An L-2-hydroxyglutarate biosensor based on specific transcriptional regulator LhgR - PubMed
- ️Fri Jan 01 2021
a Sequential images of mTFP (top), Venus (middle), and Venus/mTFP emission ratio (bottom, pseudocolored) of single HEK293FT cell expressing LHGFR0N3C. 10 mM
l-2-HG was added at time point zero (min). Elapsed time (in minutes) after the addition of
l-2-HG is shown at the top of the images. Scale bar, 10 μm. b Time course of the emission ratio changes inside the region of interest (ROI) depicted from the top-left image of a. c Normalized dose-response curve of LHGFR0N3C expressed in HEK293FT cells with increasing concentrations (10 nM to 10 mM) of
l-2-HG. Cells were permeabilized with 10 μM digitonin. The emission ratio of non-permeabilized HEK293FT cells under physiological conditions is indicated with a black dash line. d Responses of LHGFR0N3C expressed in HEK293FT cells to exogenously added 1 mM glutarate,
l-2-HG,
d-2-HG, and glucose. Cells were permeabilized with 10 μM digitonin. All data were normalized to the control (ratio in the absence of any tested compounds). e Identification of the function of L2HGDH in
l-2-HG catabolism by LHGFR0N3C. The emission ratio was measured after co-transfecting siRNA targeting L2HGDH and LHGFR0N3C for 48 h. ***a: P = 0.0002, **b: P = 0.0058, ***c: P = 0.0002, *d: P = 0.0303. f Detection of hypoxia-induced
l-2-HG accumulation by LHGFR0N3C. Emission ratio changes were recorded after LHGFR0N3C-expressing HEK293FT cells cultured in normoxia or hypoxia in the absence and presence of 5 mM dimethyl-2-ketoglutarate (DMαKG) for 24 h. The emission ratio was normalized to normoxic conditions without DMαKG. **a: P = 0.0029, **b: P = 0.0030, ***c: P < 0.0001, ***d: P = 0.0002, **e: P = 0.0016, **f: P = 0.0068, *g: P = 0.0126, *h: P = 0.0139. g Identification of the functions of LDHA and MDH2 in
l-2-HG anabolism by LHGFR0N3C. HEK293FT cells were cultured in the presence of 5 mM DMαKG. The emission ratio was normalized to the normoxic conditions treated with negative siRNA. **a: P = 0.0011, *b: P = 0.0400, ***c: P = 0.0002, ***d: P = 0.0004, **e: P = 0.0021, ***f: P = 0.0002. Inconsistent initial emission ratios were detected in HEK293FT cells under different conditions. The normalized emission ratios were thus used to monitor the changes of
l-2-HG in different HEK293FT cells. All data shown are means ± s.d. (n = 3, 3, 4, 4, and 4 independent experiments for c, d, e, f, and g). *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference (P ≥ 0.05); one-way ANOVA test with Tukey’s Multiple Comparison Test for e and g; two-tailed t-test for f.