In situ enzyme staining reaction conditions.

In situ enzyme staining reaction conditions.</p

Intense UGDH and diaphorase enzyme histostaining activity in transformed A549 cells.

By in situ enzyme staining, A549 cells showed relatively high (A) 99.8% diaphorase/GR staining with 0.1 mM NADH, (B) 93.1% UGDH enzyme staining and (C) 12.1% background NBT staining in the absence of UDP-Glc compared to primary human FLS. Scale bars: 100 μm.</p

Nuclear UGDH enzyme histostaining was observed in selected FLS cells.

(A, B) UGDH enzyme histostaining was occasionally detected in the nuclear or perinuclear compartment in sub-confluent cells in the middle of the culture well, and (C, D) was mainly cytosolic in more confluent cells at the edge of the well. (A, C) bright field and (B, D) matching epifluorescence image of the same field showing Hoechst-stained nuclei of FLS stimulated here with 1 ng/mL each IL1β + TGF-β1 and stained for UGDH enzyme activity. Scale bars: 20 μm. (TIF)</p

UDP-xylose and peroxide inhibited UGDH but not diaphorase staining activity in cultured monolayer FLS.

Cells were from a 23-year old male donor (passage 4). FLS stained for UGDH (A: bright field; B: phase contrast) or UGDH with UDP-xylose (C: bright field; D: phase contrast), diaphorase (E), or diaphorase with UDP-xylose (F). In panel (G), quantitative histomorphometry was used to measure the relative staining intensity in the absence or presence of UDP-xylose or peroxide. Symbols: *pvs UGDH; ** p (TIF)</p

TLC demonstration that NADH and GSH reduce NBT to the same formazan product with different kinetics.

Arrowheads show the origin and final solvent front; UV light was absorbed in the TLC plate by NADH and NBT at the origin, and by diformazan at the arrow. Reduced formazan had an Rf value ranging from 0.42 to 0.44 [38] depending on whether the lane was in the middle or edge of the TLC plate. In the absence of enzyme, GSH reduced NBT after 60 minutes but NADH only reduced NBT after 24 hours. Diaphorase or GR was necessary and sufficient for NADH to reduce NBT within 1 hour. See (S1 File) for original TLC plate images. (TIF)</p

Injured rabbit knee infrapatellar synovium showed higher diaphorase/GR and UGDH activity, and enhanced synovial fluid HA levels in 2 out of 3 rabbit knees compared to contralateral intact knees.

Panels show 20x magnification images of synovial membrane from regions with maximal UGDH induction, enzyme stained in situ for diaphorase (A, C), or UGDH (B, D), in intact (A, B) and contralateral injured knees (C, D). Boxed insets in panels B1-3, D1-3 show measured synovial fluid HA levels (mg/mL). (E) Quantitative histomorphometry of % stain (median, min-max, N = 3). Panels C4, D4 show low-magnification image of male rabbit infrapatellar synovium with dotted boxes showing regions illustrated in panels C3 & D3, respectively. Symbols and abbreviations: rabbit ID and sex are shown on the upper left; arrows (A-B) are regions in intact knees where UGDH staining was detected; BV: Blood vessel. Scale bars show 50 μm or 100 μm, as indicated.</p

Hypothetical model representing 2-enzyme UGDH activity staining mechanisms in cultured cells and unfixed cryosections.

Boxed molecules are enzyme staining components, encircled factors and dashed lines represent activities modulated in vivo by cytokines. Oxidative stress induced by inflammatory cytokines (TGF-β1+IL-1β) can lead to peroxide generation and reversible inactivation of UGDH unless GSH scavenges the free radicals to sustain UGDH activity. High endogenous GSH can produce background staining by directly reducing NBT and by-passing UGDH. Abbreviations: PDGF: platelet derived growth factor; GR: glutathione reductase; GSH: glutathione; H2O2: peroxide; NEM: N-ethyl maleimide (thiol alkylating agent). In the upper panel, Dark blue cells: UGDH-stained cell compartments where cytosolic diaphorase, or nuclear glutathione reductase activity is present; dashed lines: freeze-thaw permeabilized cell membranes.</p

UGDH protein showed detectable nuclear staining in selected cells.

(A-D) Phase contrast and (E-H) matching epifluorescent images of UGDH immunostain and (I-L) DAPI-counterstained fluorescent nuclei (I-L) of FLS (P4) extracted from a synovial biopsy from a 23-year old male donor and cultured under different cytokine conditions as indicated, fixed in acetone, and immunostained for UGDH with red substrate detection which is fluorescent. Symbols: small white arrows: cytosolic UGDH immunostain; open arrows: detectable nuclear UGDH immunostain. Scale bars: 20 μm. (TIF)</p