Fig 2: A combination of IL-24wt and HMGA1-siRNA showed greater inhibition of AKT activation in lung cancer cellsA. Western blotting showed that a combination of HMGA1 siRNA and IL-24wt produced a greater reduction in HMGA1, pAKTT308 and pAKTS473 expression compared with all other treatments. Beta-actin was used as a protein loading control. Differences in the expression of the proteins against untreated control were determined by semi-quantitative analysis and represented in graphical format (p<0.001). Bars denote standard deviation (SD). B. H1299- IL-24wt cells were untransfected or transfected with pIRES-HMGA1 plasmid, followed by treatment with or without 1 µg/ml doxycycline. Cells were subjected to western blot analysis. Expression of pIRES-HMGA1 plasmid abrogated the inhibitory effect of IL-24wt on HMGA1 and the associated effect on its downstream target PPP2R2A. Beta-actin was used as a protein loading control. Differences in the expression of the proteins were determined by semi-quantitative analysis and represented in graphical format (p<0.05). Bars denote standard deviation (SD).

Fig 3: miR-221 directly targets PPP2R2A(A) MG-63 and SaoS-2 cells were transfected with 50 nM of control or miR-221 mimics for 48 h, followed by Western blot analysis of PPP2R2A expression. (B) Luciferase assay showing miR-221 binding to the 3'-UTR of PPP2R2A. (C) Negative correlation between miR-221 and PPP2R2A mRNA in OS tumors. Data are presented as mean ± SD. Columns, mean of three independent experiments; bars, SD; **, P<0.01.

Fig 4: RIPK3 suppresses ERK phosphorylation by activating PP2A function. A) Western blot analysis of ERK phosphorylation after transiently expression of human RIPK3 and human LCK in HEK293T cells. HEK293T cells were transfected with GST-LCK and/or Flag-RIPK3 expression constructs. B) Computational docking model for RIPK3 (olive) and PP2A predicted using ClusPro. The PP2A comprises three subunits (A subunit; blue, B55a subunit; green, and C subunit: magenta). C) Western blot analysis after immunoprecipitation of human RIPK3 and human PP2A subunits in HEK293T cells. HEK293T cells were transfected with GFP-PPP2CA or GFP-PPP2R2A and/or Flag-RIPK3 expression constructs. Cells were harvested at 24 h after transfection. D) The endogenous RIPK3 interacted with PPP2R2A in response to anti-CD3/CD28 stimulation. E) In vitro kinase activity of RIPK3 toward PPP2R2A with 32P-labeled ATP. To avoid false positive artifacts in the in vitro kinase assay, we included conditions with or without ATP. GST-RIPK3 (amino acid 2–328) and GST-PPP2R2A recombinant proteins were purified from sf-9 cells. F) Total thymocytes from Ripk3+/+ and Ripk3-/- mice were stimulated with anti-CD3 (1 µg mL-1) and anti-CD28 (2 µg mL-1) for indicated time. Cell lysates were immunoprecipitated with anti-ERK antibody. Immunocomplexes and cell lysates were analyzed by immunoblotting.

Fig 5: Attenuation of HMGA1 by IL-24wt inhibits AKT activation in H1299-IL24 lung cancer cellsA. Western blotting analysis showed that expression of IL-24wt protein in H1299-IL24 cells reduced the expression of phosphorylated (p) AKTT308 and pAKTS473, and increased PPP2R2A expression at 24 h and 48 h after doxycycline treatment. B. Western blotting analysis showed that expression of IL-24wt protein in HCC827 and A549 cells reduced the expression of phosphorylated (p) AKTT308 and pAKTS473, and increased PPP2R2A expression. Beta-actin was used as a protein loading control. Differences in the expression of the proteins were determined by semi-quantitative analysis and represented in graphical format (p<0.05). Bars denote standard deviation (SD).