![Figure 3. Knockdown of the IgL CR reduces immunoglobulin secretion and cell viability. (A) Five human MM cell lines that secrete IgL were treated with si[IGLCCR] targeting the CR of the IgL mRNA or si[-] control, and the residual MFI at 20 hours after knockdown is shown. Mean ± SD; n = 3 observations per cell line. In all (n = 15), the mean MFI of intracellular IgL after si[IGLCCR] treatment was 55% ± 23% of control indicating a 45% average reduction in less than a day. (B) The concentrations of secreted IgL in culture supernatants from these 5 human MM cell lines after treatment with si[IGLCCR] or si[-] control were measured by ELISA. Cultures were charged with 106 cells per mL, and supernatant was obtained 24 hours later for measurement. All cells treated with si[IGLCCR] secreted less IgL. Mean ± SD is shown for 3 independent repeats per cell line with P values obtained with 2-tailed paired Student t test. (C) This graph shows the percent reductions in secreted IgL with si[IGLCCR] treatment. Mean ± SD is shown for 3 independent repeats per cell line. The mean percent reduction for all 15 observations was 45% ± 23%. (D) The concentrations of secreted IgH in culture supernatants from 3 human MM cell lines that secrete intact IgGλ after treatment with si[IGLCCR] or si[-] control were measured by ELISA. Mean ± SD is shown for 3 independent repeats per cell line with P values obtained with 2-tailed paired Student t test. The mean reductions in secreted IgH were 45% ± 9%, 43% ± 22%, and 14% ± 8% for ALMC1, ALMC2, and EJM cells, respectively. (E) ALMC1 as IgGλ-producing cells were compared with MM.1S as IgL-only producing cells for evidence of activation of the UPR and change in the expression of NOXA by qPCR. At 20 hours after treatment, the relative fold changes in expression of the UPR markers GRP78, CHOP, and XBP1S, and of NOXA, with si[IGLCCR] are substantial in cells producing IgGλ but not IgL only. Assays were performed 3 times with cDNA from 3 independent repeat experiments. Mean ± SD is shown with P values obtained with 2-tailed unpaired Student t test. Dotted line is qPCR control. (F) Caspase 3/7 activity was measured with the GloMax bioluminescence assay in 2 myeloma cell lines that produce intact IgGλ (ALMC1 and EJM) and 2 that produce only IgL (MM.1S and OPM-2) after treatment with si[IGLCCR] or si[-]. Reductions in IgL in cells producing intact IgGλ were associated with significantly increased caspase 3/7 activity, whereas in cells producing only IgL there was no increase in caspase 3/7 activity associated with IgL reductions. (In OPM-2 cells, there was an apparent decrease in caspase 3/7 activity with IgL knockdown.) Mean ± SD of the changes in caspase 3/7 activity and P values are shown, the latter obtained with 2-tailed paired Student t test (n = 3 for each cell line). (G) Cell viability and proliferation were measured by MTT assay in 2 myeloma cell lines that produce intact IgGλ (ALMC1 and EJM) and in 2 that produce only IgL (MM.1S and OPM-2) after treatment with si[IGLCCR] or si[-]. Reductions in IgL in cells producing intact IgGλ were associated with significantly decreased cell viability and proliferation, whereas in cells producing only IgL there was no decrease compared with si[-] controls. Mean ± SD of the changes in MTT readouts and P values are shown, the latter obtained with 2-tailed paired Student t test (n = 3 for each cell line).