Journal: Frontiers in Immunology

Article Title: A tumor immune microenvironment gene expression signature for predicting prognosis, immunotherapy efficacy, and drug candidates in triple-negative breast cancer

doi: 10.3389/fimmu.2025.1676768

Figure Lengend Snippet: The immune microenvironment–reprogramming function of NCD is mediated by JAK2 inhibition. (A) Volcano plot of DEGs in MDA-MB-231 cells treated with or without 5 μM NCD. (B) KEGG enrichment analysis of DEGs. (C) Western blot of JAK2, p-JAK2, STAT3, and p-STAT3 in MDA-MB-231 cells treated with increasing doses of NCD (n=3). (D) Quantification of JAK2, p-JAK2, STAT3, and p-STAT3 proteins corresponding to (C) (n = 3). (E) Western blot of JAK2, p-JAK2, STAT3, and p-STAT3 in 4T1 cells treated with increasing doses of NCD (n=3). (F) Quantification of JAK2, p-JAK2, STAT3, and p-STAT3 proteins corresponding to (E) (n = 3). (G) Protein expression levels of JAK2 after knockdown of JAK2 in MDA-MB-231 cell line (n=3). (H) Quantification of JAK2 protein levels corresponding to (G) (n=3). (I) mRNA expression levels of JAK2 , CXCL10 , CXCL11 , EBI3 , FLT3LG after knockdown of JAK2 in MDA-MB-231 cell (n=3). (J) Protein stability of JAK2 under thermal gradient with or without NCD (n=3). (K) CETSA Melting Curve (n=3). (L) SPR sensorgrams of JAK2 binding to NCD. (M) Affinity analysis and KD calculation of JAK2-NCD interaction. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Membranes were blocked and then incubated with the following primary antibodies: Stat3 (124H6) Mouse mAb, Phospho-Stat3 (Tyr705) (D3A7) XP ® Rabbit mAb, Jak2 (D2E12) XP ® Rabbit mAb, Phospho-Jak2 (Tyr1007/1008) Antibody (all from Cell Signaling Technology), and Alpha Tubulin Monoclonal antibody (Proteintech).

Techniques: Inhibition, Western Blot, Expressing, Knockdown, Binding Assay