Accuri c6 machine

Freshly isolated tumor, spleen and BM samples from each group were passed through a 40µm-cell strainer to make single cells. Cells were labeled with antibodies (Bio Legend) against immune cell antigens such as CD45, CD4, CD8, Gr1, CD11b, F4/80, CD68, CD206 (mannose receptor, M2), and CD86 (mature dendritic cells, M1) in the tumor as well as in spleen and BM. CSC antigens such as CD133 and CD44 were analyzed on tumor cells (CD45−). Immunogenicity or presence of MHC1 (H2Ld-H2Db) on GL261 tumor cell line was analyzed using specific antibody (BioLegend). Flow cytometry data were acquired using Accuri C6 machine (BD Biosciences) and analyzed by BD Accuri C6 software.

A549, 293T or 293T (+ pCMV Shh) cells were labeled with Shh antibody (Abcam 53281, 1:100) or therapeutic test antibodies (1:100) for 1 hour at room temperature after serum blocking. Following two PBS washes, a FITC-conjugated secondary antibody (Abcam 97029, 1:100) or an Alexa-Fluor 647 (Invitrogen, 1:1000) was used to label cells. Samples were screened by flow cytometry using an Accuri™ C6 machine (BD Biosciences). For fluorescence activated cell sorting, samples were sorted using an S3e™ Cell Sorter (Bio-Rad) and the Shh+ population was collected. For all experiments, cells processed without primary antibody served as a negative control. Experiments were performed in duplicates or triplicates.

MDA-MB-231 cancer cells (1.5 × 10⁶ cells) were cultured in a 6-well plate for 24 h and treated with DMSO as control or dihydroconiferyl ferulate (50 μM) for 24 h [63 (link)]. The cells were harvested, dissociated, and incubated with monoclonal antibody antihuman CD44 (FITC-conjugated) and monoclonal antibody antihuman CD24 (APC-conjugated) antibodies (BD) for 45 min at 4 °C. After washing with 1X PBS, the CD44⁺/CD24⁻ cell populations were examined using an Accuri C6 machine (BD San Jose, CA, USA). The Annexin V Apoptosis Detection kit with PI (BD) was used to measure apoptosis of the mammospheres treated with dihydroconiferyl ferulate (50 μM) following the manufacturer’s protocol. Mammospheres were collected and dissociated with 0.05% trypsin-EDTA 1X (Corning). Briefly, 1 × 10⁶ cells were incubated with Annexin V (FITC) and PI in a binding buffer at room temperature (RT), protected from light for 30 min, and the cells were examined via flow cytometry at the Jeju Center of Korea Basic Science Institute (core facility center, Jeju, South Korea). Mammospheres derived from MDA-MB-231 cells cultured with or without dihydroconiferyl ferulate (50 µM) were divided into single cells, and an equal number of cancer cells were seeded into six-well plates. The number of cells was counted at 24, 48, and 72 h to measure the growth of the mammospheres.

HCT116 cells were seeded at 1 × 10⁵ cells/well in a six-well plate and grown for 2 days. In case of inducing the expression of OsTIR1(WT or F74G) under the control of the Tet promoter (Supplementary Fig. 1F), cells were treated with 0.5 µg/mL of doxycycline for 24 h, and then 100 µM IAA or 1 µM 5-Ph-IAA was added. For detecting EGFP and Clover signals after ligand treatment, cells were trypsinized and fixed in 4% methanol-free paraformaldehyde phosphate buffer (FUJIFILM Wako Pure Chemical Corporation) at 4 °C overnight. Fixed cells were washed and resuspended in PBS containing 1% BSA. To detect OsTIR1-V5 in Supplementary Fig. 1F, fixed cells were treated with anti-V5 antibody (Invitrogen, #R960-25) and subsequently stained with Alexa Fluor 647 anti-mouse IgG (ThermoFisher, #A-21236). For measuring the DNA signal after ligand treatment, cells were trypsinized and fixed in 70% EtOH. Fixed cells were washed, resuspended in PBS containing 1% BSA, 50 μg/ml of RNase A, and 40 μg/ml of propidium iodide, and incubated at 37 °C for 30 min. Flow cytometric analysis was performed on a BD Accuri C6 machine (BD Biosciences) using FCS4 Express Cytometry software (DeNovo Software). Ten thousand cells were analyzed from each sample, except in the case of Supplementary Fig. 1F, in which 50,000 cells were analyzed.