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. 2020 Jun 12;10(16):7409–7421. doi: 10.7150/thno.44782

Figure 6.

Figure 6

Generation and expansion of HERS spheroids rely on the HIF-1 pathway. (A) RT-qPCR analysis of expression of representative genes in the HIF1 pathway. The expression of Hif1a, Arnt, Pgk1, Pdk1, and Slc2a1 was relatively higher in the HERS spheroids groups, consistent with the results of RNA-seq analysis. (B) Immunofluorescence staining was used to verify the expression of HIF-1a protein. The HIF-1a (green) signal was located in many of the nuclei of cells in HERS spheroids, but in only a few nuclei in the 2D monolayer HERS cells. (C) HIF-1a expression can be detected in HERS tissues. (D) DMOG (a HIF-1 activator) and BAY (a HIF-1 inhibitor) were applied to perform gain- and loss-of-function assays. The sphere-formation rate (E) and the diameter of spheroids (F) were severely inhibited by HIF-1 inhibitor and enhanced by HIF-1 activator, indicating that the HIF-1 pathway is important for the formation and expansion of HERS spheroids. In addition, the expansion of 2D monolayer HERS cells was also inhibited by BAY (G). *** P < 0.001; ** P < 0.01; * P < 0.05.