Figure 4. GS drives continued cell proliferation under ammonia stress.

(A) Immunoblot comparison of time-dependent HIF expression levels over time after treatment with 10 mM of NH₄Cl in aggressive (HEYA8, TOV-21G, TOV-112D and CD90⁺ PEO1) and indolent (CD90⁻ PEO1, OVTOKO, OV-56 and OVCAR5) OVC cell lines. The values of the GI₅₀ of NH₄Cl as determined in Figure 1A are shown in the middle. Right panel bar chart shows the ratio of HIF-1α to β-actin levels loading as determined by immunoblot. Band intensities were measured using Image J. (B) Gene expression microarray heatmap showing more than 3,500 genes up- or down-regulated in CD90⁺ cells compared to CD90⁻. Right table shows selected genes up-regulated in the CD90⁺ subpopulation, which are components of the metabolic pathways as indicated. (C) The GI₅₀ of NH₄Cl (upper panel) and GS protein expression (lower panel) were determined for the PEO1 CD90⁺/CD90⁻ system and CD90⁺ cells with two independent GS-KD and control knockdown (NT; non-targeting). (D) Effect of GS-KD on HIF-1α activation levels upon 10 mM NH₄Cl treatment. (E) Anchorage-independent colony formation was assessed in CD90⁺ PEO1 cells with shNT or GS-KD. (F) In vivo tumor growth measured using luciferase bioluminescence and IVIS Imaging (n = 5 for shNT and shGS #1; n = 4 for shGS #2). β-actin was used for loading control in (C and D). Error bars indicate s.e.m. ^*P < 0.05; ^**P < 0.01 (Student’s t-test).