Fig 2: Cyclin-dependent kinase–like 3 (CDKL3) promotes the growth of osteosarcoma (OS) cells.(A) Comparison of CDKL3 expression levels by qRT-PCR analysis between adjacent non-tumor tissues and OS tissues derived from four OS patients. Three OS and three non-OS tissues from each patient were collected and analyzed. Two housekeeping genes (ACTB and GAPDH) were included in this study to normalize the CDKL3 expression. (B) Cell growth analysis of U2OS cells transfected with indicated siRNAs. (C) Cell growth analysis of U2OS and Saos-2 cells with CDKL3-shRNA silencing (n = 5). (D) Colony formation assay of cells conducted in (C). Scale bar = 1 cm. (E) Representative colony images and quantitative analysis from (D) (n = 3). Scale bar = 200 μm. Error bars represent SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

Fig 3: Generation of CDKL3-KO cells.(A) Schematic diagram showing the KO strategy of CDKL3. The sgRNA and PAM sequences were marked in purple and green, respectively. (B) Mapping of nucleic acid sequence and its corresponding amino acids on Sanger sequencing.

Fig 4: 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 5: Cyclin-dependent kinase–like 3 (CDKL3)-KO inhibits osteosarcoma (OS) cell growth.(A) Fold change of U2OS and Saos-2 cell growth at indicated time courses after cocultured with CDKL3-KO cells. (B) Representative images of FACS data from cell growth competition assay, U2OS, and Saos-2 cells (GFP+) were cocultured with CDKL3-KO U2OS and Saos-2 cells. Error bars represent SD (n = 4). *P < 0.05, ***P < 0.001, two-tailed t test.