Fig 2: Expression levels of CDK4- and CDK6-producing effect to ribociclib sensitivity.(A) CDK6 overexpression cell lines (MCF7-C6) were established from MCF-7 by the stably transfected CDK6 expression vector. Cell lines–transfected control vector (MCF7-C6 Ctrl) was simultaneously established as a negative control. (B) Western blotting demonstrated the protein expression levels of CDK4 and CDK6 in MCF7-C6 Ctrl and MCF7-C6 cell lines. (C) Cell proliferation assay of MCF-7, MCF7-C6 Ctrl, MCF7-C6, and MDA-MB-231 cell lines treated with ribociclib for 3 days was measured relative to the negative control (treated with DMSO). The results are expressed as mean ± SD of three independent experiments; * P < 0.05. (D), MCF7-C6 Ctrl and MCF7-C6 cell lines were treated with DMSO or ribociclib (500 nM) for 24 h and measured by fluorescence-activated cell sorting (FACS) cell-cycle analysis. (E), two kinds of siRNA for CDK4 (siRNA1, siRNA2) or nonspecific control siRNA (scr) were transfected for 24 h with MCF7-C6 Ctrl and MCF7-C6 V1 cell lines. Protein expression levels of CDK4 and CDK6 were analyzed by western blotting with β-tubulin as a protein loading control. (F) Cell proliferation of MCF7-C6 Ctrl -scr, -siRNA1, -siRNA2, MCF7-C6 V1-scr, -siRNA1, and -siRNA2 treated with ribociclib (100 nM) for 3 days was measured relative to dishes treated with DMSO as a negative control. The results are expressed as mean ± SD of two independent experiments; * P < 0.05.

Fig 3: ZC3H15 is involved in the G1-to-S phase transition of melanoma cell cycle. (a) The cell cycle of MV3 and A375 cells after ZC3H15 silencing was analyzed by flow cytometry, and the percentage of indicated cells in different phases was determined. (b, c) The expression of proteins related to G0/G1 phase including CDK4, CDK6, CCND1, and p21 was detected by Western blot in ZC3H15-knockdown and ZC3H15-rescued cells. The data are represented as the mean ± SD; ∗∗P < 0.01.

Fig 4: Aminoquinol treatment inhibited tumor growth in vivo in BALB/C nude mice xenografted with Huh7 cells. BALB/C nude mice xenografted with Huh7 cells were treated with aminoquinol (35 mg/kg), 5-Fu (10 mg/kg), aminoquinol (35 mg/kg) plus 5-Fu (10 mg/kg) and PBS for 21 days daily by intraperitoneal injection (A) Tumor weight at 21 days after treatment (B) Tumor volumes during the experiment (C) The body weight of the mice during the experiment (D–K) The representative pictures and quantified data of immunohistochemistry staining of the xenografted tumor tissues for the expressions of (D) Rb, (E) CDK4, (F) CDK6, (G) ki67, (H) PI3K, (I) AKT, (J) P-AKT, and (K) mTOR. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from the control PBS treatment group.

Fig 5: Activation of PDGFR signal transduction pathway-related kinases and targets in PDGFRA-mutated HEK293T cells. (A–F) The levels of pGSK3βSer9 were decreased in 14D6 cells (A,B). The expression of the PDGFR signal transduction pathway targets, cyclinD1 (A,C), c-Myc (A,D), CDK4 (A,E), and CDK6 (A,F), was increased in 14D6 compared to isogenic control cells. The amounts of phosphorylated proteins were normalized to those of total proteins. *, p < 0.05; **, p < 0.01. For uncropped blot source of (A), see Supplemental Fig. S13.