The binding potency of M4K compounds to ALK2 has been assayed in biochemical assay and cellular assays in HEK293 or C2C12 myoblast cell lines. However, the potency of ALK2 inhibition by M4K compounds has not determined directly in DIPG patient-derived cell lines. While no major deviation from existing assay data is expected, direct experimental evidence is essential. DIPG cells will be trypsinized and resuspended in TSM-base medium without any growth factor for one-hour starvation. Subsequently, equal volume of TSM-base with 2X Activin A (200ng/mL) and M4K compounds or DMSO vehicle control will be added. After one-hour treatment, the cells will be pelleted and lysed in buffer with protease and phosphatase inhibitors for Western Blot analysis. For other related studies, please refer to my opennotebook blog. https://openlabnotebooks.org/evaluating-the-inhibition-of-alk2-phosphorylation-of-smad1-5-by-m4k-lead-compounds-in-dipg-patient-derived-cells-su-dipg-iv-hsjd-dipg-007-hsjd-dipg-018-and-su-dipg-xxi/