Tumor-associated macrophages (TAMs) with or without HRS knockdown (KD)

To study the mechanism by which HRS regulates macrophage polarization, we performed bulk RNA sequencing on TAMs with or without HRS KD. Overall design: Enriched human monocytes were obtained from healthy volunteers by Human Immunology Core at the University of Pennsylvania. Human macrophages (Mf) were generated by incubating human monocytes with GM-CSF (10 ng/mL) for 5 days. Briefly, to produce conditioned medium (CM), WM9 were seeded at 70-80% confluence in 15-cm plates and cultured in RPMI1640 medium supplemented with 100 IU penicillin, 100 µg/ml streptomycin and 2% FBS for 3 days. The supernatant was then collected and concentrated 40-fold using centricons (Millipore, Inc.). To make the final CM, concentrated media were diluted at a 1:80 in complete RPMI 1640 or DMEM. TAMs were subsequently induced by incubating with CM for 48 hrs. TAMs with or without HRS KD were collected, and the total RNA was extracted using a PureLink RNA mini kit (Invitrogen) and the RNA-seq was performed by Novogene. The ClusterProfiler package (version 3.18) was then used for GO enrichment analysis.