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Fig. 4 | Journal for ImmunoTherapy of Cancer

Fig. 4

From: Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1

Fig. 4

p38-STAT3 signaling in BMDMs and protein fraction in TRAPs are essential for induction of PD-L1 and IL-10. a BMDMs were exposed to TRAPs (10 μg/ml) at indicated time points. Cell lysates were analyzed for p38, p-p38, STAT3 and p-STAT3 by western blot. GAPDH was used as a loading control. b BMDMs were pretreated with p38 inhibitor SB203580 (3 μM) for 1 h, and then co-incubated with TRAPs (10 μg/ml) for 4 h. Expression of STAT3 and p-STAT3 was detected by western blot. c BMDMs were exposed to SB203580 at described concentrations for 1 h, and followed by incubation with TRAPs (10 μg/ml) for 72 h. PD-L1 expression was determined by flow cytometry, and (d) the production of IL-10 was assessed by ELISA. e BMDMs were pretreated with STAT3 inhibitor Stattic (1 and 3 μM) for 1 h, and then stimulated with TRAPs (10 μg/ml) for 72 h. PD-L1 expression and (f) IL-10 secretion was determined by flow cytometry and ELISA, respectively. g TRAPs were pretreated with Proteinase K for 2 h at 55 °C, DNase I for 1 h at 37 °C or RNase for 3 h at 37 °C, respectively, followed by incubation with BMDMs for 72 h. PD-L1 was evaluated by flow cytometry, and (h) IL-10 was tested by ELISA. Data are shown as mean ± SEM, and are representative of three independent experiments. ***p < 0.001 by unpaired t test. (d, f and h)

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