Fig 2: Skp2 knockdown promotes apoptosis and inhibits proliferation of AML cells via inhibition of RIP1. (A) Successful knockdown of Skp2 in U937 cells. The expression levels of p53 and GSK3β were increased, whereas those of p90, SOX2, Akt and pSer 9-GSK3β, were decreased. (B) Attenuation of U937 cell proliferation ability upon Skp2 knockdown was detected by CCK-8. (C) Flow cytometry analysis of annexin V-PI-stained U937 cells with successful Skp2 depletion. (D) Lysates from THP1, KG1α, NB4, and U937 cells transfected with control or Skp2 siRNA for 48h were subjected to Western blot analysis for the expression of RIP1, Skp2, and GAPDH (loading control). Data are presented as the mean ± SD of three independent experiments and analyzed by Student's t-test, ***p< 0.001, ****p<0.0001.

Fig 3: Skp2 regulates the expression and function of RIP1 through transcriptional regulation and K63-linked ubiquitination. (A) Cells were treated with 25 µM CHX for 2h, 4h and 6h to analyzed the stability of RIP1 regulated by Skp2; RIP1 protein levels were detected by Western blotting. (B) Cells were treated with 5 µM MG132 for 2h,4h and 6h to determine whether RIP1 was degraded via the ubiquitin-proteasome system (UPS) regulated by Skp2. Western blotting was performed to evaluate the protein levels of RIP1. (C) Co-immunoprecipitation analysis showing a reduction in K63-linked ubiquitination but no significant total ubiquitination of RIP1 upon Skp2 knockdown. (D) RT-qPCR analysis showing that RIP1 mRNA reduction in NB4 and U937 cells upon Skp2 knockdown. Data represented the mean ± SD of three independent experiments and analyzed by Student's t-test, *p< 0.05, **p< 0.01, ***p< 0.001.

Fig 4: Inhibition of RIP1 promotes the differentiation of AML cells but RIP1 is required for ATRA-induced differentiation. (A) Co-immunoprecipitation analysis showing the physical interaction between endogenous RIP1 and RARα proteins in AML cells. (B) NB4-sh-NC and NB4-sh-Skp2 cells were transfected with PML-RARα siRNA respectively, following harvested the cells for Western blot analysis. (C-D) NB4 and U937 cells were treated with Nec-1, ATRA, and a combination of the two agents for 48h, followed by immunoblotting analysis (C) and collected to analyzed CD11b-positive cells by flow cytometry assay (D). (E) RT-qPCR experiments determined the quantification mRNA levels of CD11b, CD14 and C/EBPβ following treatment with Nec-1, ATRA, and Nec-1 plus ATRA for 24h in NB4 and U937 cells. (F) RT-qPCR experiments determined the quantification mRNA levels of RIP1 following treatment with Nec-1, ATRA, and Nec-1 plus ATRA for 24h in NB4 and U937 cells. Data presented as the mean ± SD of three independent experiments. P values were determined by Student's t-test. *p< 0.05, **p< 0.01, ***p< 0.001.

Fig 5: Skp2 and RIP1 expression in AML patients. (A-B) Analysis of Skp2 and RIP1 mRNA expression in AML BM and normal peripheral blood cells; datasets were obtained from the GEO database (PB: peripheral blood; PBSC: peripheral blood stem cells, CD34+). (C) The Skp2 and RIP1 mRNA expression in AML BM were download from TARGET, We used Spearman's correlation analysis to describe the correlation between Skp2 and RIP1. A p-value of less than 0.05 was considered statistically significant. Data analysed by Student's t-test,*p< 0.05, **p< 0.01.