Figure 1.
DHR induces human plasma cell apoptosis.
Human plasma cell lines LP1, OPM2, KMS11 and U266 were treated with DHR for 24 h. Induction of apoptosis in human plasma cells by DHR was assessed by Annexin V-FITC and propidium iodide (PI) double staining followed by analysis on a flow cytometer.
Figure 2.
DHR induces human plasma cell death by activating apoptotic pathway.
A, Human plasma cells LP1 and KMS11 were treated with DHR at the indicated concentrations for 24 h. Cell lysates were then prepared and subject to immunoblotting assay against apoptosis-associated proteins caspase-3, -8 and -9. GAPDH were used as a loading control. B, LP1 and KMS11 cells were treated for 24 h with DMSO, DHR, z-VAD-fmk or DHR+Z-VAD-fmk, followed by caspase-3 analysis by Western blotting. C, LP1 and KMS11 cells were treated for 24 h with DMSO, DHR, z-VAD-fmk or DHR+Z-VAD-fmk, followed by Annexin-V-FITC/PI staining and flow cytometric analyses. Pro-casp: pro-caspase; Cle-casp: cleaved caspase.
Figure 3.
DHR dysregulates mitochondrial resident proteins in human plasma cells.
Human plasma cell lines LP1 and KMS11 were treated with DHR at the indicated concentrations for 24 h. Cell lysates were then prepared and subject to immunoblotting assay against Bim (A), Bcl-2 (B) and Mcl-1 (C).β-actin was used as a loading control.
Figure 4.
DHR leads to mitochondrial membrane potential collapse in human plasma cells.
LP1 cells were treated with DMSO, 10 or 20 µM of DHR for 24 h, stained by TMRM alone (A) or in combination with Annexin V-FITC (B) followed by flow cytometric analysis. (C) LP1 cells were treated with 10 µM of DHR for indicated time periods, followed by TMRM and Annexin V-FITC staining and flow cytometric analysis.
Figure 5.
DHR activates the ER stress signaling in human plasma cells.
A, LP1 and OPM2 cells were treated with DHR for 24 h, the expression of GRP78 was then detected by immunoblotting. (B) and (C) LP1 and OPM2 cells were treated DHR for different concentrations and time periods before subject to ATF4 and CHOP analysis. (D) ER-associated caspase-12 was evaluated after exposed to DHR at indicated concentrations for 24 h.
Figure 6.
DHR activates p38 MAP kinase but suppresses JNK kinase in human plasma cells.
LP1 and OPM2 cells were treated with DHR at indicated concentrations followed by immunoblotting analyses for p38 (A) and JNK (B) against specific antibodies. GAPDH was used as an internal loading control.
Figure 7.
DHR activates p38 phosphorylation which is important for DHR-induced human plasma cell apoptosis.
(A) Human plasma cell lines LP1, OPM2, RPMI-8226, and U266 were treated with 10 µM of DHR for 30 or 60 min, followed by p38 phosphorylation analysis. The relative phosphorylated level of p38 to GAPDH was calculated by Quality One software. (B) LP1 cells were treated by DHR at indicated time points to examine p38 activation. (C) LP1 and OPM2 were pre-treated with p38 inhibitor SB203580 (2 µM) for 30 min, followed by DHR (20 µM) for 30 min. (D) LP1 and OPM2 were pretreated with SB203580 (2 µM) for 30 min, followed by DHR (20 µM) for 12 or 24 h. Cell lysates were analyzed by specific antibodies. GAPDH was used as an internal loading control. Pro-casp-3: pro-caspase-3; Cle-casp-3: cleaved caspase-3.