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. 2022 Sep 20;13(9):806. doi: 10.1038/s41419-022-05243-4

Fig. 3. Ciliobrevin D (CilioD) treatment phenocopies kinesin family member 3A (KIF3A) knockdown in the AT/RT cell lines BT‐12 and CHLA‐266.

Fig. 3

BT‐12 and CHLA‐266 cells were seeded in complete medium. The next day, complete medium was replaced with medium lacking serum and the cells were treated with 30 µM CilioD or dimethylsulfoxide (DMSO) as a negative control. a CilioD treated BT‐12 and CHLA‐266 cells were incubated with 10 μM EdU for 6 h. Then, cells were fixed and EdU (modified thymidine analogue) was detected using Alexa Fluor™ 488 azide. Cells were counterstained with 4´,6‐diamidino‐2 phenylindole (DAPI). The percentage of proliferating cells was calculated based on the number of EdU‐labeled cells in relation to the number of DAPI‐labeled cells. b Quantification of colonies formed by CilioD treated BT‐12 and CHLA‐266 cells following plating of 100 (BT‐12) or 500 (CHLA‐266) cells per 10 cm dish and 17 (BT‐12) or 24 (CHLA‐266) days of incubation. c, d Cell cycle profiles of CilioD treated BT‐12 (c) and CHLA‐266 (d) cells. Cells were fixed and stained with propidium iodide (PI). DNA content was analyzed using flow cytometry. e To assess cells undergoing apoptosis, cells were stained with Annexin V and PI, and analyzed using flow cytometry. Shown are bar graphs representing the fold change in apoptotic cells following treatment with 30 μM of CilioD for 24 h or DMSO as a negative control in the AT/RT cell lines BT‐12 and CHLA‐266. Values shown represent mean ± SEM of three biologically independent replicates. nd: not detected, ns: not significant, *p < 0.05; **p < 0.01; ***p < 0.001 (t-test).