Figure 6.
Figure 6. Amyloidogenic LC expression is an intrinsic cellular stressor. Effect of expression of amyloidogenic (AL) and nonamyloidogenic (MM) LC on PC growth, PI sensitivity, and autophagy. (A) The non-immunoglobulin-expressing NS0 murine plasmocytoma cell line was lentivirally engineered to express amyloidogenic and nonamyloidogenic immunoglobulin LCs under control of an inducible promoter, and clones selected and characterized for LC expression. Effective induction of LC expression and secretion by doxycycline (1 µM for 3 days) in 2 representative clones is shown in 1 representative immunoblot analysis. (B) AL and MM NS0 clones were treated with 1 µM doxycycline or left untreated for the indicated days. The number of viable cells upon induction of LC production by doxycycline and in untreated cultures was determined every 24 hours by trypan blue exclusion. The effect of LC induction on cell growth as compared with parallel, untreated cultures was expressed as percentage of cell number change. The histogram shows the average of 2 AL (A2-1 and A2-11) and 2 MM (M1-11 and M1-19) NS0 clones. (C) AL (A2-1) and MM (M1-19) NS0 clones were induced to express the respective LC for 3 days and the effect on cell growth of the indicated doses of Btz administered for the last day evaluated as in panel B. (D) Analysis of cell viability 3 days post-LC induction in the experiment in panel C. (E) A2-1 cells were induced to express the amyloidogenic LC for 3 days and the effect on cell viability of treatment with the distal autophagic inhibitor, leupeptin (Leu, 20 µM for the last 48 hours) and Btz (10 nM for the last 24 hours), alone or in combination, assayed by trypan blue exclusion (average of 4 independent experiments). (F) AL (A2-1) and MM (M1-19) NS0 clones were induced to express the respective LC for 3 days and the effect on cell growth of the indicated dose of leupeptin for the last 48 hours evaluated as in panel B. (G) The effect of 3 days induction of AL and MM LC on the accumulation of autophagic substrate SQSTM1/p62 assessed by immunoblotting. Top panel: one representative image; histogram: quantification of 3 independent experiments. Results are expressed as mean ± standard error of the mean (SEM) of at least 3 independent experiments. *P < .05; ***P < .001 (2-tailed Student t test).

Amyloidogenic LC expression is an intrinsic cellular stressor. Effect of expression of amyloidogenic (AL) and nonamyloidogenic (MM) LC on PC growth, PI sensitivity, and autophagy. (A) The non-immunoglobulin-expressing NS0 murine plasmocytoma cell line was lentivirally engineered to express amyloidogenic and nonamyloidogenic immunoglobulin LCs under control of an inducible promoter, and clones selected and characterized for LC expression. Effective induction of LC expression and secretion by doxycycline (1 µM for 3 days) in 2 representative clones is shown in 1 representative immunoblot analysis. (B) AL and MM NS0 clones were treated with 1 µM doxycycline or left untreated for the indicated days. The number of viable cells upon induction of LC production by doxycycline and in untreated cultures was determined every 24 hours by trypan blue exclusion. The effect of LC induction on cell growth as compared with parallel, untreated cultures was expressed as percentage of cell number change. The histogram shows the average of 2 AL (A2-1 and A2-11) and 2 MM (M1-11 and M1-19) NS0 clones. (C) AL (A2-1) and MM (M1-19) NS0 clones were induced to express the respective LC for 3 days and the effect on cell growth of the indicated doses of Btz administered for the last day evaluated as in panel B. (D) Analysis of cell viability 3 days post-LC induction in the experiment in panel C. (E) A2-1 cells were induced to express the amyloidogenic LC for 3 days and the effect on cell viability of treatment with the distal autophagic inhibitor, leupeptin (Leu, 20 µM for the last 48 hours) and Btz (10 nM for the last 24 hours), alone or in combination, assayed by trypan blue exclusion (average of 4 independent experiments). (F) AL (A2-1) and MM (M1-19) NS0 clones were induced to express the respective LC for 3 days and the effect on cell growth of the indicated dose of leupeptin for the last 48 hours evaluated as in panel B. (G) The effect of 3 days induction of AL and MM LC on the accumulation of autophagic substrate SQSTM1/p62 assessed by immunoblotting. Top panel: one representative image; histogram: quantification of 3 independent experiments. Results are expressed as mean ± standard error of the mean (SEM) of at least 3 independent experiments. *P < .05; ***P < .001 (2-tailed Student t test).

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