Fig 2: TRPM2 knockdwon markedly reduced p47 phox phosphorylation, TXNIP expression and Trx activity under HG in U937 monocytes.(A) Representative immunoblots and graphs for protein expressions of p47 phox, p22phox, gp91pox, TXNIP or GAPDH in the presence of BAPTA-AM (2.5, 5, 10 μM), or EGTA (0.5, 1, 5 mM) under low glucose (LG; 5.5 mM glucose) or high glucose (HG; 30 mM glucose) (n = 4–5). (B) Representative immunoblots and graphs for protein expressions of p47 phox, TXNIP or GAPDH or β-actin in the presence of GAPDH- or TRPM2-siRNA under HG (n = 4). (C) Quantitative PCR was performed on TXNIP mRNA in GAPDH- (GA si) or TRPM2-siRNA-treated cells under LG or HG, and it was normalized to LG + GAPDH-siRNA (n = 4). (D) TRX activity was determined by the insulin disulfide reduction assay, and was normalized to LG + GAPDH-siRNA. The cells were stimulated with HG for 24, 48, 72 h in the presence of GAPDH- or TRPM2-siRNA (n = 4). Data were shown as mean ± S.E.M. (C,D) *P < 0.05 and ***P < 0.001 vs. LG + GAPDH-siRNA; #P < 0.05 and ###P < 0.001 vs. HG + GADPH-siRNA.

Fig 3: TRPM2 interacted with p47 phox during HG stimulation in U937 monocytes.(A,B) Immunofluorescence images showing the location of the (A) FM4-64 (1 uM; cell membrane marker) and subcellular p47 phox, or (B) subcellular p47 phox and TRPM2, in fixed cells by using confocal microscopy. The cells were pre-treated with 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 µM) under low glucose (LG; 5.5 mM glucose;) or high glucose (HG; 30 mM glucose). The percentage of co-localization of p47 phox with (A) FM4-64, or (B) TRPM2, was calculated as the average volume of the overlapping areas (n = 4–5). (C,D) Representative immunoblots showing the immunoprecipitation results for the interaction of TRPM2 with p47 phox or phosphorylated-Ser345 p47 phox under high glucose (10, 20, 30 mM glucose) for 48 h (n = 4). Data were shown as mean ± S.E.M. (A,B) **P < 0.01 vs. LG; ##P < 0.01 vs. HG.