Fig. 4. PGD status influences MondoA trafficking.

a MondoA was concentrated in the nucleus of PGD^low 38Per peritoneal control cells by IF (left panel). In contrast, MondoA was distributed more equally between the nuclei and cytosol in PGD^high liver and lung metastases from the same patient (38Lv, 38Lg). b ChIP assays also detected reduced MondoA enrichments at the TXNIP promoter in the liver and lung metastases. c PGD inactivation by Crispr/Cas (sgRNAs #1, #2) concentrated MondoA into the nucleus of PGD^high 38Lg cells, as compared to wild-type controls. d PGD Crispr/Cas also increased MondoA enrichments at the TXNIP promoter by ChIP assays in 38Lg. e siRNAs targeting MondoA (siMLXIP #1, #2) knocked down MondoA in 38Lg cells (top panels) and prevented upregulation of TXNIP during Crispr/Cas PGD inactivation (middle panels). MondoA knockdown also rescued surface GLUT1 (bottom panels). f ChIP assays detected increased MondoA enrichments at the TXNIP promoter across the indicated PGD^high subclones treated with PGD inhibitor (PGDi: 6AN). g (Top panels) Treatment of GFP-expressing PGD^high 38Lg cells with excess (20 mM) 6PG concentrated MondoA in the nucleus by IF. This effect was rescued by depleting the excess 6PG with PGD overexpression⁴. Measurements of intracellular 6PG concentrations (right plots) confirmed that 6PG accumulated in treated GFP-expressing cells and that the excess was depleted in exogenous PGD-(over)expressing cells. Bottom panels: G6P control treatments failed to concentrate MondoA into the nucleus under any condition. For IF experiments: green, antibody signal; blue, Hoechst signal; n = 3 biological replicates, error bars: s.e.m., indicated p values calculated by two-tailed t tests, scale bars: 20 µm. For ChIP experiments: n = 3 technical replicates, error bars: s.d.m., indicated p values calculated by two-tailed t tests, results are representative of two biological replicates.