Levels of cellular Ca²⁺ and PKCα in NRF2i U937 cells.

<p>(A) Levels of ER stress markers in SCi and NRF2i U937 cells. Protein levels of phosphorylated PERK and EIF2α were assessed using a western blot analysis. Levels for un-spliced and spliced XBP-1 mRNA were quantified in SCi and NRF2i using RT-PCR analysis. (B) Cellular level of Ca²⁺ was monitored in SCi and NRF2i U937 cells. Cells were incubated with Fluo-4 AM (2 μM) for 30 min. Five min after the addition of vehicle (ethanol) or PMA (10 ng/mL), green fluorescence from Ca²⁺-reacting dye was monitored. The fluorescent intensity was quantified using the ZEN software. A confocal microscopic observation was performed with 400× magnification following 4',6-diamidino-2-phenylindole (DAPI) nuclear staining. Data represent the means ± SD of 3–4 experiments. ^aP < 0.05 compared with the vehicle control of each cell line. ^bP < 0.05 compared with PMA-treated SCi cells. (C) Western blot analysis of PKCα was performed in SCi and NRF2i U937 cells following PMA (10 ng/mL) incubation for 30 min. Similar blots were obtained in three independent experiments. (D) Immunocytochemical analysis of PKCα. SCi and NRF2i cells were incubated with vehicle (ethanol) or PMA (10 ng/mL) for 24 h, and PKCα cellular level was assessed following antibody incubation. A confocal microscopic observation was performed with 400× magnification following DAPI nuclear staining.