Fig 2: The oncogenic role of the α7-nicotinic acetylcholine receptor (α7-nAChR) is mediated in part by JAK2 activation. (A) Visualization of the molecular connectivity network among the α7-nAChR, JAK2, STAT3, SOX2, OCT4A (POU5F1), Survivin (BIRC5), Bcl-xL (BCL2L1), MCL-1, MMP2, MMP9, RhoA, and ROCK1 using STRING version 10.5. (B) Differential expressions of the α7-nAChR and JAK2 in hepatocellular carcinoma (HCC) specimens compared to non-tumor liver specimens in the GSE14323 dataset. (C) Representative images of α7-nAChR and JAK2 expression levels in HCC tissues of different TMN stages showing mild expression in stage I, moderate expression in stage II, and high expression in stage III, compared to no expression in non-tumor liver (NL) tissues. The original magnification is indicated; bar = 100 μm. (D) Photo-images showing the differential expression of JAK2 in HCC specimens from a current smoker compared to non-tumor liver specimens from a non-smoker. (E) Dot and whisker plots showing that the α7-nAChR and JAK2 are upregulated in tumor compared to non-tumor liver tissues. * p < 0.05. (F) Graph showing the positive correlation between the α7-nAChR and JAK2 in patients with HCC.

Fig 3: The α7-nicotinic acetylcholine receptor (α7-nAChR) promotes metastasis and recurrence of human hepatocellular carcinoma (HCC) by modulating the JAK2/STAT3 signaling axis. (A) Representative IHC photo-images showing the correlation of α7-nAChR expression levels on the immunoreactivity of TGR5 and JAK2 in HCC tissues. The original magnification is indicated; bar = 100 μm. (B) Nuclear co-localization of the α7-nAChR and TGR5 in HCC samples as indicated by IFC staining. DAPI served as a nuclear marker. (C) Western blot images showing the effect of silencing of the α7-nAChR on expressions of TGR5, p-JAK2, JAK2, p-STAT3 (Tyr705), p-STAT3 (Ser727), and STAT3. β-Actin served as a loading control. (D) Overall survival of JAK2 expressions. (E) Comparison of disease-free survival with low and high levels of JAK2 expression. (F) α7-nAChR/JAK2 complex formation was detected by immunoprecipitation (IP). Hep-J2 and Mahlavu cells lysate were incubated with α7-nAChR and JAK2 antibody, respectively. IgG was used as a negative control.

Fig 4: IL-10 Induces the Activation of STAT3 that Binds to the dnmt1 and dnmt3b Promoters in Colon Epithelial and Carcinoma Cells(A) WT C57BL/6 mice were treated with the 2% DSS-water cycle, as described in STAR Methods. Colon tissues were collected from mice at the indicated time points and analyzed by western blotting for STAT1 and STAT3 protein levels. ß-Actin was used as a normalization control.(B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 2 hr and analyzed for the indicated proteins by western blotting.(C) Top: structure of the Dnmt1 promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt1 transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using immunoglobulin G (IgG) control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt1 promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD.(D) Top: structure of the Dnmt3b promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt3b transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using IgG control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt3b promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD.(E and F) The human DNMT1 (E) and DNMT3b (F) promoter DNA fragments were amplified by PCR from the two indicated regions (top: P1 and P2 for DNMT1, and P3 and P4 for DNMT3b) and cloned to the pGL3 vector. pGL3 vectors containing the P1, P2, P3, or P4 DNA fragments were transiently transfected to CCD841 and HT29 cells, respectively, overnight. Cells were either untreated (control) or treated with IL-10 (100 ng/mL) for 4 hr. Cells were lysated and analyzed for luciferase activity. Bar, SD.

Fig 5: InTraSeq captures the proteomics signals and transcriptional changes during Th17 cell differentiationA. Schematic of experiment design.B. UMAP plot showing the cells of different time points. UMAP coordinates were computed using RNA data.C. UMAP plot showing the cells of different stimulations. The UMAP coordinates are the same as Figure 3B.D. UMAP plots showing the p-STAT3-Ty705 protein, the p-STAT3-Ser727 protein, and Stat3RNA expression in each stimulation. The UMAP coordinates are the same as Figure 3B.E. Average expression of p-STAT3-Ty705 protein, the p-STAT3-Ser727 protein, and Stat3RNA in every condition over time.F. A comparison of the Western blot validation results (left) and InTraSeq data (right) for selected proteins showed substantial change at 10 minutes compared to naïve CD4+ T cells.G. Dot plot depicting the expression of all proteins at every time and stimulation combination except PMA/IO treatment. Proteins were ordered and clustered using hierarchical clustering.H. Number of STAT3-bound genes that are significantly up- or down-regulated in npTh17 compared to Th0 (top) or pTh17 (bottom) at each time point.I. Expression of STAT3 direct targets of interest at the 24-hour time point. The color indicates the z-normalized average expression.J. qPCR validations of selected STAT3 direct targets of interest. Y-axis represents the ratio of a sample’s relative expression (RE) compared to the mean RE of the Th0 group. The unadjusted p-values were computed using Wilcoxon rank-sum tests.K. Expression of STAT3 direct targets of interest that were included in the InTraSeq antibody panel. The unadjusted p-values were computed using Wilcoxon rank-sum tests.L. Schematic of non-pathogenic and pathogenic Th17 differentiation pathways according to the STAT3-focused analysis.