Induction of transglutaminase 2 (TG2) expression by hypoxic stress. (a) Cell lines were cultured for 24 h under normoxic or hypoxic (1% oxygen (O2)) conditions. Cell extracts were analysed using western blotting using antibodies to TG2 and hypoxia-inducible factor-1 (HIF-1α) respectively. N, normoxia; H, hypoxia. (b) quantitative reverse transcriptase–PCR (qRT–PCR) analysis of TG2 expression. Cells were cultured in hypoxic condition (0.1% O2) for 5 h. For analysis, 1 μg of total RNA was used. (c) Cell lines were cultured in Dulbecco's modied Eagle’ s medium (DMEM) containing 5-aza-2 deoxycytidine (5-aza-dC, 5 μM) for 4 days, and changing medium every 24 h with fresh 5-aza-dC. Cell extracts were analysed using western blotting. (d) U373MG cells exposed to CoCl2(200 μM) or hypoxia (1% O2) for 24 h were fractionated into the cytosolic and the nuclear fractions. The extracts were probed with antibodies to TG2, actin, hexokinase and lamin B, respectively. (e) U373MG cells cultured on glass coverslip were incubated in the presence of 0.2 mM 5′-(biotinamido)pentylamine (BP) for 24 h under normoxic or hypoxic (1% O2) conditions. Expression and intracellular activity of TG2 were assessed using a TG2/fluorescein isothiocyanate (FITC)-labeled immunoglobulin G (IgG) and Texas Red-conjugated streptavidin, respectively. To visualize the nucleus, 4,6-diamidino-2-phenylindole (DAPI) was used. Data represent mean±s.d. based on three independent experiments. *P⩽0.05; **P⩽0.001.