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

Figure 2.

Figure 2.

Slug is induced by glutamine deprivation via ERK signaling and ATF4. (A) Relative expression of Slug, Snail, Zeb1, and Twist transcript levels measured by qPCR in the indicated PDAC cells cultured in glutamine-replete or glutamine-free medium for 24 h. Results are expressed relative to control cells in glutamine-replete conditions (+Q). Data are expressed as mean ± SEM; n = 3–5 independent experiments. (B) Immunoblots of Slug expression in the indicated PDAC cells cultured in glutamine-replete or glutamine-free medium for 24 h. α-tubulin was used as a loading control. (C) Immunoblots assessing Slug expression in response to the indicated glutamine concentrations in KPC and AsPC-1 cells. α-tubulin was used as a loading control. Slug/α-tubulin ratios relative to 4 mM Q are shown. (D) Relative Slug mRNA expression by qPCR at the indicated glutamine concentrations in KPC and AsPC-1 cells. Results are expressed relative to the 4-mM Q concentration. Data are expressed as mean ± SEM; n = 3 independent experiments. (E) Immunoblots assessing protein expression of Slug, phospho-ERK (p-ERK), and ERK in KPC cells treated with vehicle (DMSO), 2 µM Tram or 2 µM SCH772984 (SCH) in glutamine-free medium for 5 h. α-tubulin was used as loading control. The immunoblots shown are representative of three independent experiments. (F) Relative Slug mRNA expression measured by qPCR in KPC cells under the same conditions described in E. Data are expressed as mean ± SEM; n = 3 independent experiments. (G) Immunoblots showing protein expression of Slug, ATF4, p-eIF2α (Ser51), and eIF2α in KPC cells treated with vehicle (DMSO) or ISRIB at the indicated concentrations in glutamine-free medium. α-tubulin was used as loading control. (H) Immunoblots assessing Slug and ATF4 protein expression in KPC cells stably expressing a nontargeting shRNA control (shGFP) or two independent short hairpins targeting ATF4 (shATF4#1 and shATF4#2) in glutamine-replete or glutamine-free medium for 24 h. β-actin was used as a loading control. Statistical significance was determined using unpaired two-tailed Student’s t test. Results are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.