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Fig. 4 | Journal of Hematology & Oncology

Fig. 4

From: Endothelial-mesenchymal transition harnesses HSP90α-secreting M2-macrophages to exacerbate pancreatic ductal adenocarcinoma

Fig. 4

EndoMT-derived cells secrete HSP90α to induce macrophage M2-polarization. a mRNA levels of TGF-β, HSP90α, IL-13, IL-4, and C-C motif chemokine ligand 22 (CCL22) in EndoMT-derived cells. TGF-β, HSP90α, IL-13, and IL-4 mRNA expressions were significantly induced in the HUVECs treated 24 h with OPN. #P < 0.01 when compared with PBS treatment. b HSP90α protein levels in the HUVECs and EC-RF24 cells treated 24 h with PBS or 0.3 μg/ml of OPN. HSP90α protein expression was obviously induced in OPN-treated endothelial cells. c, d Secreted HSP90α levels of HUVECs and their EndoMT derivatives. Ctrl, Endo CM, and EndoMT CM were prepared as described in Materials and Methods, and subjected to analyses of secreted HSP90α levels using immunoblot analysis (c) and ELISA (d). Induction of HSP90α secretion was significantly detected in OPN-treated HUVECs. #P < 0.01 when compared with “Ctrl” or “Endo CM” group. e mRNA levels of IL-1β, TNF-α, iNOS, CD163, CD204, IL-10, TGF-β, and Arg1 in the THP-1-derived macrophages treated 24 h with control medium (Ctrl), EndoMT CM, or EndoMT CM plus control IgG or anti-HSP90α antibody. αP < 0.05 when compared with “Ctrl” group. βP < 0.05 when compared with “EndoMT CM + IgG” group. f Secreted levels of IL-1β, IL-10, and TGF-β from the THP-1-derived macrophages treated 24 h with Ctrl, Endo CM, or EndoMT CM in the absence or presence of PBS, 1 μM DMAG-N-oxide, or 10 μg/ml of IgG or anti-HSP90α antibody. THP-1-derived macrophages were pre-incubated with 1% FBS-containing medium for 16 h and subjected to indicated treatments for further 24 h. The treated macrophages were then incubated with 5 ml of fresh 1% FBS-containing medium for 24 h. The media were finally collected for ELISAs. αP < 0.05 and δP < 0.01 when compared with “Ctrl” group. βP < 0.05 and εP < 0.01 when compared with “EndoMT CM + PBS” group. γP < 0.05 and λP < 0.01 when compared with “EndoMT CM + IgG” group. g Cell-surface levels of CD163 and CD204 in the THP-1-derived macrophages treated 24 h with Ctrl or EndoMT CM plus control IgG or anti-HSP90α antibody. h mRNA levels of IL-1β, TNF-α, CD163, CD204, IL-10, and TGF-β in the THP-1-derived macrophages treated 24 h with PBS or 15 μg/ml of rHSP90α. *P < 0.05 and #P < 0.01 when compared with PBS treatment. i Secreted levels of IL-1β, IL-10, and TGF-β from the THP-1-derived macrophages treated 24 h with PBS or 15 μg/ml of rHSP90α. THP-1-derived macrophages were pre-incubated with 1% FBS-containing medium for 16 h and then added with PBS or 15 μg/ml of rHSP90α for another 24 h. The treated macrophages were further incubated with 5 ml of fresh 1% FBS-containing medium for 24 h. The media were finally collected for ELISAs. #P < 0.01 when compared with PBS treatment. j Cell-surface levels of CD163 and CD204 in the THP-1-derived macrophages treated 24 h with PBS or 15 μg/ml of rHSP90α. k mRNA expression status of Arg1 and iNOS in the THP-1-derived macrophages treated 24 h with PBS or 15 μg/ml of rHSP90α. l mRNA levels of IL-1β, TNF-α, iNOS, CD163, CD204, IL-10, TGF-β, and Arg1 in the THP-1-derived macrophages treated 24 h with LPS plus IFN-γ in the absence or presence of rHSP90α. #P < 0.01 when compared with “LPS + IFN-γ” group. m mRNA levels of IL-1β, TNF-α, iNOS, CD163, CD204, IL-10, TGF-β, and Arg1 in the mouse BMDMs treated 24 h with PBS, 20 ng/ml of IL-4, or 15 μg/ml of rHSP90α. rHSP90α significantly induced M2-polarization of BMDMs

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