Fig 2: CT inhibition of Hepa1-6 hepatoma cell proliferation. a Hepa1-6 cell proliferation was measured using 3H-TdR incorporation and presented as the average (mean ± SD) of 3H-TdR incorporation of triplicate wells. The % proliferation was calculated as (CPM with compound—CPM blank) × (CPM without compound—CPM blank) × 100. b Hepa1-6 cells treated with different concentrations of cucurbitacin I for 48 h were solubilized in 1 × SDS sample buffer at 107/ml, separated on a gradient gel, and analyzed for STAT3 by Western blot. The same membrane was stripped and re-probed with anti-GAPDH. c A CCK-8 assay was performed to assess cell viability in Hepa1-6 cells treated with different concentrations of cucurbitacin I for 48 h. All data are averages (mean ± SD). d, e Hepa1-6 cells grown in 6-well plates were treated with CT at various concentrations for 2 h (d) or at specified concentration and time (e). The samples were analyzed by Western blot for phospho-JAK2 or phospho-STAT3. The same membranes were stripped and re-probed with anti-JAK2 or anti-STAT3, respectively

Fig 3: GPR87 Activates STAT3 through Interacting with JAK2(A) Western blot analysis of GPR87, p-STAT3, total STAT3, p-JAK2, and total JAK2 expression in the indicated cells with GPR87 overexpression or knockdown.GAPDH was used as a loading control. The phosphorylation of STAT3 and JAK2 was represented as p-STAT3 and p-JAK2, respectively. (B) Western blot analysis of GPR87, p-STAT3, total STAT3, p-JAK2, and total JAK2 expression once knocking down JAK2 in the GPR87 overexpressed PDA cells. GAPDH was used as a loading control. (C) IP assay revealing that GPR87 interacts with JAK2. (D) Schematic illustration of wild-type JAK2 and truncated JAK2. (E) IP assay showing that GPR87 interacts specifically with the JH2 and JH3-7 domains of JAK2.

Fig 4: TG101209 down-regulated the JAK-STAT pathway in the T-ALL cell lines(A) The T-All cell lines HSD2 and PEER were treated with TG101209 for 48 hours and lysed for western blotting. Untreated cells were used as the control group. The expression levels of the JAK-stat pathway related proteins JAK2 (1st lane), JAK3 (2nd lane), Stat3 (3rd lane) and Stat5 (4th lane) were all decreased after TG101209 treatment. All of the samples were normalized with β-actin (bottom lane). Bands of western blotting were quantified by densitometry with Scion Image software (Image J 1.48u). All the results were analysed using SPSS11.0. The graphs were listed respectively. (B) Representative image of the FISH analysis. The PEER, which possess a JAK2 copy gain (red dots), are shown in the left. The control samples, which possesses a normal JAK2 copy number (red dots), is shown on the right. All of the samples were also analysed with the CEN9q probe as an internal reference (green dots). The nuclei were all counter-stained with DAPI (blue).

Fig 5: IFNγ production by NK cells is dependent on TYK2 kinase activity. (A) DX5+ cells were MACS-purified from spleens of WT, Tyk2K923E and Tyk2−/− mice, expanded for 7 d with IL-2, stimulated with IL-12 for indicated time points or left untreated (w/o) and further analyzed for activation of STATs and JAK2 and for IFNγ production. Levels of pSTAT3, pSTAT4 and pJAK2 were assessed by Western blot and a representative blot out of two independent experiments is shown (left panel). The production of IFNγ by NK cells was assessed by ELISA (right panel) and mean ± SEM is presented from three independent experiments (n = 3 per genotype per treatment). (B) WT, Tyk2K923E and Tyk2−/− splenocytes were stimulated with anti-NK1.1 antibody for 5 h. Intracellular IFNγ levels were assessed by flow cytometry and the percentage of IFNγ+ cells within the CD3ϵ−DX5+ population (left panel) or level of IFNγ expression (MFI, right panel) is shown as boxplots with whiskers from minimum to maximum from three independent experiments (n = 6−8 per genotype). (C) WT, Tyk2K923E and Tyk2−/− splenocytes were stimulated with anti-NK1.1 antibody for 8 h. Intracellular CCL3 levels were assessed by flow cytometry and the percentage of CCL3+ cells within the CD3ϵ−DX5+ population (left panel) or level of CCL3 expression (MFI, right panel) is shown as boxplots with whiskers from minimum to maximum from two independent experiments (n = 4 per genotype). (A, B, C) *P < 0.05, **P < 0.01, ***P < 0.001.