Macrophage Polarization Assay with KS-133

The detailed method and DNA primer sequences were described previously [7 (link)]. RAW264.7 murine macrophage-like cells and CT26.WT murine colorectal cancer cells (CRL-2638) were purchased from RIKEN BRC (Tsukuba, Japan) and American Type Cell Collection (Manassas, VA, USA), respectively. CT26 cells (4 × 10⁶) were grown in T75 flask with 20 mL of RPMI medium containing 10% FBS for 3 days. The culture medium (CT26-CM) was collected and centrifuged at 400 g for 5 min. RAW264.7 cells (2.4 × 10⁵) were incubated in 24-well plate in the presence or absence of KS-133 (KS-133 concentration = 1, 3, or 10 μM, DMSO concentration = 0.1%) with 20% CT26-CM/DMEM for 3 days. During the incubation, medium containing KS-133 was replaced every day. mRNA extraction was performed using ISOGEN (Nippon Gene, Tokyo, Japan) on day 3 after CT26-CM incubation, and cDNA was prepared. The gene expression of M1 markers (TNF-α, iNOS, CXCL10), M2 markers (Mrc-1, IL-1rn, CCL22), and β-actin was analyzed by real-time polymerase chain reaction (PCR) using a 7300 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, MA, USA,) and Light Cycler Fast Start DNA Master kit (Applied Biosystems). The relative mRNA expression was determined using the 2^−ΔΔCt method. ΔΔCt was calculated using control group data.