Fig 2: Bazedoxifene inhibits signal transducer and activator of transcription (STAT)3 phosphorylation and expression of STAT3 downstream genes, and induces apoptosis. HEPG2 (A), 7721 (B) and HUH-7 (C) cancer cell lines exhibit constitutively phosphorylated STAT3. Bazedoxifene (10, 15, 20, 25 µmol/L) inhibited the phosphorylation of STAT3 (Tyr 705), Janus kinase (JAK)1 and JAK2, induced apoptosis as evidenced by increased cleaved caspase-3. The expression of STAT3-mediated genes was decreased by bazedoxifene except for the expression of Bcl-2 in 7721 cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Fig 3: JAK2 is dispensable for NK cell survival and maturation. (A) Frequency (left panel) and total numbers (right panel) of CD3-NKp46+ NK cells in the spleens of Jak2WT and Jak2fl/flNcr1Cre mice were assessed by flow cytometry. (B) Splenic CD3-NKp46+ NK cells were analyzed for expression of CD27 and CD11b by flow cytometry. Frequency of cells in each maturation stage is show: DN (CD27-CD11b-), CD27 (CD27+CD11b-), DP (CD27+CD11b+), CD11b (CD27-CD11b+). (C) The expression of JAK2 and ß-actin was analyzed by Western blot in NK cells upon 6 days of expansion in IL-2. Scans of full blots are available in Supplementary Material. (A,B) Bar graphs represent mean ± SEM of 2–3 independent experiments; n = 5–12.

Fig 4: SOID-8 represses the JAK2/STAT3 signaling pathway in a dose- and time-dependent manner.(A) Total proteins were isolated from A2058 (left) and A375 (right) cells incubated with 2.5, 5, 10, or 20 µM SOID-8 for 24 h. Western blot was done with antibodies to total or phosphorylated (p) STAT3, JAK2 and Src using 40 µg total proteins. ß-Actin was used as a loading control. (B) Time course of inhibition of STAT3 upstream regulatory proteins JAK2 following SOID-8 treatment. A2058 cells were treated with 10 µM of SOID-8 for indicated times. Total proteins were isolated and the level of total or phosphorylated (p) STAT3, JAK2 and Src was measured by western blot.

Fig 5: Methylation-specific PCR (MSP) for SHP1, SOCS1 and SOCS3 genes and methylation density at the SOCS3 promoter, in T-LBLs with mutant JAK2. (a) Schematic depiction of the CpG-island around the transcription start site of the genes (long black arrow). Short vertical lines represent CpG dinucleotides. (b) MSP analysis. The presence of a PCR band under lanes M or U indicated methylated or unmethylated CpG islands. Normal lymphocytes (NL) and in vitro methylated DNA (IVD) were used as a positive control. (c) Methylation profile of SOCS3 in two fetal thymuses and T-LBLs harboring JAK2 genetic alterations. The presence of methylated (black squares) or unmethylated (white squares) CpG sites is indicated in 10 sequenced clones for every tumor. Methylation density is indicated as the percentage of methylated sites.