Figure 5

CNL suppresses STAT3 phosphorylation via multiple kinases including BTK. (a) (i) CNL suppresses the activity of BTK. JVM-3 cells were treated with 40 μM ghost liposomes or CNL for indicated time periods and western blotting was performed. The blots are a representative of three independent experiments. (ii) and (iii) BTK inhibitors suppress phosphorylation of STAT3 in JVM-3 cells and CLL patient cells. Cells were treated with varying concentrations of ibrutinib for indicated time periods and western blotting was performed. Graphical representation of the western blot is also shown. The blots and graphs are representative of three independent experiments or three CLL patient samples. Student’s t-test was used to perform statistical analysis, *P<0.05. The final western blot image was created by grouping different parts of the same film of the same gel as indicated by the black dividing line. (iv) Synergism analysis of CNL and ibrutinib treatments. JVM-3 cells were treated with single agents and co-treated with different doses of CNL (1–10 μM) and ibrutinib (1–2.5 μM) for 24 h and MTS assay was performed. The cell viability data were analyzed for synergism using Compusyn software. No synergism was observed with ghost nanoliposomes. (b) (i) CNL suppresses the activity of MEK1/2 kinase. JVM-3 and Mec-2 cells were treated with 40 μM ghost liposomes or CNL for indicated time periods and western blotting was performed. The blots are a representative of three independent experiments. (ii) JVM-3 cells were treated with 10 μM U0126 for indicated time periods and p-Erk levels were evaluated to confirm the effectiveness of U0126 as a MEK inhibitor. (iii) and (iv) MEK1/2 inhibitor suppresses phosphorylation of STAT3 in JVM-3 cells and CLL patient cells. Cells were treated with 10 μM U0126 for indicated time periods and western blotting was performed. The blots and graphs are representative of three independent experiments or three CLL patient samples. Student’s t-test was used for statistical analysis, *P<0.05. (c) (i) CNL suppresses the activity of PKC. JVM-3 cells were treated with 40 μM ghost liposomes or CNL for indicated time periods and western blotting was performed. The blots are a representative of three independent experiments. (ii) and (iii) PKC inhibitor suppresses phosphorylation of STAT3 in JVM-3 cells and CLL patient cells. Cells were treated with 5 μM Bis-I for indicated time periods and western blotting was performed. The blots and graphs are representative of three independent experiments or three CLL patient samples. Student’s t-test was used for statistical analysis, *P<0.05. (d) CNL does not activate phosphatases. JVM-3 cells were pretreated for 2 h with: (i) 5 nM OA; (ii) 50 μM PV, followed by 12 h of treatment with 40 μM ghost nanoliposomes or CNL. Both the inhibitors were non-toxic to cells at the specific concentration. The blots are a representative of two independent experiments. The final western blot image was created by grouping different parts of the same film of the same gel as indicated by the black dividing line.