Fig. 2: Brusatol specifically targets the PI3K/AKT signaling pathway.

a RNA-Seq analysis was performed in Brusatol-treated LCL1 cells. The heatmap illustrates 12 genes with the greatest differences after Brusatol treatment. b Signaling pathway analysis of RNA-Seq data showed the percentages of regulated genes in these indicated pathways. The numbers above the bar charts described the total genes involved in these different pathways. c Upstream regulator analysis in the IPA program showed PI3K family members are the important upstream factors. d Chemical structures of the parent Brusatol and three biotin-conjugated Brusatol analogs were shown. The synthesized C-3-biotinylated Brusatol derivatives (51048, 51052) were set as the experimental samples (+), while a C-21 biotin conjugate (51046) was set as a negative control (−). e Correlation of mass spectrometry data and upstream regulator data were conducted using the IPA program. f Real-time PCR was performed in untreated or Brusatol-treated Raji cells to verify the mRNA expression of upstream regulators (PI3K family, TP73, JUP) associated genes. The results from three independent experiments were shown. Results are the mean ± standard error of the samples (n = 3). **p < 0.01 and ****p < 0.0001 show the significant differences as compared to the control group. g Real-time PCR was performed to determine the mRNA expression of PI3K/AKT signaling members in LCL1 cells after Brusatol treatment. Results are the mean ± standard error of the samples (n = 3). *p < 0.05 shows significant differences as compared to the control group. h, i Western blot analysis was conducted to show the protein expression of PI3K/AKT signaling factors in Raji (h) or LCL1 (i) cells after Brusatol treatment. The PI3Kγ band was labeled by an asterisk. Two bands of GSK3 were GSK3α (51kD) and GSK3β (47kD), respectively.