Fig. 7: LDHA mediates ROS generation by BH4 through S-nitrosylation.

a, b S-nitrosylation of LDHA (Cys163 and Cys293) was examined by LC–MS/MS. Sequence-informative fragmentation ions are summarized based on the peptide sequence and are annotated in blue (b-ions) and red (y-ions). c S-nitrosylated LDHA in HELF cells was analyzed by a biotin switch assay followed by Western blot analysis. The sample without ascorbate was representative of endogenous biotinylated protein levels. d The mRNA levels of LDHA in parental and LDHA-KO cells. e Immunofluorescence staining for LDHA (red) and DAPI (blue) in BEAS-2B cells. Scale bar = 20 μm. f LDH activity was examined in BEAS-2B cells. g ROS levels in BEAS-2B cells were determined using a fluorescence microscope or 96-well plate reader. Scale bar = 200 μm. h LDHA-KO BEAS-2B cells were incubated with FX11 followed by 10 Gy of X-ray irradiation. ROS levels in BEAS-2B cells were determined using a 96-well plate reader. i LDHA KO BEAS-2B cells were treated with BH4 followed by 10 Gy of X-ray irradiation. ROS levels in BEAS-2B cells were determined using a fluorescence microscope or 96-well plate reader. Scale bar = 200 μm. j LDHA-KO BEAS-2B cells were incubated with the GCH1 inhibitor DAHP (50 μM) and then exposed to 10 Gy of X-ray irradiation. ROS levels were determined using a fluorescence microscope or 96-well plate reader. k LDHA-KO BEAS-2B cells were incubated with the NOS inhibitor L-NMMA (100 μM) and then exposed to 10 Gy of X-ray irradiation. ROS levels were determined using a fluorescence microscope or 96-well plate reader. BEAS-2B cells were transfected with WT LDHA or the mutants followed by 10 Gy of X-ray irradiation. l The clonogenic survival of parental and LDHA-KO BEAS-2B cells was measured. m Crystal structure of LDHA bound to NADH and D-lactate (PDB entry 4JNK). The proteins are shown in a rainbow ribbon representation. NADH is shown as sticks with carbons colored cyan and d-lactate colored magenta. The residues Cys163 and Cys293 are highlighted with balls. The residue number was corrected for the first amino acid, which was missing in the X-ray structure. n Western blot analysis of LDHA expression in LDHA-KO BEAS-2B cells transfected with the indicated vectors. o LDHA-KO BEAS-2B cells were transfected with WT LDHA or mutant LDHA (with double mutations at Cys163 and Cys298) (2C-A). S-nitrosylated LDHA was analyzed by using a biotin switch assay followed by western blotting. The sample without ascorbate was representative of endogenous biotinylated protein levels. p ROS levels in BEAS-2B cells were measured in the presence of GSNO (200 μM) using a fluorescence microscope or a 96-well plate reader. q Cells were transfected with WT LDHA or the mutants followed by radiation. The nuclear and cytoplasmic levels of LDHA were examined by Western blot analysis. r The clonogenic survival of LDHA-KO BEAS-2B cells was measured. P < 0.05 and **P < 0.01 compared with the control group.