Figure 1.
Functional titration of IKK inhibitors.
(A,B) HT29 cells were pretreated for 1 h with the indicated concentrations of TPCA-1 and Bay 11-7082 and were subsequently challenged with TNF (50 ng/ml) for 3 and 10 min (A) or with Flag-TWEAK (200 ng/ml) for 8 hours (B). Total cell lysates were finally analyzed by Western blotting with respect to TNF-induced phosphorylation and degradation of IκBα (A) and TWEAK-induced p100 processing (B). (C) HT29 cells (40,000/chamber) were grown on glass slides and were pretreated with Bay 11-7082 (30 µM) or TPCA-1 (20 µM) for 30 min. Cells were then challenged with 100 ng/ml TNF for 1 h. After immunofluorescence staining for p65, the ratio of nuclear to cytoplasmic fluorescence intensity (FI) was determined. Data shown corresponds to 95–111 analyzed cells per experimental condition derived from a total of four independent experiments. (D) HT29 cells (20,000/well, 96 well-plate, triplicate values) were pretreated with the IKK inhibitors Bay 11-7082 (30 µM) or TPCA-1 (20 µM) for 30 min and were then stimulated with 100 ng/ml TNF for 6 h. The IL8 content of supernatants was subsequently determined by ELISA. To minimize the background signal related to constitutive IL8 production, medium was changed prior to inhibitor treatment. (E) The effects of TPCA-1 and Bay 11-7082 on TNF-induced phosphorylation and degradation of IκBα were analyzed in KMS-12-BM myeloma cells as described under “A”; n.s. = non specific. (F) Equivalent analysis on TNF-induced IL8 production as described in “D” using the RPMI8226 MM cell line. For statistical analysis of data shown in C, D and F one-way ANOVA with a Tukey post-test was performed. Asterisks indicate p-values≤0.01.
Figure 2.
Effects of IKK inhibitors on MM cell viability.
(A,B) MM cell lines were challenged with either TPCA-1 (20 µM) or Bay 11-7082 (30 µM) for 4 h (A) and 18 h, respectively (B), and analyzed for viability using the MTT assay. (C) Primary MM samples (n = 11) in co-culture with BMSCs were incubated with either TPCA-1 (20 µM) or Bay 11-7082 (30 µM) for 3 days and cell death was determined by annexin V-FITC/PI staining and flow cytometry (relative values with respect to the DMSO-treated controls shown; horizontal lines indicate the mean). Data were analyzed using a two-tailed, paired t-test. Asterisk indicates a p-value <0.01. (D) MM.1S and KMS-12-BM cells were challenged with 30 µM Bay 11-7082 for the indicated time. Triplicate values were taken and cellular viability was determined using the MTT assay.
Figure 3.
The mechanism of Bay 11-7082-induced MM cell death involves necrosis.
(A) MM.1S and KMS-12-BM cells were treated with 30 µM Bay 11-7082 and analyzed by time-lapse video microscopy. Pictures shown represent typical stages of cells undergoing Bay 11-7082-induced cell death within 3 h. (B) Cells were pretreated for 30 min with BHA (50 µM), necrostatin-1 (90 µM) or remained untreated and were then challenged with 30 µM Bay 11-7082 for 2 h. Cells were finally photographed (B, arrows indicate swollen cells and the plasma membrane). (C) MM cells were either left untreated or pretreated for 1 h with BHA (50 µM), necrostatin-1 (90 µM), z-VAD-fmk (100 µM) or both necrostatin-1 and z-VAD-fmk. Cells were then challenged with 15 µM Bay 11-7082 for 1 h (KMS-12-BM) or 2 h (MM.1S) followed by annexin V-FITC/PI staining and FACS analysis. (D) MM.1S and KMS-12-BM cells were pretreated in triplicates for 30 min with the indicated combinations of BHA (50 µM), necrostatin-1 (90 µM) and z-VAD-fmk (100 µM), exposed for 2 h to 30 µM Bay 11-7082 and then analyzed for viability using the MTT assay. For statistical analysis a one-way ANOVA with a Tukey post-test was performed. Experimental settings that display significant protection against Bay 11-7082 induced cell death (p-values≤0.01) are indicated by asterisks.
Figure 4.
Functional titration of MLN4924 and effects on MM cell viability.
(A) HT29 and KMS-12-BM cells were pretreated for 1 h with the indicated concentrations of MLN4924 and challenged with TNF (50 ng/ml) for 3 and 10 min. Phosphorylation and degradation of IκBα were subsequently analyzed by Western blotting of total cell lysates. (B) HT29 cells (20,000/well, 96 well-plate, triplicate values) and RPMI8226 cells (50,000/well, 96 well-plate, triplicate values) were pretreated for 30 min with different concentrations of MLN4924. Cells were then stimulated with 100 ng/ml TNF for 6 h and the IL8 content of supernatants was determined by ELISA. For statistical analysis a one-way ANOVA with a Tukey post-test was performed. Groups showing significant inhibition (p-values≤0.01) of TNF-induced IL8 production are indicated by asterisks. (C) HT29 cells were pretreated for 1 h with the indicated concentrations of MLN4924 and then stimulated with Flag-TWEAK (200 ng/ml) for 18 h. The levels of p52, p100 and NIK were analyzed by Western blotting of total cell lysates. (D) MM cell lines were treated for 18 h with 10 µM MLN4924 and assayed for viability using the MTT assay. Significant (unpaired, two-tailed t-test, p-values≤0.01) induction of cell death is highlighted by asterisks. (E) Primary MM samples (n = 11) in co-culture with BMSCs were treated with 10 µM MLN4924 for 3 days and cell death was assessed by annexin V-FITC/PI staining and FACS analysis.
Figure 5.
Knockdown of IKK1, IKK2 or both in MM cells.
(A) Western blots showing depletion of IKK1 and IKK2 in L363 cells after single or combined transfection with stealth siRNAs for the respective targets. Scram denotes non-specific stealth siRNA control. Samples prepared at day 2 post-electroporation from cells co-transfected with an expression plasmid for CD4Δ and purified by CD4 microbead selection of strongly transfected cells. (B) Viability (AlamarBlue, left panel) and survival (annexin V, right panel) assays measured at day 4 post-electroporation for the sample preparations shown in “A”. All values calculated relative to the measurement obtained for the non-specific siRNA control at 2.5 µM concentration (i.e. the concentration present in single stealth siRNA transfections). (C) Overview of the viability and survival effects of IKK1 and IKK2 knockdown in MM cell lines JJN3, L363 and MM.1S. Mean values (calculated with respect to the respective non-specific siRNA controls) and standard deviations shown.