Fig. 5: Pyridoxine facilitates GSH synthesis via the PKM2-Nrf2 pathway. | Nature Communications

Fig. 5: Pyridoxine facilitates GSH synthesis via the PKM2-Nrf2 pathway.

From: Pyridoxine induces glutathione synthesis via PKM2-mediated Nrf2 transactivation and confers neuroprotection

Fig. 5

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.

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