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/123/22/10.1182_blood-2013-10-535187/4/3440f3.jpeg?Expires=1735558567&Signature=F7A5F6AborMAcB9r42ph1nKUFObC9Sac4Esa7N1M-i1~Xcz85j70ayfxo0K0PWCpCL85VqQhGdNlGtqkDvUH-9-BzXEsR6679wJrkWBn4ZtExhAQknUJLm8iR3FkTSYAGzHo2lgBPvUh9H1k2gh-ddr~piwq3Kd7XZOF~GSNYa4mo6~RKhzNcIjgmaorkZi~BY47yLaHWPmmVxbUw8HOcnTVWRgMo7-z3RLo8jUqW8IDgNqyN1qjhjm9dz-hg2DdtYtkUf294UMM21m-V4eyYx7782R2OMmRkkZpYKhouhiFsK2jqajeN4tK8iZiVGKOVjsprxR02UT-iD56ubaWiQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Knockdown of the IgL CR reduces immunoglobulin secretion and cell viability. (A) Five human MM cell lines that secrete IgL were treated with si[IGLCCR] targeting the CR of the IgL mRNA or si[-] control, and the residual MFI at 20 hours after knockdown is shown. Mean ± SD; n = 3 observations per cell line. In all (n = 15), the mean MFI of intracellular IgL after si[IGLCCR] treatment was 55% ± 23% of control indicating a 45% average reduction in less than a day. (B) The concentrations of secreted IgL in culture supernatants from these 5 human MM cell lines after treatment with si[IGLCCR] or si[-] control were measured by ELISA. Cultures were charged with 106 cells per mL, and supernatant was obtained 24 hours later for measurement. All cells treated with si[IGLCCR] secreted less IgL. Mean ± SD is shown for 3 independent repeats per cell line with P values obtained with 2-tailed paired Student t test. (C) This graph shows the percent reductions in secreted IgL with si[IGLCCR] treatment. Mean ± SD is shown for 3 independent repeats per cell line. The mean percent reduction for all 15 observations was 45% ± 23%. (D) The concentrations of secreted IgH in culture supernatants from 3 human MM cell lines that secrete intact IgGλ after treatment with si[IGLCCR] or si[-] control were measured by ELISA. Mean ± SD is shown for 3 independent repeats per cell line with P values obtained with 2-tailed paired Student t test. The mean reductions in secreted IgH were 45% ± 9%, 43% ± 22%, and 14% ± 8% for ALMC1, ALMC2, and EJM cells, respectively. (E) ALMC1 as IgGλ-producing cells were compared with MM.1S as IgL-only producing cells for evidence of activation of the UPR and change in the expression of NOXA by qPCR. At 20 hours after treatment, the relative fold changes in expression of the UPR markers GRP78, CHOP, and XBP1S, and of NOXA, with si[IGLCCR] are substantial in cells producing IgGλ but not IgL only. Assays were performed 3 times with cDNA from 3 independent repeat experiments. Mean ± SD is shown with P values obtained with 2-tailed unpaired Student t test. Dotted line is qPCR control. (F) Caspase 3/7 activity was measured with the GloMax bioluminescence assay in 2 myeloma cell lines that produce intact IgGλ (ALMC1 and EJM) and 2 that produce only IgL (MM.1S and OPM-2) after treatment with si[IGLCCR] or si[-]. Reductions in IgL in cells producing intact IgGλ were associated with significantly increased caspase 3/7 activity, whereas in cells producing only IgL there was no increase in caspase 3/7 activity associated with IgL reductions. (In OPM-2 cells, there was an apparent decrease in caspase 3/7 activity with IgL knockdown.) Mean ± SD of the changes in caspase 3/7 activity and P values are shown, the latter obtained with 2-tailed paired Student t test (n = 3 for each cell line). (G) Cell viability and proliferation were measured by MTT assay in 2 myeloma cell lines that produce intact IgGλ (ALMC1 and EJM) and in 2 that produce only IgL (MM.1S and OPM-2) after treatment with si[IGLCCR] or si[-]. Reductions in IgL in cells producing intact IgGλ were associated with significantly decreased cell viability and proliferation, whereas in cells producing only IgL there was no decrease compared with si[-] controls. Mean ± SD of the changes in MTT readouts and P values are shown, the latter obtained with 2-tailed paired Student t test (n = 3 for each cell line).
