Fig 1: HMGB1 secretion is regulated by autophagy-mediated secretion in macrophages. (A) RAW264.7 cells were pretreated with 100 nM Wo, 20 nM Baf, 20 μM CQ for 2 h, and treated with 250 ng/mL LPS for 24 h. WCLs and supernatants were immunoblotted with anti-HMGB1, anti-LC3, and anti-ACTB antibodies. (B) RAW264.7 cells were pretreated with 2 μM GW (GW4869) for 2 h and treated with 250 ng/mL LPS and 50 μM H2O2 for 24 h. WCLs and supernatants were immunoblotted with anti-HMGB1, anti-IL1B, and anti-ACTB antibodies. (C) RAW264.7 cells were pretreated with 100 nM Wo, 20 nM Baf, 20 μM CQ for 2 h, and treated with 250 ng/mL LPS for 24 h. TNF levels were measured in the culture supernatants by ELISA. All results were compared to those of LPS treatment alone. (D) RAW264.7 cells were pretreated with 2 μM GW4869 for 2 h and then treated with 250 ng/mL LPS and 50 μM H2O2 for 24 h. TNF level in the supernatants was measured by ELISA. (E) THP-1 cells were pretreated with 2 μM GW4869 for 2 h and treated with 20 μM CQ and 300 ng/mL LPS for 24 h. WCLs and supernatants were immunoblotted with anti-HMGB1, anti-IL1B, and anti-ACTB antibodies. (F) BMDMs were pretreated with 2 μM GW4869 for 2 h, and treated with 250 ng/mL LPS for 24 h. WCLs and supernatants were immunoblotted with anti-HMGB1, anti-IL1B, anti-CASP1, and anti-ACTB antibodies. (G) WT BMDM and gorasp2−/- BMDM cells were pretreated with 0.5 μM GA for 2 h and treated with 250 ng/mL LPS for 24 h. WCLs and culture supernatants were immunoblotted with anti-GORASP2, anti-HMGB1, and anti-ACTB antibodies. Culture supernatants were concentrated with Centricon filters. Data are presented as the mean ± SEM from at least three independent experiments. n.s: not significant, *p < 0.05, **p< 0.01, ***p < 0.001, one-way ANOVA followed by Tukey honestly significant difference posthoc test for multiple comparisons
Fig 2: MVB formation is crucial in HMGB1 secretion. (A) HEK293T cells were transiently transfected with shRNA control (SHC001), shHGS (TRCN0000379833), and shTSG101 (TRCN0000315042) for 24 h. The cells were treated with 20 μM CQ, 350 nM PMA, and 25 ng/mL TSA for 24 h and with EBSS for 14 h. Culture supernatants and WCLs were immunoblotted with anti-HMGB1, anti-HGS, anti-TSG101, and anti-ACTB antibodies. All results were compared to that for the shRNA control for each treatment. (B, C) HEK293T cells pretreated with 2 μM GW4869 for 2 h, and treated with 350 nM PMA and 25 ng/mL TSA for 24 h, and EBSS for 14 h (B); 100 nM Wo, 20 nM Baf, and 20 μM CQ for 24 h (C). WCLs and supernatants were immunoblotted with anti-HMGB1 and anti-ACTB antibodies. (D, E) HEK293T cells were transfected with HSP90AA1-Flag and GORASP2-MYC (D); HA-ARF1Q71L (E) for 24 h. The cells were treated with 2 μM GW4869 for 24 h. Culture supernatants and WCLs were immunoblotted with anti-HMGB1, anti-HA, and anti-ACTB antibodies. (F) HEK293T cells were transiently transfected with shRNA control, shRAB7A (TRCN0000229643), shRAB8A (TRCN0000048216), shRAB11A (TRCN0000073021), and shRAB27A (TRCN0000005295) for 24 h. The cells were treated with 20 μM CQ for 24 h. Culture supernatants were immunoblotted with anti-HMGB1 antibodies. All results were compared to those of the shRNA control treated with CQ. Data are presented as the mean ± SEM from at least three independent experiments. n.s.: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey honestly significant difference posthoc test for multiple comparisons
Fig 3: GORASP2 increases HMGB1 secretion. (A, B) HEK293T cells were transfected with GORASP2-MYC, shRNA control (SHC001) or shGORASP2 (#1; TRCN0000129348 and #2; TRCN0000127611). The cells were incubated in EBSS media for 14 h. WCLs were immunoblotted with anti-MYC, anti-HMGB1, anti-GORASP2, and anti-ACTB antibodies. The culture supernatants were concentrated with Amicon Centricon filters. (C) GORASP2-MYC and HSP90AA1-Flag were transfected into HEK293T cells. WCLs were immunoblotted with anti-MYC, anti-Flag, anti-HMGB1, and anti-ACTB antibodies. Culture supernatants were concentrated with Centricon filters. Results were compared to those for co-expressed HSP90AA1 and GORASP2/GRASP55. (D) GORASP2-MYC was transfected and treated with 0.5 μM GA for 24 h. WCLs were immunoblotted with anti-MYC, anti-HMGB1, and anti-ACTB antibodies. Culture supernatants were concentrated with Centricon filters. Data are presented as the mean ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey honestly significant difference posthoc test for multiple comparisons
Supplier Page from Abcam for Anti-GORASP2/GRASP55 antibody