Lmk 235

6-week-old C57BL/6 mice were from Charles River Laboratories (Wuhan, China). The study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Panc02 cells (5 × 10⁶ in 100 µL 1 × PBS) were injected s.c. into the right flank of mice. The volume of the allograft was calculated by the formula (L × W² × 0.5). After the allografts reached a size of 50 mm³, mice carrying same types of tumors were divided randomly (simple randomization) into different groups and treated with anti-PD-1 (BioXcell, Clone RMP1-14) or IgG (BioXcell, Clone 2A3) with or without the co-treatment of LMK235 (MedChemExpress, HY-18998). Mice were euthanized at day 24 (mice meet the standard for euthanize before the end-day because of unexpected reasons like ulceration or infection were excluded from the analysis). Tumors were collected for flow cytometry analysis. KPC derived PDAC cells were cultured as described previously 26 (link). 10⁵ KPC derived PDAC cells were injected orthotopically into C57BL/6 mice. Mouse was euthanized when it meets the end-point standard required by ethics committee. And the tumor was collected for immune-fluorescence and FACS.

LMK-235(Cat# HY-18998) and Vorinostat (Cat# HY-10221) were purchased from MedChemExpress (MCE, USA). KYSE150 and TE-1 cells, treated with LMK-235 for 48 hr, were plated at 2×10³ cells/well into 96-well plate in five replicates. Cell proliferation was measured using CCK-8 kit (Dojindo, Japan) at the following day (day 0) and every 24 hr after (up to day 4). Optical density (OD) was read at 450 nm by SpectraMax M5e Microplate Reader (Molecular Devices, CA, USA).
For EdU (5-Ethynyl-2´-deoxyuridine) incorporation assay, the above-mentioned cells were seeded into glass bottom dish (35 mm) at 5×10⁴ cells/dish in triplicate. After incubation with 10 mM EdU (RiboBio Co., Ltd, China; Cat# C10310-3) for 5 hr, nuclei were counterstained with Hoechst 33342. The percentage of proliferative cells (EdU-positive) was measured within three random fields under LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany) using 20×objective.