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    • Plant material and growth conditions Arabidopsis thaliana

    2024-03-14

    Plant material and growth conditions. Arabidopsis thaliana ecotype Columbia was grown on a mixture of soil, vermiculite, and perlite (4:2:1) with a 16h light/8h dark Rifapentine in a temperature-controlled growth room (22°C), unless indicated otherwise. Molecular cloning and plant transformation. The cyclin D1 cDNA was cloned following a yeast two-hybrid screening method [21]. The binary vector was introduced into Agrobacterium tumefaciens strain LBA4404 and used to transform Arabidopsis plants by vacuum infiltration [22]. Histochemical analyses of β-glucuronidase (GUS) expression were performed as described previously [23]. Kinase activity. Recombinant cyclin D1 affinity columns were used to purify the corresponding kinase domain(s) from the lysate of Arabidopsis suspension cultured cells. Purified p22ack1 protein was added to the kinase reaction mixture (50mM Tris–HCl, pH 7.4, 250mM NaCl, 50mM NaF, 5mM EDTA, 100μM ATP, and 5.0μCi [γ-32P]ATP) and 32P-labeled histone H1 was detected by autoradiography. RNA extraction and RT-PCR. Total RNA was extracted from leave tissues using TRI reagent (Sigma) in accordance with the manufacturer’s instructions. Reverse transcription reactions were carried out using RT-PCR kit (Takara, Japan) with 2μg of total RNA for 5min at 55°C. We then used 1μl of the reverse transcription reaction as a template in a 20μl PCR with 5U of Taq DNA polymerase. The PCR consisted of 24 cycles of 30s at 90°C, 30s at 50°C, and 90s at 72°C. The primers (ACK1) used were: 5′ GAGAAAAGACTT-GTTCGATGGTTCTCATA 3′ and 5′ TGAATTGCTTCTTCTTATCGTCT-TGACTC 3′. In situ hybridization with dioxigenin-labeled RNA probes. Ten micrometer tissue sections were fixed to dimethylsilane-treated microscope slides in 3.7% formaldehyde, 5% acetic acid, and 50% ethanol by overnight incubation at 45°C, cleared in Histo-clear, and embedded in paraffin. To remove paraffin, the slides were immersed twice in 100% xylene for 10min and twice transferred into 100% ethanol for 5min to remove xylene. Slides were then dehydrated in with the following ethanol series [100%–95% 85%–70%–50%–30%–15% PBS (50mM phosphate buffer, pH 7.0, 130mM NaCl in DEPC treated water)] for 5min at each step. The slides were then incubated in l00μg/ml protease K solution in 100mM Tris–HCl, pH 7.5, 50mM EDTA at 37°C for 20min, and washed in PBS twice, 5min each. The slides were immersed immediately in fresh 1% triethanolamine, pH 8.0, 0.5% acetic anhydride mixture. After 10min, the slides were washed in PBS twice, 5min each. The slides were then dehydrated in the ethanol series (15%–30%–50%–85%–95%–100%), washed once with 100% ethanol, and vacuum-dried. Comparable tissue sections were hybridized with different probes (p22ack1, cyclin D1). Dioxigenin (DIG)-labeled RNA probes were made by in vitro transcription in accordance with manufacturer’s instructions. Hybridized slides were washed for 1h at room temperature in 2× SSC and 0.01M DTT and for 1h at 48°C in 0.1× SSC, 0.01M DTT, and 50% formamide. Slides were then dipped into photographic emulsion and exposed for 3–5 days before development.
    Results and discussion
    Acknowledgments
    ACK1 (Activated Cdc42Hs-associated Kinase 1) is a nonreceptor tyrosine kinase that was first identified by its interaction with Cdc42 and was suggested to function as an effector of Cdc42, Rac, and FAK in several integrin signaling pathways. ACK1 has also been identified as a regulator of endocytosis via direct association with clathrin. ACK1 is ubiquitously expressed, with highest expression levels in the brain, and has been shown to be overexpressed in a number of human tumor cell lines potentially predisposing them to become metastatic. Recently it was suggested that ACK1 activity is required for the survival of v-RAS-transformed murine fibroblasts. Moreover, activation of ACK1 has been shown to stimulate prostate tumorigenesis. These findings suggest that inhibition of ACK1 could be a potential new point of intervention in preventing the onset and spread of cancer. The development of a potent and selective ACK1 inhibitor would allow the role of ACK1 in tumor progression to be probed. Toward this end, herein we report on a new class of ACK1 inhibitors.