for 2 h and then stimulated without or with LPS for 12 h. The supernatants and cells were harvested for NO expression, Nos2 mRNA level and positive ratio of iNOS by aforementioned methods ( n = 3). (N–O) Ngp NC /RAW and Ngp. HI /RAW cells were transfected with scramble siRNA or STAT1 siRNA, respectively, followed by 12-h stimulation with PBS or LPS. The supernatants and cells were harvested for concurrent measurement of NO levels and Nos2 mRNA expression via Griess assay and qRT-PCR ( n = 3). The data represent the mean ± SEM of at least three independent experiments. Statistical analysis was performed using one-way ANOVA to compare the results. * p < 0.05

Article Snippet: STAT1 antibody (bsm-33270 M) for LSCM was obtained from Bioss (Beijing, China).

Techniques: Expressing, RNA Sequencing, Phospho-proteomics, Microarray, Western Blot, Protein-Protein interactions, Binding Assay, Co-Immunoprecipitation Assay, Generated, Control, Transfection, Griess Assay, Quantitative RT-PCR

Journal: Science Advances

Article Title: Intratumoral antigen-presenting cell activation by a nanovesicle for the concurrent tertiary lymphoid structure de novo neogenesis

doi: 10.1126/sciadv.adr1299

Figure Lengend Snippet: ( A ) Mechanisms of ADU-S@M1–mediated APC activation. Figure created with BioRender.com . ( B ) Relative amounts of miRNA155 in different EV formulations. ( C to E ) Western blot and real-time polymerase chain reaction (RT-PCR) analysis of p-STAT1 and p-STAT3 in M2 cells with indicated treatments. ( F ) Western blot analysis of p-65, p-TPK1, and p-IRF3 in M2 cells with indicated treatments. ( G and H ) RT-PCR detection of IFN-β and CXCL10 in M2 cells with indicated treatments. ( I and J ) Proportions of CD86-positive and CD206-positive cells (gated on CD11b + cells) in different groups. ( K ) Western blot analysis of p-65, p-TPK1, and p-IRF3 in DCs with indicated treatments. ( L and M ) RT-PCR detection of IFN-β and CXCL10 in DCs with indicated treatments. ( N ) Fluorescence imaging of M2 cells with indicated treatments. Green, iNOS; blue, cell nucleus. Scale bar, 50 μm. ( O and P ) Expression levels of CD80 and MHC II (gated on CD11c + cells) in DCs with indicated treatments. ( Q ) Schematic illustration of the transwell assays. Figure created with BioRender.com . ( R ) In vitro cytotoxicity of M2 cells against B16F10-OVA cells after indicated treatments. ( S and T ) Proportions of H-2Kb OVA positive macrophages (gated on CD11b + cells) and DCs (gated on CD11c + cells). ( U and V ) Proportions of CD69 + T cells (gated on CD8 + T cells) in upper (U) and bottom (V) chambers. Data in (B), (D), (E), (H) to (K), (O) and (P), and (S) to (V) were presented as mean values ± SD ( n = 3). Statistically significant differences were identified by one-way ANOVA. **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05. n.s., not significant; MFI, mean fluorescence intensity.

Article Snippet: Anti–p-STAT1 antibody, anti–p-STAT3 antibody, anti–p-IRF3 antibody, anti–p-TBK1 antibody, and anti–p-p65 antibody were purchased from Bioss (China).

Techniques: Activation Assay, Western Blot, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Imaging, Expressing, In Vitro