Water-soluble tetrazolium (WST) dyes, such as WST-1 and WST-8, are widely used in cell proliferation and anti-cell-growth drug screen assays. However, the underlying determinants for WST reduction are still largely unknown. In addition, application of tetrazolium-based assays to cellular glucose metabolism studies has not been fully explored. In the present study, we show here that WST-8 reduction is dependent on cellular glucose metabolism. In order to minimize the variance of live cell number during stimulation, we treated cells with different stimuli and performed tetrazolium-based assays within 6 hours. Withdrawal of medium glucose supply greatly attenuated WST-8 reduction but not intracellular ATP levels, while re-adding glucose reconstituted WST-8 reduction, indicating the effect was not due to cell death. The role of glucose on WST-8 reduction is specific since glutamine, fructose or galactose did not substitute for the effect of glucose on WST-8 reduction. Furthermore, inhibition of glucose transporters, intracellular glucose metabolic enzymes or EGFR-PI3K-Akt signaling also attenuated WST-8 reduction. In an attempt to screen inhibitors targeting cellular glucose metabolism from hyperglycemia-associated drugs, it turned out that HIV protease inhibitor, ritonavir, could largely block WST-8 reduction, but not cellular ATP level. Interestingly, ritonavir has been shown to acutely block glucose transport in vitro and in vivo. Taken together, our studies not only demonstrate an essential role of cellular glucose metabolism on WST-8 reduction, but also propose a novel application of tetrazolium-based assays in screening for inhibitors of cellular glucose metabolism when used in combination with ATP assay.

Bumblebee queens have remarkable spermathecae that store sperm for year-round reproduction. The spermathecal gland is regarded as a secretory organ that could benefit sperm storage. Queen mating provokes substantial physiological, behavioral, and gene expression changes. Here, the transcriptomes of spermathecae were compared between virgins and mated queens of the bumblebee, Bombus terrestris L., at 24 h post mating. Differentially expressed genes were further validated by real time quantitative PCR and immunofluorescence assay. In total, the expression of 11, 069 and 10, 862 genes were identified in virgins and mated queens, respectively. We identified that 176 differentially expressed genes between virgin and mated queen spermathecae: 110 (62.5%) genes were upregulated, and 66 (37.5%) genes were downregulated in mated queens. Most of the differentially expressed genes validated by RT-qPCR were concentrated on immune response [i.e., leucine-rich repeat-containing protein 70 (35.8-fold), phenoloxidase 2 (41.9-fold), and defensin (4.9-fold)] and sperm storage [i.e., chymotrypsin inhibitor (6.2-fold), trehalose transporter Tret1 (1.7-, 1.9-, 2.4-, and 2.4-fold), and heterogeneous nuclear ribonucleoprotein A3 (1.2-, and 2.6-fold)] functions in the spermathecae of mated queens. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) was hypothesized to promote the mating behavior according to RT-qPCR and immunofluorescence assay. The expression levels of most upregulated immune genes were decreased significantly at 3 days post mating. In conclusion, the external sperm transfer into spermathecae led to the significantly upregulated immune response genes in bumblebees. These gene expression differences in queen spermathecae contribute to understanding the bumblebee post mating regulatory network.