Fig. 5: Feedback activation of JAK1-STAT3 signaling via downregulation of PTPN11 expression.
From: Combined Inhibition of PI3K and STAT3 signaling effectively inhibits bladder cancer growth
A Venn diagram showed overlapping of the negative regulators of JAK-STAT pathway and down-regulated genes in resistant cells screened by RNA-seq. B The relative mRNA levels of PTPN11 in 5637 and UM-UC-3 cells with or without copanlisib treatment. C Knockdown of PTPN11 by two different siRNAs in 5637 and UM-UC-3 cells were verified by qPCR. D Western blot analysis of phosphorylated STAT3 and total STAT3 protein levels after transfection with indicated PTPN11 siRNAs in 5637 or UM-UC-3 cells. E Overexpression of PTPN11 by pEnCMV-PTPN11 plasmid in 5637 and UM-UC-3 cells were verified by qPCR. F Western blot analysis of phosphorylated STAT3 and total STAT3 protein levels after transfection with pEnCMV-PTPN11 plasmid in 5637 or UM-UC-3 cells. Representative images (G) and quantification (H) of apoptosis assay in the effect of PTPN11 overexpression in 5637 or UM-UC-3 cells on copanlisib sensitivity. I CCK8 assay explored the effect of PTPN11 overexpression in 5637 or UM-UC-3 cells on copanlisib sensitivity. J The synergistic score between copanlisib and napabucasin was determined using Loewe and HSA synergy analysis in cells overexpression of PTPN11. K Relative luciferase activity of C/EBPβ upon PI3K inhibition in 5637 or UM-UC-3 cells. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. L Putative binding site of C/EBPβ in PTPN11 promoter region. M ChIP assay analyzed the recruitment of C/EBPβ at PTPN11 promoter in 5637 and UM-UC-3 cells treated with copanlisib. Data are presented as mean ± SD of independent samples with individual data points shown; P values were assessed by two-tailed Student t test (B, C, E, H, L, M) or equivalent ANCOVA (I) in comparison with the vehicle (DMSO) group (B, H, L) or the Scramble group (C, E, M); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
