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. 2021 Sep 27;10(10):2550. doi: 10.3390/cells10102550

Figure 7.

Figure 7

Analysis of cell signals involved in the bortezomib-induced HMGB1 release from RAW264.7 macrophages. (A-C) Lack of effects of pyrrolidine dithiocarbamate (A), SB203580 (B), STO-609, pyridone 6, and SP600125 (C), inhibitors of NF-κB, p38MAPK, CaMKK, JAK, and JNK, respectively, on the HMGB1 release from RAW264.7 cells stimulated with bortezomib. Pyrrolidine dithiocarbamate at 100 μM (A), SB203580 at 1 μM (B), STO-609 at 10 μg/mL, pyridone 6 at 30 nM, or SP600125 at 1 μM (C) was added 1 h before stimulation with bortezomib at 10 nM for 12 h, and the extracellular HMGB1 was detected by Western blotting. (D,E) Effects of Z-VAD-FMK, a pan-caspase inhibitor, and Z-IETD-FMK, a caspase 8-specific inhibitor, on the HMGB1 release from RAW264.7 cells stimulated with bortezomib. Z-VAD-FMK at 20 μM (D) or Z-IETD-FMK at 20 μM (E) was added 1 h before stimulation with bortezomib at 10 nM for 12 h (D,E) or paclitaxel at 1 μM for 18 h (D), and the extracellular HMGB1 was detected and quantified by Western blotting. Typical photographs are shown on the top above the columns of quantified data. Parentheses indicate molecular targets of each inhibitor (A–E). (F,G) Increase in protein levels of cleaved products of caspase-8, p43 (containing p18) and p18 following stimulation with bortezomib at 10 nM for 3–9 h (F) or for 9 h (G). The cleaved products were detected (F) and quantified (G) by Western blot analysis. (H,I) Detection of apoptotic cells after stimulation of bortezomib at 10 nM for 12 h. Nuclear fragmentation or condensation accompanying apoptosis (arrows) was detected by Hoechst 33342 staining (H), and the proportion (%) of apoptotic cells in a visual field was calculated (I). Scale bars indicate 50 μm. Data show the mean with S.E.M for 7 (D left), 5 (D right, E), or 8 (G) different experiments and for 20 visual fields from 5 different experiments (I). V, vehicle; BTZ, bortezomib; PDTC, pyrrolidine dithiocarbamate; SB, SB203580; P6, pyridone 6; SP, SP600125; PCT, paclitaxel. ** p < 0.01 vs. V (G, I) or V + V (D,E). † p < 0.05, †† p < 0.01 vs. V + BTZ (D left, E) or V + PCT (D right).