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. 2021 Dec 24;10:e62645. doi: 10.7554/eLife.62645

Figure 2. Conditioned media (CM) from cancer-associated fibroblasts (CAFs) and wild-type pancreatic ductal adenocarcinoma (PDA) cells support proliferation of GFAT1 knockout cells.

(A) Representative wells from a colony-forming assay in parental TU8988T and GFAT1 knockout clonal line D10 in 10 mM N-acetyl-glucosamine (GlcNAc), base media (DMEM), or base media supplemented 1:3 (25%) with CAF CM, boiled CAF CM, or CAF CM subject to freeze-thaw (F/T). (B) Quantitation of colonies from data in A (n = 3). (C) Representative wells from a colony-forming assay in parental TU8988T and GFAT1 knockout clonal line D10 in 10 mM GlcNAc, DMEM, or base media supplemented 1:3 (25%) with CM from wildtype TU8988T cells, boiled TU8988T CM, or TU8988T CM subject to F/T. (D) Quantitation of colonies from data in C (n = 3). (E) Quantitation of colony-forming assay data of parental and GFAT1 knockout clonal TU8988T lines in base media, positive control GlcNAc, wildtype TU8988T CM diluted 1:2 (33%) or used directly (100%) (n = 3). (F) Representative wells and quantitation of colony-forming assay data of parental and GFAT1 knockout clonal TU8988T lines in base media, positive control GlcNAc, and wildtype TU8988T CM subject to F/T and diluted 3:1 (75%) (n = 3). (G) Quantitation of colony-forming assay data of parental and GFAT1 knockout clonal TU8988T lines in base media, positive control GlcNAc, or wildtype TU8988T, HPAC, or MiaPaCa2 CM subject to F/T and diluted 3:1 (75%) (n = 3). (H) GlcNAc dose response curve presented as relative colony number for parental and GFAT1 knockout TU8988T cells (n = 3). (I) Mean of absolute quantitation of GlcNAc in various CM by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) (n = 3). Error bars represent mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 2.

Figure 2—figure supplement 1. Rescue activity of conditioned media (CM) and N-acetyl-glucosamine (GlcNAc) in GFAT1 knockout cells.

Figure 2—figure supplement 1.

(A) Quantitation of colony-forming assay data of parental MiaPaCa2 and GFAT1 knockout clonal line M11 in base media (DMEM), positive control GlcNAc, wildtype TU8988T CM diluted 1:2 (33%) or used directly (100%) (n = 3). (B,C) Quantitation of proliferation assay data of (B) parental MiaPaCa2 and GFAT1 knockout clonal line M11 and (C) parental HPAC and GFAT1 knockout clonal line H7 in base media (DMEM), positive control GlcNAc, or wildtype TU8988T, HPAC, or MiaPaCa2 CM diluted 3:1 (75%) that was subjected to freeze-thaw (F/T) (n = 3). Data represent crystal violet extracted from cells at endpoint and measured by optical density (OD) at 590 nm. (D) GlcNAc dose response curve presented as relative colony number for parental TU8988T cells and GFAT1 knockout clonal line B9 (n = 3). Error bars represent mean ± SD. n.s., non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.