Intracellular unpaired IgH triggers the UPR and can cause apoptosis. (A) IBs from ALMC1 cells (top) treated with si[IGLC] or si[-] control over a 30-hour period show reduction in IgL and persistence of IgH intracellular proteins as do IBs of EJM cells (bottom) treated in the same way. (B) IBs of GRP78 and IgH IPs from ALMC1 and EJM cells treated with si[IGLC] or si[-] control show the coimmunoprecipitation of IgH with GRP78, as well as the marked decrease in IgL and increase in GRP78 that occur at 30 hours after si[IGLC] treatment. (C) Real-time PCR (qPCR) for markers of the UPR was performed with cDNA from ALMC1 and EJM cells treated with si[IGLC] or si[-] control for the indicated times. In si[IGLC]-treated cells, there were significant increases in all 3 UPR markers at 8 and 16 hours. Mean ± SD is shown with P < .01 compared with control by 2-tailed paired Student t test (n = 3). Three independent repeat qPCRs were performed in duplicate wells. (D) IBs for 3 markers of the UPR were performed with whole cell lysates from cells treated with si[IGLC] or si[-] control for the indicated times. As this typical IB shows, in si[IGLC]-treated but not control cells there were significant increases in all 3 UPR markers. (E) Real-time PCR (qPCR) for proapoptotic BH3 family members was performed with cDNA from cells treated with si[IGLC] or si[-] control for the indicated times. In si[IGLC]-treated ALMC1 cells, there was a three- to-fourfold increase in NOXA expression maintained at 8, 16, and 24 hours. The increase in NOXA expression in EJM cells, however, was lower than in ALMC1 cells and also lower than the increase in PUMA expression. Two independent repeat qPCRs were performed in duplicate. (F) ALMC1 but not EJM cells treated with si[IGLC] displayed significantly increased levels of mitochondrial depolarization at 24 hours as represented by a significant decrease in the percentage of si[IGLC] relative fluorescence units (RFUs) of green JC-1 monomer compared with si[-] by flow cytometry. Increased levels of mitochondrial depolarization were not present when si[IGLC]-treated cells were cultured with 50 μM Z-VAD-FMK. Mean ± SD is shown with *P = .01 by 2-tailed paired Student t test (n = 3). (G) ALMC1 cells treated with si[IGLC] displayed increased levels of apoptosis manifest as annexin-V positivity at 24 hours. Viable cells were reduced to 64% with si[IGLC] treatment. Apoptosis did not occur when cells treated with si[IGLC] were cultured with a pan-caspase inhibitor (Z-VAD-FMK, 50 µM). (H) IB comparison of ALMC1 and EJM cells undergoing IgL knockdown shows that CHOP is markedly increased in ALMC1 compared with EJM cells. This IB is representative of 3 performed. (I) IP of myeloid cell leukemia sequence 1 (MCL-1) in ALMC1 and EJM cells treated with si[IGLC] indicates by IB that more NOXA is pulled down with MCL-1 from ALMC1 than from EJM cells. Normalized levels of MCL-1 and NOXA in si[IGLC] lanes are shown as percentages of si[-] levels. This IP/IB is a representative of 2 performed.