Journal: Nature Communications
Article Title: Pyridoxine induces glutathione synthesis via PKM2-mediated Nrf2 transactivation and confers neuroprotection
doi: 10.1038/s41467-020-14788-x
Figure Lengend Snippet: a , b Purified recombinant PKM2 (rPKM2) was mixed with 863 natural products individually. ▵Tm values analyzed by NanoDSF ( a ). rPKM2 dimer and tetramer formation analyzed by BN-PAGE (left) and densitometric analysis of the dimer/tetramer ratio (right) ( b ). c Chemical structures of compound no. 194 (vindoline) and no. 324 (pyridoxine). d , e Primary astrocytes were stimulated with different concentrations of pyridoxine or vindoline for 12 h. Immunofluorescence of GSH and PI analyzed by high-content screening ( d ). The relative intensity of GSH signals (upper) and the percentage of PI-positive nuclei (lower) ( e ). Scale bar, 100 µm. f – h Primary astrocytes were stimulated with 5 µM pyridoxine for 12 h. PKM2 dimer and tetramer formation analyzed by BN-PAGE and total PKM2 detected by immunoblotting with actin as a loading control ( f ). PKM2 and Nrf2 proximity ligation signals ( g ). Immunoblot analysis of PKM2 in cell lysates immunoprecipitated with a Nrf2 antibody ( h ). Immunoblotting: three independent experiments. Scale bar, 10 µm. i – l Primary astrocytes transfected with Pkm2 -specific siRNA were stimulated with 5 µM pyridoxine for 12 h. The amount of anti-Nrf2-immunoprecipitated DNA analyzed by qRT-PCR with primers flanking the Gclc and Gclm promoter regions ( i ). Gclc and Gclm mRNA detected by qRT-PCR ( j ). Immunoblotting with actin as a loading control ( k ) and GSH levels ( l ). Immunoblotting and qRT-PCR: three independent experiments; GSH assay: six independent experiments. m , n Primary neurons were stimulated with 5 µM pyridoxine for 12 h. Gclc and Gclm mRNA detected by qRT-PCR (m) and GSH levels ( n ). qRT-PCR: three independent experiments; GSH assay: six independent experiments. Data are presented as the mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, NS, not significant. One-way ANOVA with Dunnett’s multiple comparisons test ( b ) or with Tukey’s multiple comparisons test ( i , j and l ). Student’s two-tailed unpaired t -test ( m , n ). Source data are provided as a Source Data file.
Article Snippet: First, we performed an initial screen of 863 natural products purchased from TargetMol by intrinsic tryptophan differential scanning fluorimetry (nanoDSF) to explore molecules that may directly bind with PKM2.
Techniques: Purification, Recombinant, Nano Differential Scanning Fluorimetry, Immunofluorescence, High Content Screening, Western Blot, Ligation, Immunoprecipitation, Transfection, Quantitative RT-PCR, GSH Assay, Two Tailed Test
