Fig 2: Cyclin-dependent kinase–like 3 (CDKL3) critically regulates Akt activation in osteosarcoma (OS).(A) Akt and mTOR activation under regular or starvation conditions in parental and CDKL3-KO U2OS cells. Upon CDKL3 KO, Akt and mTORC1 activation were significantly alleviated. The numbers below each blotting strip is intensity quantification by ImageJ. (B) The expression patterns of mTORC1 and FoxO downstream target genes confirm that CDKL3 regulates both pathways at the transcription level. Cells were cultured under the regular growth condition. (C) Overexpression of CDKL3 in U2OS cells causes hyper-activation of Akt in the presence and absence of FBS. (D) General working mechanism of rapamycin, AZD5363, and MK-2206. (E) U2OS cell growth under different conditions (n = 3). (F) Co-immunoprecipitation (Co-IP) reveals the physical interaction between HA-CDKL3 and Myc-Akt1. (G) Endogenous CDKL3 coexists with endogenous Akt shown by co-IP. (H) Schematic diagrams of Akt1 constructs for mapping. (I) Co-IP of HA-CDKL3 with different Akt1 constructs shows that the Akt kinase domain is indispensable for CDKL3 interaction. (I, J) Reverse IP of CDKL3 and Akt1 confirms the findings in (I). Error bars indicate SD (n = 3). *P < 0.05 parental DMSO versus CDKL3−/− DMSO, or parental rapamycin, or parental AZD5363, or parental MK-2206; **P < 0.01 CDKL3−/− DMSO versus CDKL3−/− rapamycin, or CDKL3−/− AZD5363, or CDKL3−/− MK-2206; ##P < 0.01 parental MK-2206 versus CDKL3−/− MK-2206. Statistical significance of the differences was estimated by unpaired two-tailed t test.Source data are available for this figure.

Fig 3: Apoptotic cell analysis of cyclin-dependent kinase–like 3 (CDKL3)-KO osteosarcoma (OS) cells.(A) FACS analysis of parental and CDKL3-KO U2OS and Saos-2 cell apoptosis. Magenta circle represents apoptotic cell population. Error bars represent SD (n = 3). (B) Caspase3 and Caspase8 immunoblotting to detect apoptosis status for parental and CDKL3-KO U2OS cell under normal or starvation conditions. Solid and hollowed triangles represent uncleaved and cleaved Caspase3 and Caspase8, respectively.Source data are available for this figure.

Fig 4: Knockdown of cyclin-dependent kinase–like 3 (CDKL3) inhibited osteosarcoma (OS) cell growth.(A) qRT-PCR analysis in U2OS cells transfected with siRNAs. Error bars indicate SD (n = 3). (B) Western blotting confirming the knockdown of CDKL3 in U2OS cells. (C) qRT-PCR analysis in targeted cells transfected with shRNAs. Error bars indicate SD (n = 5). (D) Fluorescence photomicrographs of Saos-2 cells in consecutive 5 d after shCDKL3 infection at a magnification of 100×. Scale bar = 150 μm. (E) Fold change of cell count in consecutive 5 d, which equals to cell counts divided by the cell counts in the first day after infection. Control: Saos-2 cells infected with lentivirus containing control shRNA. shCDKL3: Saos-2 cells infected with lentivirus specific interfering of CDKL3 (n = 3). Error bars indicate SD (n = 3 or n = 5). ***P < 0.001, two-tailed t test.Source data are available for this figure.

Fig 5: HA-CDKL3 co-immunoprecipitates endogenous Akt.Source data are available for this figure.