The raw count data was background subtracted, base 2 log transformed, centered so each gene had a similar mean or median, and scaled so each gene has a similar standard deviation. cycle distribution with accumulation of G1- and loss of G2/M-phase cancer cells. release, and induce apoptosis . To understand the role of CDK11 in melanoma, we evaluated levels of CDK11 in benign melanocytes and melanoma cell lines. Next, we investigated the effects of CDK11 downregulation on melanoma cell viability, clonal survival and tumorsphere formation as well as on various (S)-Tedizolid signaling pathways and cell cycle distribution. Our data presented herein demonstrate that CDK11 is highly expressed in both BRAF- and NRAS-mutated melanoma cell lines. Loss of CDK11 induces cell cycle dysfunction and death of BRAF- and NRAS-mutant melanoma cell lines. Overall, our data indicate the dependence of melanoma cells on CDK11 expression for survival. 2. Results 2.1. CDK11A and CDK11B mRNA Expression in Non-Transformed Melanocytes and Melanoma Cell Lines We determined steady (S)-Tedizolid state mRNA expression levels for both CDK11 genes in cultured cells, comparing several BRAF- and NRAS-mutant melanoma cell lines and using adult primary human epidermal melanocytes as a reference control (Table 1). Data from quantitative real-time reverse transcriptase PCR (qRT-PCR) are summarized in Table 2. CDK11 mRNA levels were lower in malignant cells compared to primary melanocytes in all of the melanoma cell lines tested, except for CDK11A mRNA in WM39 cells. We include the data for MYC as an example for a gene generally showing higher mRNA expression levels in melanoma cells relative to non-transformed melanocytes. Table 1 Characteristics of melanoma and melanocyte cell lines. 0.05. 2.4. Loss of CDK11 Expression Has a Negative Impact on the Ability of Melanoma Cells to Form Colonies and Tumorspheres We used a clonal survival assay in A375 and WM1366 cells, each transfected one time with 30 nM siCDK11 or siControl siRNAs or left untreated. Forty-eight h after transfection, the cells were collected, counted and plated in Rabbit Polyclonal to SYTL4 triplicate into 35 mm plates. After 7 days of incubation, the cell colonies were stained with crystal violet and counted. Down-regulation of CDK11 protein expression resulted in a more than 75% reduction in colony formation compared to either siControl treated or untreated cells in both BRAF- and NRAS-mutant cell lines (Figure 3A). Open in a separate window Figure 3 Down-regulation of CDK11 inhibits clonal survival and tumorsphere formation in melanoma cells. A375 and WM1366 cells were transfected with 30 nM siRNAs as indicated in the legends and as described in materials and methods. (A) For clonal survival analysis, cells were plated onto 35 mm plates 48 h post-transfection and colonies were stained and counted seven days after plating. Left: The chart presents means SD from three experiments with three replicate plates each. ^ = 0.0001. Right: Representative crystal violet stained colonies on 35 mm plates. Cell lines are indicated to the left of images and siRNA transfections are indicated below plate images. (B) For tumorsphere formation, cells were plated into 96-well ultra-low attaching plates 48 (S)-Tedizolid h post-transfection and images captured 96 h after plating. Left: The chart presents means SD from three experiments with three areas each. ^ = 0.0001. Right: Representative tumorsphere images. Cell lines are indicated to the left of images and siRNA transfections are indicated below plate images. We next employed tumorsphere formation assays in A375 and WM1366 cells. Cells were transfected in the same manner as the clonal survival assays. Forty-eight h after transfection, cells were collected, counted, and plated in triplicate into ultra-low attachment plates. After 96 h, images of the tumorspheres were captured and measured. Transfection of A375 and WM1366 cells with control siRNAs resulted in the formation of robust tumorspheres that were comparable to tumorsphere size and morphology in untreated cells. Downregulation of CDK11 (S)-Tedizolid caused much smaller tumorspheres to form, which were also less dense and loosely formed (Figure 3B). 2.5. Effects of CDK11 Signaling Reduction on Melanoma Growth Pathways We examined the effects.