Fig. 3

IL-6 is associated with activation of STAT3/5 and resistance to PI3K inhibitors. a Cytokine expression and phospho-kinase levels in parental B/T-cell lymphoma cell lines were examined by ELISA and Western blot analysis, respectively. The bands on the Western blots were quantified via densitometry. b Decreased IL-6 production was monitored by ELISA. STAT5 and STAT3 expression were examined by Western blot analysis; β-actin was included as a loading control. c IL-6 knockdown sensitized cell lines with acquired resistance to copanlisib or duvelisib. Resistant and parental lymphoma cells were transfected with siIL-6 or control siRNA. Cells were transiently transfected with siRNAs and then treated with copanlisib or duvelisib for 72 h, after which viability was assessed by CCK-8 assay. d Constitutively active STAT3 or IL-6 induced duvelisib resistance. Cells were transiently transfected with an expression plasmid for constitutively active STAT3 (STAT3-CA) or with a control vector. Cells were pre-treated with IL-6 (2 or 10 ng/ml) 2 h before duvelisib treatment. Median inhibitory concentration (IC₅₀) values were calculated using GraphPad Prism 5 software (GraphPad). Data represent mean values ± SD of three independent experiments. e STAT inhibitor-sensitized lymphoma cells with acquired resistance to copanlisib or duvelisib. OCI-Ly1-copanlisib and HH-duvelisib resistant cells were treated with copanlisib (1 μM) or duvelisib (1 μM) in the presence or absence of SH-4-54 for 72 h. Cell viability was evaluated by trypan blue staining. P-values were determined by Student’s t-test and one-way repeated-measures ANOVA. Triple asterisk indicates statistically significant difference at P ≤ 0.005, double asterisk significant at P ≤ 0.01