PDI1A depletion correlates with a more reduced ER. (A) Bar diagram of fluorescent lifetime (FLT) of roGFPiE in HEK 293 T cells co-transfected with different shRNAs directed to PDI1A and an empty shRNA vector control. FLT of untreated (UNT) thapsigargin (TG) and DTT treated cells is included as a reference. (B) Immunoblot of endogenous PDI1A in the cells above, with tubulin serving as a loading control. The PDI1A to tubulin ratio (set to 100 in the vector control sample) is provided under each sample. (C) Scatter diagram of correlation of FLT to PDI1A expression in the samples above (R2 = 0.637, P value = 0.013, n = 7). (D) Bar diagram of fluorescent lifetime (FLT) of roGFPiE in wild type mouse embryonic fibroblasts (CRT+/+ MEF) and in mouse embryonic fibroblasts lacking calreticulin (CRT−/− MEF) . Where indicated, the cells were exposed to thapsigargin (TG, 0.8 μM for five minutes) or DTT (2 mM, five minutes). Shown is the mean ± SD of FLT (n = 46). (**P <0.001, one way ANOVA for the difference in change in FLT upon TG exposure between the +/+ and −/− cells). ANOVA, analysis of variance; DTT, dithiotreitol; ER, endoplasmic reticulum; PDI1A, protein disulfide isomerase; SD, standard deviation.