Fig 1: Release of HMGB1 from IL-1ß-stimulated astrocytes. ELISA confirmed that HMGB1 was undetectable in normal astrocyte-conditioned medium (nACM). IL-1ß (0.1 ng/ml) stimulation caused astrocytes to release HMGB1 into stimulted astrocyte-conditioned medium (sACM). HMGB1 shRNA attenuated the release of HMGB1 into the ACM (HMGB1 shRNA sACM). A control shRNA (control shRNA sACM) had no effect on HMGB1 release into IL-1ß-stimulated ACM. *P<0.001 vs. nACM; #P<0.001 vs. sACM.
Fig 2: Analysis of signaling pathways involved in HMGB1-mediated NS/PC proliferation by CCK-8 assay (A and D) and western blot analysis (B and C). NS/PCs were cultured for 96 h in NS/PC culture medium containing 7 ng/ml HMGB1. (A) Compared to an IgG control, blockade of RAGE with an anti-RAGE antibody significantly inhibited HMGB1-induced NS/PC proliferation. *P<0.001. (B) Western blot analysis showed that the blockade of RAGE reduced p-JNK levels in NS/PCs. *P<0.001. (C) Exposure of NS/PCs to HMGB1-containing culture medium rapidly increased p-JNK levels in NS/PCs; the JNK inhibitor, SP600125, reduced JNK phosphorylation in the NS/PCs. *P<0.001 vs. vehicle group; #P<0.001 vs. 0 µM group. (D) SP600125 (10 µM) significantly reduced the ability of HMGB1 to enhance NS/PC proliferation. *P<0.001 vs. vehicle group; #P<0.001 vs. 0 µM group.
Fig 3: (A) Western blot analysis and (B) double-immunofluorescence labeling of HMGB1 expression in IL-1ß-stimulated astrocytes. (A) HMGB1 protein expression was upregulated in IL-1ß-stimulated astrocytes. HMGB1 shRNA specifically suppressed HMGB1 protein expression in IL-1ß-stimulated astrocytes, as a control shRNA had no effect on HMGB1 protein expression in IL-1ß-stimulated astrocytes. *P<0.001 vs. unstimulated astrocytes; #P<0.001 vs. stimulated astrocytes. (B) Double-immunofluorescence labeling of astrocytes for GFAP (blue) and HMGB1 (red). HMGB1 expression was upregulated in IL-1ß-stimulated GFAP+ astrocytes. HMGB1 shRNA successfully suppressed HMGB1 expression in IL-1ß-stimulated GFAP+ astrocytes. There was no difference in HMGB1 expression between IL-1ß-stimulated GFAP+ astrocytes and IL-1ß-stimulated control shRNA astrocytes.
Fig 4: The effects of astrocyte-derived HMGB1 on NS/PC proliferation were measured by the CCK-8 assay. Relative absorbance values of viable NS/PCs were measured by CCK-8 assay at (A) 72 h and (B) 96 h. The proliferation of NS/PCs in sACM, control shRNA sACM and HMGB1 culture media was increased following 72 and 96 h of exposure. Compared with the sACM, the proliferation of NS/PCs in HMGB1 shRNA sACM significantly decreased after 72 and 96 h. There were no intra-time point differences in NS/PC proliferation between the vehicle (NS/PC culture medium only) and control (normal ACM with IL-1ß) groups at either the 72- or 96-h timepoint. *P<0.01, **P<0.001 vs. vehicle group; #P<0.05, ##P<0.01 vs. sACM group.
Fig 5: Astrocyte-derived HMGB1 on NS/PC proliferation was measured by analyzing the cell cycle. The cell cycle kinetics of NS/PCs were analyzed by flow cytometry at (A) 72 h and (B) 96 h. The proliferative capacity of NS/PCs was described as the proliferation index (PI): PI = (S + G2/M)/(G0/G1 + S + G2/M). The PIs of the sACM group, control shRNA sACM group and HMGB1 group were increased after 72 and 96 h. Compared with the sACM group, the PI of the HMGB1 shRNA sACM group was significantly decreased after 72 and 96 h. There were no intra-time point differences in PI between vehicle (NS/PC culture medium only) and control (normal ACM with IL-1ß) groups at either the 72 or 96-h time point. *P<0.01, **P<0.001 vs. vehicle group; #P<0.05, ##P<0.001 vs. sACM group.
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