Fig 2: (A) and (B) Evaluation of PLC-γ1 and phosphotyrosine in TCR-induced microclusters after αCD3 stimulation in Jurkat T cells transfected with empty mTurq2-C1, Cish WT-mTurq2-C1, Cish-D/BC*-mTurq2-C1 or Cish-SH2*-mTurq2-C1 as indicated. (A) Representative confocal images from transfected Jurkat cells dropped on to stimulatory coverslips, fixed after three minutes, immunostained for PLC-γ1 and phosphotyrosine (pY), and the areas of the clusters were calculated (B). Statistical significance was determined by two-tailed Student’s t test. *P ≤ 0.05, ***P ≤ 0.001.

Fig 3: (A) Representative Ca2+ flux as assessed by fluorometric evaluation after αCD3 stimulation of indicated transduced CD8+ T cells. Kinetic of the ratio indo-blue to indo-violet over time shown and assessed by flow cytometry. (B) Calcium responses were integrated and quantified relative to Cish WT+ empty vector. Error bars represent SD from the mean (n = 3). (C) To evaluate cells positive for intracellular expression of TNF-α or IFN-γ, indicated transduced CD8+ T cells were re-stimulated with anti-CD3 coated antibody (at indicated concentration) and soluble CD28 (2 μg/ml) in presence of Golgi stop for 6 hrs. Cells were then labeled for surface Thy1.1 and intracellular TNF-α or IFN-γ. The flow cytometry analysis of Thy1.1+ T cell populations is shown. (D) Absolute cell numbers of Thy1.1+ T cells positive for intracellular TNF-α or IFN-γ. Bars show mean + SEM out of at least 4 independent experiments. Statistical significance was determined by two-tailed Student’s t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (NS = Non-stimulated).

Fig 4: (A) Schematic diagram of Cish constructs used in our experiments. (B) 293 T cells were transfected with Tagged plasmids expressing PLC-?1-YFP, Ubiquitin-HA, and Cish-FLAG constructs (WT, SH2* and D/BC*) where indicated, in the presence of the proteasome inhibitor, MG-132. PLC-?1-YFP (GFP antibody) was immunoprecipitated and blotted for HA and YFP. Whole-cell lysates were blotted for Cish and Cish and YFP PLC-?1. Numbers below the band indicate PLC?1 ubiquitin quantification. A value of 1 was arbitrarily set for PLC?1 ubiquitination in the presence of the Cish WT construct. (C) Same as in (B) with no proteasome inhibitor, MG-132. Cish (Flag M2) was immunoprecipitated and blotted for YFP (PLC-?1) and Cish. Whole-cell lysates were blotted for YFP. Numbers below the band indicate PLC?1 binding to Cish quantification, a value of 1 was arbitrarily set for PLC?1 binding in the presence of the Cish WT construct. (D) Immunoprecipitation of FLAG-tagged Cish and immunoblotting of PLC-?1 and Cish, in indicated transduced CD8+ T cells with and without CD3 stimulation (5 min). Whole lysates were blotted for PLC-?1 and Flag M2.

Fig 5: The effects of NNK on the STAT3-mediated pro-inflammation genes. OEC-M1 and SCC-25 cells were transfected with miR-944 inhibitors (AM) for 12 h, and followed by 20 µM of NNK treatment for another 72 h. The RNA level of COX-2 and iNOS in cells (A), protein level of CCL3, CCL5 and IL-1ß in cultured medium (B) were measured by qRT-PCR or ELISA. (C) Migration and invasion ability were measured by transwell assays in OEC-M1 and SCC-25 cells. (D) OEC-M1 and SCC-25 cells were treated with 20 µM of NNK for 72 h, and the TP63 expression level was measured by qRT-PCR analysis. (E) Correlation analysis of miR-944 and TP63 in human OSCC patients (n = 33) by qRT-PCR analysis. (F) Proposed model for comprehensive NNK-induced upregulation of miR-944 in stimulating STAT3 activation and pro-inflammatory genes expression through suppression of CISH. All data are represented as mean ± SD; **, p < 0.01; ***, p < 0.001.