Fig. 4: TPM4 phase separation is correlated with elevated glycolysis.
From: TPM4 condensates glycolytic enzymes and facilitates actin reorganization under hyperosmotic stress
a Representative images of TPM4 and NADH signals by Peredox probe in MDA-MB-231 cells expressing TPM4-AcGFP before (isosmotic) and after (hyperosmotic) 100 mM sorbitol treatment for 3 min. Scale bar, 20 μm. b Line scan plot showing the intensity distribution of TPM4 (green) and NADH signals (magenta) along the yellow line shown in a under hyperosmotic condition. c Representative images of ATP signals by MaLionR probe in MDA-MB-231 cells expressing TPM4-AcGFP before (isosmotic) and after (hyperosmotic) 100 mM sorbitol treatment for 3 min. Scale bar, 20 μm. d Line scan plot showing the intensity distribution of TPM4 (green) and ATP signals (magenta) along the yellow line shown in c under hyperosmotic condition. e Left: representative images of TPM4 and NADH signals in MDA-MB-231 cells expressing TPM4-mCherry-Cry2 upon blue light exposure. Dashed boxes are zoomed in on the right. Scale bar, 20 μm. Right: line scan plot showing the intensity distribution of TPM4 and NADH signals along the yellow line. f Quantification of relative lactate level in cells expressing non-optoDroplet-TPM4 (TPM4-AcGFP) or optoDroplet-TPM4 (TPM4-mCherry-Cry2) before and after blue light exposure for 10 min. n = 3 independent experiments; error bar: mean with SEM; ns = 0.0898, **P = 0.0015 by paired t-test. g Lactate production gradually rises with an increasing percentage of cells displaying TPM4 condensates. The x-axis shows the percentage of cells with TPM4 condensates (n = 100 cells per condition, repeated three times independently) across different experimental conditions, including control, 100 mM sorbitol and 250 mM sorbitol hyperosmotic treatment. The y-axis shows levels of lactate produced by cells under the same conditions (n = 3 independent experiments; error bar: mean with 95% CI). h Left: quantitative curves of ECAR in MDA-MB-231 cells treated with Seahorse basic medium (isosmotic) or 100 mM sorbitol (hyperosmotic) (n = 3 independent experiments; error bar: mean with SEM). Sorbitol was added at time point a, and ECAR reaches its highest level at time point b. Right: quantification of ECAR in isosmotic and hyperosmotic groups at time points a and b. n = 3 independent experiments; error bar: mean with SEM; isosmotic ns = 0.8752, hyperosmotic ***Pab = 0.0007 by two-way ANOVA.
