Journal: iScience

Article Title: Sleep deprivation-induced sympathetic activation promotes pro-tumoral macrophage phenotype via the ADRB2/KLF4 pathway to facilitate NSCLC metastasis

doi: 10.1016/j.isci.2025.112321

Figure Lengend Snippet: KLF4 is associated with sleep deprivation-induced ADRB2 modulation of macrophage prompting on proliferation, migration, and invasion of LLC-LUC cells (A) Differentially expressed Top20 genes in metastatic lung tissues of NC group and sleep deprivation group. (B) The expression of top 5 upregulated genes in tumor tissues was analyzed by qRT-PCR. (C) KLF4 expression was detected by ADRB2-specific agonist ISO and ADRB2 antagonist ICI118,551 in macrophages treated with IL-4 in vitro . (D) The effect of ADRB2 agonist on CD206 expression in IL-4-induced KLF4-knockdown macrophages. (E) EdU assays showed the proliferation of tumor cells in the coculture system with 30% CM. CM derived from IL-4 and IL-4 + ISO induced supernatant of macrophages with KLF4 low and high expression. (F) The migration ability of tumor cells was determined by wound healing assays in the indicated coculture medium. (G) Transwell assays demonstrated invasion of tumor cells in the indicated coculture system. (H) Representative IHC images of p-JAK1 and p-STAT6 in xenograft tumors. (I) The phosphorylation of JAK1 and STAT6 expression in RAW264.7 treated with ADRB2 agonist or antagonist via Western blotting. (J) Immunoblotting of KLF4 in RAW264.7 treated with different conditions. Data represented as mean ± SD. Statistical analysis was performed using Student’s t test or Mann-Whitney U test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: After blocking the membranes with 5% BSA for 1 h, they were incubated with primary antibodies against KLF4 (Proteintech, Wuhan, China), mouse monoclonal antibody against JAK1 (Proteintech), rabbit polyclonal antibody against p-JAK1 (ImmunoWay, USA), mouse monoclonal antibody against STAT6 (Proteintech) and rabbit polyclonal antibody against p-STAT6 (ImmunoWay) for 2 h, followed by three washes with Tris-buffered saline containing Tween-20 (TBST), and then incubated with appropriate secondary antibodies.

Techniques: Migration, Expressing, Quantitative RT-PCR, In Vitro, Knockdown, Derivative Assay, Phospho-proteomics, Western Blot, MANN-WHITNEY

Journal: iScience

Article Title: Sleep deprivation-induced sympathetic activation promotes pro-tumoral macrophage phenotype via the ADRB2/KLF4 pathway to facilitate NSCLC metastasis

doi: 10.1016/j.isci.2025.112321

Figure Lengend Snippet: NE level is elevated in NSCLC patients with sleep deprivation and is associated with a poor prognosis (A) The concentration of NE in serum of the NSCLC patients, determined by ELISA assay. (B) Representative IHC images and quantitative analysis of ADRB2 in the NSCLC patients. (C) Representative IHC images and quantitative analysis of KLF4 in the NSCLC patients. (D) Investigation into the association between ADRB2 expression and immune cell infiltration in patients diagnosed with LUAD. (E) Analysis of the correlation between KLF4 and immune cell infiltration in LUAD patients. (F–H) Correlation analysis between ADRB2, KLF4, JAK1, and STAT6 expression. (I and J) Correlation analysis between KLF4, JAK1, and STAT6 expression. (K and L) Correlation analysis between JAK1, STAT6, and pro-tumoral macrophages. Data represented as mean ± SD. Statistical analysis was evaluated using Student’s t test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: After blocking the membranes with 5% BSA for 1 h, they were incubated with primary antibodies against KLF4 (Proteintech, Wuhan, China), mouse monoclonal antibody against JAK1 (Proteintech), rabbit polyclonal antibody against p-JAK1 (ImmunoWay, USA), mouse monoclonal antibody against STAT6 (Proteintech) and rabbit polyclonal antibody against p-STAT6 (ImmunoWay) for 2 h, followed by three washes with Tris-buffered saline containing Tween-20 (TBST), and then incubated with appropriate secondary antibodies.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: iScience

Article Title: Sleep deprivation-induced sympathetic activation promotes pro-tumoral macrophage phenotype via the ADRB2/KLF4 pathway to facilitate NSCLC metastasis

doi: 10.1016/j.isci.2025.112321

Figure Lengend Snippet:

Article Snippet: After blocking the membranes with 5% BSA for 1 h, they were incubated with primary antibodies against KLF4 (Proteintech, Wuhan, China), mouse monoclonal antibody against JAK1 (Proteintech), rabbit polyclonal antibody against p-JAK1 (ImmunoWay, USA), mouse monoclonal antibody against STAT6 (Proteintech) and rabbit polyclonal antibody against p-STAT6 (ImmunoWay) for 2 h, followed by three washes with Tris-buffered saline containing Tween-20 (TBST), and then incubated with appropriate secondary antibodies.

Techniques: Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Phagocytosis Assay, Sequencing, Software