Fig. 6. Trim21-mediated CDK2 autophagic degradation in cancer cells.

A THP1 cells transfected with either Trim21-HA plasmid or empty vector were treated with HHT as indicated for 9 hours, and the protein levels of CDK2 and Trim21 were analyzed by western blot with β-actin as the loading control. B THP1 cells transfected with either Trim21-HA-ΔC plasmid or empty vector were treated with HHT as indicated for 9 hours, and the protein levels of both CDK2 and Trim21 were analyzed by western blot with GAPDH as the loading control. C THP1 cells were transfected with siRNA targeting either Trim21 or GFP, and after 48 hours treated with HHT as indicated for 9 hours. The protein levels of CDK2 and Trim21 were analyzed by western blot with GAPDH as the loading control. D Mononuclear cells isolated from a primary AML patient were transfected with siRNA targeting either Trim21 or GFP. Forty-eight hours after transfection, cells were treated with HHT as indicated for 12 hours, and the protein levels of CDK2 and Trim21 were analyzed by western blotting with GAPDH as the loading control. E HEK293 cells were transfected with either CDK2-GFP or CDK2-3As-GFP as well as mRuby2-Trim21; then cells were either treated with HHT (50 ng/mL) or DMSO for 9 hours, and the localizations of CDK2 (green) and Trim21 (red) were determined by fluorescence microscopy (upper panel). Scale bar, 20 μm. The positive cell ratio with colocalization between CDK2-GFP and mRuby2-Trim21 after HHT treatment in three different experiment replicates was analyzed (lower panel). Data represent the mean ± SD from three independent biological samples for each group. P values are indicated by two-tailed unpaired Student’s t test. F A putative model of Trim21-mediated autophagic degradation of CDK2 protein induced by HHT. All the results shown here were representative of three independent experiments. Source data are provided as a Source data file.