Fig 1: IL7R induced gemcitabine resistance via the JAK1/STAT5 signaling pathway. (A) Representative dot plots and statistical analysis of the frequency of CTRL (left), PLD1-OE (middle 1), IL7R-KD (middle 2), and PLD1-OE+IL7R-KD (right). (B) Representative dot plots and statistical analyses of the frequency of CTRL (left), IL7R-OE (middle 1), PLD1-KD (middle 2), and PLD1-KD+IL7R-OE (right). (C) Immunohistochemical (IHC) staining of tissues from 24 patients with PDAC using anti-PLD1 and -IL7R antibodies. Pearson correlation analysis between PLD1 and IL7R IHC scores. The bubble size represents the patient number with indicated IHC staining (from small-to-large; n = 1, n = 2, n = 3). (D) IHC results revealed that higher expression of IL7R in PDAC tissues was correlated with shorter OS (log-rank test). (E, F) The levels of p-JAK1, p-STAT5, and BCL-2 expression in MIA PaCa-2, EV, WT, nuclear localization signal mutation (NLM), and KRM cell lines using Western blot analysis. (G-H) MIA PaCa-2 cell line was treated with gemcitabine, a combination of gemcitabine and JAK1-IN-9, or STAT5-IN-1. Cytotoxicity was analyzed using flow cytometry. The data are expressed as the mean ± SEM from three independent experiments. *P < 0.05; **P < 0.01 (one-way ANOVA); ns, not significant.
Fig 2: NPM1 and PLD1 bind at the promoter region of IL7R. (A) Heatmap showing the results of CTRL and PLD1-OE cell line RNA-seq. (B) Changes in gene expression profiling upon changes to PLD1 expression in the indicated cell lines. qPCR was performed to detect the transcriptional change in the genes selected from RNA-seq analysis. Relative expression is shown as a fold-change relative to GAPDH. (C, D) Binding of PLD1 to the promoters of IL7R, GSTT2, and NLRP1 in MIA PaCa-2-PLD1 determined by ChIP. Experiments were repeated three times independently. Representative data are shown. (E) Venn diagram in which PLD1 was identified as one of the core intersections of RNA-seq and CHIP-seq analyses. (F) Scatter plots indicated that NPM1 binds with the promoters of IL7R in CHIP-seq data. (G) Binding of NPM1 and PLD1 to the promoters of IL7R in empty vector, PLD1-WT, and PLD1-KRM by CHIP. (H) Binding of NPM1 and PLD1 to the promoters of IL7R in empty vector, PLD1-WT, and PLD1-KRM by RE-CHIP. (I) Binding of PLD1 to the promoter of IL7R was determined by chromatin immunoprecipitation in the NPM1 KD cell line. IgG was used as a negative control. Anti-GAPDH was used as a positive control. Representative results are shown. (J-M) The HEK 293T (left) and MIA PaCa-2 cells (right) were transfected with control or pCDH-PLD1 in conjunction with the luciferase reporter pGL3-empty vector, WT pGL3-IL7R-promoter, or pGL3-IL7R-promoter with EBS1 mutation. The results are expressed as fold-change relative to the corresponding cells transfected with the control vector after normalization of firefly luciferase activity according to Renilla luciferase activity. Experiments were independently repeated three times. Data are presented as the mean ± SD. Paired Student’s t-test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
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