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Fig. 6 | Journal for ImmunoTherapy of Cancer

Fig. 6

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. 6

PRODH Knockdown of PCC Weaken the Effect on T Cells. (a) Human primary CD3+ T cells were pretreated with PCM or PRODH knockdown-PCM then stimulated for 3 days with anti-CD3/CD28 beads. Shown is the percentage of cell proliferation by flow cytometry. (b) Human primary CD3+ T cells were pretreated with PCM or PRODH knockdown-PCM then stimulated for 3 days with anti-CD3/CD28 beads.. Shown is the levels of ROS by flow cytometry. (c) Human primary CD3+ T cells were pretreated with PCM or PRODH knockdown-PCM then stimulated for 3 days with anti-CD3/CD28 beads. Shown is the levels of mitochondria ROS by flow cytometry. (d) Jurkat cells treated with PCM or PRODH knockdown-PCM for 24 h. Shown is the percentage of cell proliferation by CCK-8 assay. (e-f) Jurkat cells treated with PCM or PRODH knockdown-PCM for 24 h. Shown is the levels of total and mitochondria ROS by flow cytometry.(g-i) Human primary CD3+ T cells were pretreated with PCM or PRODH knockdown-PCM then stimulated for 3 days with anti-CD3/CD28 beads. Supernatants from cell cultures were analyzed for cytokines levels using commercially available ELISA kits, including IL-2, TNF-α, IL-17A. (j-k) Jurkat cells treated with PCM or PRODH knockdown-PCM for 24 h. Shown is the ATP concentration and activity of CIII (l) The mRNA expression of SHP1 in Jurkat cells by qPCR after treatment with PCM or PRODH knockdown-PCM. (m) Western bolt showing the protein expression of SHP1 in Jurkat cells after treatment with PCM or PRODH knockdown-PCM. (n-p) Western bolt showing the cytoplasm, mitochondria and nuclear protein expression of SHP1 in Jurkat cells after treatment with PCM or PRODH knockdown-PCM. (q) The localization of SHP-1 in Jurkat cells after treatment with PCM or PRODH knockdown-PCM by immunofluorescence. DAPI was used for marking nuclear. All experiments were repeated at least three times. Error bars are SEM of biological replicates and *p < 0.05; **p < 0.01; ***p < 0.001

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