Fig 2: Predicted miRNAs can target F8 and downregulate FVIII expression in mammalian cells. (A) Relative overexpression of miRNAs (scrambled control: SC = 1.035 ± 0.026, N = 14; miR-144 = 64.00 ± 1.54, N = 6; miR-19b = 1.562 ± 0.154, N = 6; miR-186 = 8.790 ± 1.468, N = 8) and (B) relative expression of F8 mRNA (SC = 1.004 ± 0.019, N = 16; miR-144 = 0.9089 ± 0.0354, N = 12; miR-19b = 0.879 ± 0.028, N = 12; miR-186 = 0.751 ± 0.073, N = 12) in LCL cells transfected with miRNAs expression vectors compared to control cells (scrambled control, SC). Q-PCR results of miRNAs and F8 were normalized to RNU6 and GAPDH, respectively. (C) Relative FVIII level in LCL cells collected 72 h after transfection with miRNAs: miR-144, miR-19b, and miR-186 expression vectors compared to control cells (SC) determined by Western blot from transfected samples along with recombinant FVIII (rFVIII). Multiple species were detected: Single chain FVIII (FVIII:SC) at about 267 kDa, heavy chain polypeptides (FVIII:HC) generated after FVIII single chain cleavage within the B domain with apparent MW range between 90 and 200 kDa; light chain (FVIII:LC) with apparent MW of 80 kDa and loading controls; and β-actin and GAPDH detected at approximately 42 and 35 kDa, respectively. The blots were cropped to improve clarity of the results. Full-length blots are presented in Supplementary Figure S3. (D) Quantification of FVIII heavy chain in cells transfected with miR-144 and miR-19b (SC = 1.011 ± 0.0127, N = 8; miR144 = 0.955 ± 0.085, N = 8; miR-19b = 0.523 ± 0.0623, N = 8) from (C). (E) Quantification of FVIII heavy chain in cells transfected with miR-186 (SC = 0.989 ± 0.0472, N = 8; miR186 = 0.717 ± 0.002, N = 8) from (C). Bands normalized to β-actin and GAPDH. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Tph2 mRNA level (a) and protein (b) levels. Maoa mRNA level (c) and protein (d) levels in the hippocampus of experimental and control mice. Gene expression is presented as the number of cDNA copies per 100 cDNA copies of Polr2a. Protein levels were assessed in chemiluminescence relative units and normalized to GAPDH chemiluminescence relative units. N = 7–9.

Fig 4: Effects of C-ANP4–23 treatment on oxidative stress indexes in kidneys of Dahl salt-sensitive rats. Concentrations of MDA, lipofuscin (A) and Nox (B) in kidneys of Dahl salt-sensitive rats at 6 weeks after a normal-salt or a high-salt diet with vehicle or C-ANP4–23 injection. (C) Dahl salt-sensitive rats with a normal-salt or a high-salt diet plus vehicle or C-ANP4–23 injection for 6 weeks were subjected to Western blot analysis using antibodies against Nox4. The expression was normalized to the housekeeping protein GAPDH. ∗P < 0.05 compared with DS + NS + V group. †P < 0.05 compared with DS + NS + C group. ‡P < 0.05 compared with DS + HS + V group. C-ANP4–23: C-atrial natriuretic peptide (ANP)4–23; MDA: Malondialdehyde; Nox: Nicotinamide adenine dinucleotide phosphate oxidase; Nox4: Nicotinamide adenine dinucleotide phosphate oxidase 4; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; DS + NS + V: Dahl salt-sensitive rats with normal-salt diet and vehicle injection; DS + NS + C: Dahl salt-sensitive rats with normal-salt diet and C-ANP4–23 injection; DS + HS + V: Dahl salt-sensitive rats with high-salt diet and vehicle injection; DS + HS + C: Dahl salt-sensitive rats with high-salt diet and C-ANP4–23 injection; prot: protein.

Fig 5: Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) immunohistochemical staining of the cells in the maculae flavae and lamina propria of the human adult vocal fold mucosa. Cells in the human maculae flavae strongly expressed GAPDH. On the other hand, stratified squamous epithelium and interstitial cells in the lamina propria of the vocal fold mucosa sparsely expressed GAPDH. (*: Border between posterior macula flava and lamina propria of the vocal fold mucosa)