Supplementary Material for: Effects of Simvastatin on inflammatory response and biological behavior of adamantinomatous craniopharyngioma

Introduction: To investigate the autoinflammatory effect and biological behaviour of simvastatin (SIM) on adamantinomatous craniopharyngioma (ACP) cells. Methods: Craniopharyngiomas imaging, intraoperative observations, and tumour histopathology were employed to investigate the correlation between esters and craniopharyngiomas. Filipin III fluorescent probe verified the validity of SIM on the alternations of synthesized cholesterol in craniopharyngioma cells. The cell counting kit-8 (CCK8) assay detected the impacts of SIM on cell proliferation and determined the IC50 value of tumour cells. Reverse transcription polymerase chain reaction (RT-PCR) measured the expression of inflammatory factors. Flow cytometry technique detected the cell cycle and apoptosis, and cell scratch assay judged the cell migration. Meanwhile, Western blot was adopted to determine the expression of proteins related to inflammation, proliferation, and apoptosis signalling pathways. Results: In the ACP tumour parenchyma, many cholesterol crystalline clefts were observed, and the deposition of esters was closely associated with craniopharyngioma inflammation. After simvastatin intervention, a reduction in cholesterol synthesis within ACP was noted. RT-PCR analysis revealed SIM inhibited the transcription of inflammatory factors in ACP cells. Western blot analysis demonstrated SIM inhibited nuclear factor-kappa B (NF-κB) p65 activation expression while promoted the expressions of Cl-caspase-3 and P38 MAPK. CCK8 assay indicated a decrease in ACP cell activity upon SIM treatment. Scratch assay signified that SIM hindered ACP cell migration. Flow cytometry results suggested that the drug promoted ACP cell apoptosis. Conclusion: SIM suppressed the inflammatory response to craniopharyngiomas by inhibiting craniopharyngioma cholesterol synthesis, inhibited proliferation of ACP cells, and promoted their apoptosis.