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. 2020 Jun 8;217(9):e20200388. doi: 10.1084/jem.20200388

Figure S3.

Figure S3.

Slug regulates EMT and survival upon glutamine starvation. (A) Relative Slug mRNA expression by qPCR in KPC Slug knockdown cells compared with control (shLuc) in glutamine-replete or glutamine-free medium for 24 h. Results are expressed relative to shLuc/+Q condition. Data are shown as mean ± SEM; n = 5 independent experiments. (B) Relative expression of the indicated EMT-related genes in KPC Slug knockdown compared with shLuc in glutamine-free medium for 24 h. Results are expressed relative to shLuc. Data are shown as mean ± SEM; n = 3 independent experiments. (C) Chart showing enriched pathways among the genes up-regulated in Slug-depleted KPC cells compared with control (shLuc) in glutamine-free conditions. The graph was generated with Metascape. (D) Relative expression of the indicated genes in KPC Slug knockdown compared with shLuc in glutamine-free medium for 24 h. Results are expressed relative to shLuc. The results are representative of three independent experiments. Data are presented as the mean ± SEM of three replicates. (E) Relative cell number assessed by Syto60 staining in KPC shLuc, shSlug#1, or shSlug#2 cells incubated in medium containing 0.05 or 0.2 mM glutamine for 3 and 5 d. Representative data are shown for n = 3 independent experiments with three to five replicates each. (F) Relative cell number assessed by crystal violet staining in KPC shLuc, shSlug#1, or shSlug#2 cells incubated in glutamine-free medium or medium containing 0.2 mM glutamine for 5 or 6 d. Data are normalized to day 0 for each cell line and are shown relative to the shLuc control. Results are expressed as mean ± SEM; n = 5 independent experiments. (G) Relative cell number assessed by crystal violet staining in KPC shLuc, shSlug#1, or shSlug#2 incubated in complete medium for 48 h. Results are expressed as mean ± SEM; n = 4 independent experiments. ns, not significant. Statistical significance was determined using unpaired Student’s t test in A and one-way ANOVA followed by Dunnett's test for multiple comparisons in B–G. All data are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.001.