Fig 2: Nuclear Translocation of UBQLN2 Is Unaffected by Disease Mutation, and HSP70 Clients Induce HSP70-UBQLN2 Interaction, Related to Figures 4 and 5(A) Wild-type of P520T knock-in MEFs were heat shocked and fractionated as indicated and no difference was observed in the nuclear localization as a result of the disease mutation.(B) HEK293 cells were treated with the broad spectrum kinase inhibitor Staurosporine (1 µM) or the p38 (BIRB-0796 and VX-745; 1 µM) and JNK (JNKIN8; 10 µM) kinase inhibitors for 1h prior to heat shock and showed that kinase signaling is not regulating the inducible interaction of HSP70 and UBQLN2.(C) HEK293 cells were treated with the ubiquitin E1 inhibitor MLN7243 (10 µM) for 1h prior to heat shock and demonstrated that ubiquitylation or ubiquitin signaling is not involved in regulating the inducible interaction between HSP70 and UBQLN2.(D) UBQLN2 does not bind non-specifically to GST in the presence or absence of denatured luciferase. GST or GST-HSP70 and purified UBQLN2 was incubated at 42°C in the presence or absence of Luciferase, as indicated. This was followed by GST pulldown, and Western blot for associated UBQLN2.(E) Luciferase was denatured at 95°C for 5 min and found to stimulate the binding of untagged recombinant UBQLN2 to GST-HSP70 upon GST-pulldown, unlike native luciferase.(F) R6/2 brain extracts but not WT brain extracts were found to be able to stimulate the interaction of recombinant untagged UBQLN2 with GST-HSP70 in GST pulldown experiments.(G) HEK293 cells stably expressing inducible UBQLN2 WT or PXXP deletion mutants were found to both equally interact with endogenous HSP70 after heat shock, indicating that the PXXP motif does not directly mediate the interaction.

Fig 3: Heat Shock Generates Insoluble Ubiquitin-Positive Aggregates and Does Not Inactive Proteasomes, Related to Figure 1(A) UBQLN2 is not pelleted when cells are heat shocked post lysis. Cell lysates were incubated at 37 or 42°C and then fractionated into soluble (S) and pellet (P) fraction. This indicates that UBQLN2 itself does not aggregate as a result of high temperature.(B) UBQLN2 levels are not upregulated in response to heat shock. HSP70 and GAPDH were used as a positive and negative controls, respectively.(C) Heat shock aggregates are insoluble in up to and including 1% SDS but are solubilized in 2% SDS. Blotting of soluble and pellet fractions with anti-ubiquitin and UQBLN2 antibodies confirmed dissolution of the aggregates in 2% SDS.(D) Proteasomes are active after heat shock. To confirm that proteasome activity was not affected by heat shock, we incubated U2OS and MEFs at the indicted temperatures for 2h. Cells were then harvested and cell lysates were incubated with the proteasome inhibitor MG132 or DMSO, followed by incubation with a fluorescent proteasome-activity probe, as indicated. The presence of fluorescently labeled beta-subunits at the same intensity under both heat stress and normal temperature, indicate that proteasome activity is not significantly affected by heat shock.(E) U2OS cells were treated with control or UBQLN2 siRNA and subjected to heat shock for the indicated times. Analysis of the pellet fraction revealed that insoluble ubiquitylated aggregates are generated within 5 min of heat shock, but that depletion of UBQLN2 does not noticeably alter the accumulation of these aggregates at any of the indicated time points.

Fig 4: Identification of hBMSC-derived exosomes. (a) Identification of morphological characteristics of hBMSC-derived exosomes by transmission electron microscopy. The exosome concentrations were 1.5 to 1.6 × 109/ml. (b) Western blot demonstrated characteristic markers of hBMSC-derived exosomes: HSP70 and CD9. (c) Size distribution of exosomes with a ZetaView analysis system.

Fig 5: IRE induces ICD of tumor cells. A, The expression and the quantification of integrated optical density of the protein bands of intracellular DAMPs of Panc-1 and Pan02 treated by electric fields with different field strengths. The expression of HMGB1, HSP70, and calreticulin increased significantly along with the increasing strength of electroporation. B, The analysis of released HMGB1 in TSN of Panc-1 treated with electric fields with different field strengths by enzyme-linked immunosorbent assay (ELISA). C, The analysis of released HMGB1 in TSN of Pan02 treated with electric fields with different field strengths by ELISA. The levels of secreted HMGB1 in TSN of Panc-1 and Pcan02 increased significantly along with the increasing strength of electroporation. One-way analysis of variance (ANOVA) with Bonferroni comparison test was performed. *P < .05, **P <.01, ***P < .001