Fig 2: 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).

Fig 3: Modulation of miR-222-3p enhances the inhibitory activity of IL-24wt on HMGA1 signaling in H1299-IL24wt cellsA. Induction of IL-24wt downregulates miR-222-3p and -5p expression in H1299-IL-24wt cells (p<0.05). B. IL-24wt shows greater inhibition of miR-222-3p expression when combined with genetic knockdown of HMGA1 (p<0.05). C. The expression of IL-24wt in the presence of miR-222-3p-Luc plasmid produced an increase in luciferase activity compared with the miR-222-3p-Luc plasmid alone group (p<0.0001). D. Treatment of H1299-IL-24wt cells with miR-222-3p inhibitor increased PPP2R2A protein expression. E. IL-24wt combined with miR-222-3p inhibitor exhibited a greater increase in PPP2R2A expression (p<0.05). F. IL-24 expression did not reduce miR-222-3p expression in cells overexpressing miR-222-3p mimic (p<0.05). Further, we observed increased PPP2R2A expression after IL-24wt treatment in miR-222-3p-overexpressing cells when compared with miR-222-3p mimic alone group (p<0.0001). Beta-actin was used as a protein loading control.

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: 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.