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. 2021 Feb 23;11:622244. doi: 10.3389/fonc.2021.622244

Figure 3.

Figure 3

Establishment and characterization of radioresistant hypoxic organoids. (A) Representative confocal microscopic images of normoxic (21% oxygen) and hypoxic (1% oxygen) organoids of various sizes stained by Green Hypoxia Reagent (GHR), top panel shows nuclear staining by DAPI (blue), hypoxia staining by GHR (green), and bright field imaging (gray scale); magnification 400×, Scale bar 100 µm. (B) Schematic of high-dose radiation regimen to establish radioresistant organoids. (C) Relative cell viability of normoxic and hypoxic organoids relative to respective control untreated organoids at day 14 (D14), n = 3, ns, not significant, ****p < 0.0001. (D) Representative confocal microscopic images of normoxic and hypoxic organoids without and with radiation (5Gy) showing proliferation of cells (anti-Ki67 staining-red) in the periphery; magnification 400×, hypoxia staining by GHR (green), BF, Bright field image, Scale bar 100 µm. (E) Expression of HIF-1α in different NPC samples quantified by RT-PCR, fold-changes are relative to normoxic control organoids and normalized to changes in the GAPDH gene expression, n = 3.