Fig 2: TDP-43 loss decreases Btn1a1 and Xdh mRNA stability.a, b Primary MECs isolated at pregnancy day 17.5 (P17.5) (a) and lactation day 1 (L1) (b) from wild-type (WT) and Tardbp-/- mice were treated with actinomycin D for the indicated time, then Btn1a1, Xdh, and Gapdh mRNA expression levels were analyzed by qRT-PCR. Data were normalized to 18S rRNA levels in each experiment and represented as a percentage of mRNA levels measured at time 0 h (before actinomycin D addition) using a semi-logarithmic scale. Half-lives (t1/2) were calculated as time of each mRNA to decrease to 50% of its initial abundance. c qRT-PCR analysis of Btn1a1 and Xdh mRNA expression in differentiated HC11 cells treated with actinomycin D after overexpression of control (Flag), Flag-TDP-43 full-length (Flag-FL), and Flag-TDP-43 C-term (Flag-C-term). d Upper: schematic of reporter constructs containing green fluorescent protein (GFP) gene fused with Btn1a1 and Xdh mRNA 3'-UTRs. GFP-Btn1a1-mutUTR and GFP-Xdh-mutUTR were generated by deletion mutations of UG-enriched sequences in Btn1a1 (position 2845–2864, 2935–2972, 3319–3326, and 3365–3372 nt) and Xdh (position 4523–4538 nt) 3'-UTRs, respectively. Lower: GFP expression levels were measured by western blotting 72 h after co-transfection with GFP reporter and sh-TDP-43. sh-TDP-43-1 and sh-TDP-43-2 represent independent shRNAs used. Data are shown as the means ± SD of three independent experiments. Source data are provided as a Source Data file.

Fig 3: TDP-43 directly binds to Btn1a1 and Xdh mRNA.a mRNA structures of Btn1a1 (upper) and Xdh (lower) showing TDP-43-binding motif in 3'-UTRs. Deep green boxes represent coding region. Light green boxes represent non-coding region. b, c RNA immunoprecipitation (RIP) assay for analysis of interaction between TDP-43 protein and Btn1a1 or Xdh mRNA in MECs and differentiated HC11 cell line using TDP-43 antibody. d, e MECs (d) and differentiated HC11 (e) cellular extracts were incubated with in vitro-transcribed biotin-labeled control (pcDNA3.1 vector), Btn1a1, or Xdh mRNA fragments for biotin RNA pull-down followed by western blot analysis. Control RNA used biotinylated RNA without TDP-43-binding site (ugugug). f Structures of TDP-43 and their mutants used in RIP assays. TDP-43 protein contains an N-terminal domain (N-term), two RNA-recognition motifs (RRM1 and RRM2), a glycine-rich domain (GRD), and a C-terminal domain (C-term). g Western blot of overexpressed Flag-TDP-43 or its mutants in HC11 cell line. h RIP assay using Flag antibody in differentiated HC11 cells after overexpression of Flag-TDP-43 or Flag-mutants of TDP-43. i Schematic of mouse Btn1a1 or Xdh mRNA fragments used for RNA pull-down assays. j Binding between TDP-43 protein and fragmented Btn1a1 and Xdh mRNA. Four biotinylated Btn1a1 mRNA fragments or six biotinylated Xdh mRNA fragments were incubated with HC11 cellular extract, and interaction between TDP-43 and each fragment was examined by RNA pull-down followed by western blot analysis. Data are shown as the means ± SD of three independent experiments. Source data are provided as a Source Data file.

Fig 4: The effects of nitrate were dependent on XOR but independent of iNOS. NO levels in medium loaded with LPS, various concentrations of sodium nitrate (10, 100, and 500 µM), and the iNOS inhibitor L-canavanine (1 mM) (a) or the XOR inhibitor allopurinol (100 µM) (b). The mtROS (c) and ??m (d) after L-canavanine (1 mM) and allopurinol (100 µM) treatment. Data are expressed as mean ± SEM (n=3); p < 0.01 (**) and p < 0.001 (***).

Fig 5: Nitrate modulated mitochondrial function via NO and did not alter the expression of iNOS or XOR. The mtROS (a) and ??m (b) after addition of the NO scavenger Carboxy-PITO (1 mM) (n = 4). The mRNA expression of iNOS (c) and XOR (d) in medium loaded with LPS and various concentrations of sodium nitrate (10, 100, and 500 µM). The protein expression of iNOS (e) and XOR (f) in medium loaded with LPS and various concentrations of sodium nitrate (10, 100, and 500 µM). Data are expressed as mean ± SEM (n = 3); p < 0.05 (*) and p < 0.001 (***).