NEM abolished UGDH enzyme histostaining but only partly inhibited diaphorase staining when added to live cell cultures.

(A) Quantitative histomorphometry of staining intensity for FLS stained for NAD+ only, UGDH, or diaphorase/GR, with and without NEM. NEM was either included in the staining solution (“in stain”) or added to cell cultures at 1 mM NEM for 2 hours before enzyme staining (“added to live cells”). Representative images of histostained FLS from one female donor, showed no stain for (B1-B3) the negative control (NAD+ only), NEM-sensitive staining for (C1-C3) UGDH (UDP-Glc and NAD+), and NEM-resistant staining for (D1-D3) “low diaphorase/GR”, (E1-E3) “high diaphorase/GR stain”; (B1-E1) no NEM, (B2-E2) NEM “added to stain”, or (B3-E3) NEM “added to live cells”. In panel A, significant differences are noted with letters and symbols (*, ***). The graph in Panel (A) shows the median, 25% quartile range (box), min-max, distinct cultures with 2 female human donors and 1 male donor FLS (n = 4–7). Scale bars: 50 μm.