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Fig. 2 | Journal for ImmunoTherapy of Cancer

Fig. 2

From: 1-Pyrroline-5-carboxylate released by prostate Cancer cell inhibit T cell proliferation and function by targeting SHP1/cytochrome c oxidoreductase/ROS Axis

Fig. 2

PCM Inhibit T cell ROS and ATP Production and Inhibit Activity of CIII. (a) Human primary CD3+ T cells were pretreated with PCM or two normal cells media then stimulated for 3 days with or without anti-CD3/CD28 beads. Intracellular total ROS levels were measured by FACS after incubation with the ROS-reactive fluorochorome DCFH. The geometric mean of DCF fluorescence intensity was used to determine the rate of ROS generation. The right side of bar graph is the representative result by flow cytometry. (b) Jurkat cells were treated with PCM or two normal cells media for 24 h. Shown is the levels of ROS by flow cytometry. The right side of bar graph is the representative result by flow cytometry. (c) Human primary CD3+ T cells were pretreated with PCM then stimulated for 3 days with or without anti-CD3/CD28 beads. Shown is the levels of mitochondria ROS by flow cytometry. The right side of bar graph is the representative result by flow cytometry. (d) Jurkat cells were treated with PCM or two normal cells media for 24 h. Shown is the levels of mitochondria ROS by flow cytometry. The right side of bar graph is the representative result by flow cytometry. (e) Jurkat cells were treated with PCM or two normal cells media for 24 h. Shown is the ATP concentration by microplate reader. (f) Jurkat cells were treated with PCM or two normal cells media for 24 h. Shown is the relative activity of CI by microplate reader (g) Jurkat cells were treated with PCM or two normal cells media for 24 h. Shown is the relative activity of CII by microplate reader. (h) Jurkat cells were treated with PCM or two normal cells media for 24 h. Shown is the relative activity of CIII by microplate reader. (i, j) The mRNA expression of NOX2 in CD3+ T cells and Jurkat cells after treatment with PCM or two normal cells media by qPCR. (k) Western bolt showing the protein expression of NOX2 in CD3+ T cells and Jurkat cells. An antibody to β-actin was used as a loading control. All experiments were repeated at least three times. Error bars are SEM of biological replicates and *p < 0.05, ***p < 0.01

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