Dulbecco phosphate buffered saline (dpbs)

To detect the cell surface expression of xlMC3R.L and xlMC3R.S by xlMRAPs, the expression of xlMC3Rs and xlMRAPs on cell membrane was measured by cell surface ELISA. Concisely, cells were cultured in 24-well plates, covered by poly-d-lysine solution (Sangon), and transfected with xlMC3R and xlMRAPs at different ratios of 1:0, 1:1, 1:3, 1:6. 24 h later. DPBS (Sangon) and 4% (w/v) paraformaldehyde (Sangon) were used for washing and fixing cells, respectively. To block nonspecific binding, cells were incubated with 5% (m/v) non-fat milk (Sangon) in DPBS (Sangon) for 1 h. These cells were incubated with HA-tag rabbit mAb (CST) or FLAG-tag rabbit mAb (diluted 1:4000) (CST) in DPBS (Sangon) supplemented with 5% (m/v) non-fat milk (Sangon) for 2 h, washed with DPBS (Sangon), and incubated with the secondary HRP-conjugated antibody (1:7500) (ABclonal Biotech) for 1 h. The tetramethylbenzidine (TMB) solution (Beyotime) was added to the plate for 30 min. The reaction was stopped by adding 2 M H₂SO₄, and optical density was detected at 450 nm by a Spectramax iD3 Multi-Mode Microplate reader. Experiments were replicated independently at least three times.

H9‐CMs were grown in 6‐well plates to 80% confluence, detached with TrypLE (Gibco) and then pelleted at 12 000 rpm for 3‐5 minutes at 4°C. After washing with DPBS (Sangon Biotech), the pellets were re‐suspended in 50‐100 μL lysis buffer. Lysates were placed on ice for 30 minutes, and then, the supernatants were collected after centrifuging at 12 000 rpm for 5 minutes. Protein concentration was measured using a BCA kit (Pierce). Western blot was performed using standard protocol with the following antibodies: caspase 3 (Cell Signaling Technology), phosphorylated P38 (Cell Signaling Technology), total P38 (Cell Signaling Technology), total c‐Myc (Cell Signaling Technology), phosphorylated Akt (Cell Signaling Technology) and total Akt (Cell Signaling Technology).

Fresh tumor tissue samples were washed with DPBS (Hyclone, Logan, UT, USA) and cut into 2–4 mm³ slices. The tissue samples were suspended in 1640 medium (Gibco Tour, Grand Island, NY, USA) and transferred to a gentleMACS C tube containing an enzyme mix. This tube was connected to the gentleMACS Octo Dissociator, and the appropriate procedure was performed. The digested tissue was then passed through a 70-μm strainer, and the cell suspension was centrifuged at 300 g for 7 min. After removal of the supernatant, the cells were subsequently resuspended in erythrocyte lysis buffer and incubated at 4 °C for 10 min to remove the erythrocytes. The cells were centrifuged at 300 g for 10 min at 4 °C, and the supernatant was removed without disturbing the cell precipitation. Finally, 5 mL of frozen wash buffer (0.04% BSA(Sangon Biotech, Shanghai, China) in DPBS) was added to the cells and the mixture was stirred with gentle puffing to resuspend the cell precipitate. The cell viability in all samples was >80% and determined via trypan blue (Sigma, Darmstadt, Germany) staining. The final cell concentration was adjusted to 700–1200 cells/μL for single-cell library preparation.