Figure 2.
TG02 inhibits PI-induced HSF1 S326 phosphorylation and PI-induced HSR in myeloma cells. (A) MM.1s cells were cotreated with TG02 and either bortezomib (left) or carfilzomib (right). Protein lysates were collected at 9 hours for western blot analysis, and cells were analyzed at 24 hours for apoptosis. Apoptosis was measured by annexin V and PI staining and flow cytometry. Western blot data are representative of 4 independent experiments. (B) TG02 and PI combination leads to an additive effect on apoptosis in MM.1s cells. Experimental setup was as described in panel A. Apoptosis was measured as described previously. P values are calculated by paired Student t test. *P < .05; **P < .01. (C) TG02 inhibits bortezomib-induced HSF1 S326 phosphorylation and bortezomib-induced HSR in H929 cells and HSF1 S326 phosphorylation in U266 cells. H929 or U266 cells were cotreated with TG02 and bortezomib. Western blot data are representative of 4 independent experiments. (D) TG02 and PI combination leads to an additive effect on apoptosis in H929 cells but not U266 cells. Experimental setup was as described in panel C. Apoptosis was measured as described previously. P -values are calculated by paired Student t test. *P < .05; **P < .01. (E) TG02 inhibits carfilzomib-induced HSF1 S326 phosphorylation and bortezomib-induced HSR in patient samples. CD138+ (>90%: PS10001496; >75%: PS10001225-2) cells from freshly isolated patient samples were cotreated with TG02 and carfilzomib. Experimental design as previously. (F) CDK9 is not responsible for bortezomib-induced S326 phosphorylation, and its silencing does not sensitize cells to bortezomib-induced apoptosis. MM.1s cells were treated with a nonsilencing control (-), CDK9 (C), or HSF1 (H) siRNA for 24 hours followed by bortezomib treatment of either an additional 9 hours for western blot analysis (top) or 24 hours for flow cytometry analysis (bottom). Data are representative of 3 independent experiments. Apoptosis was measured as described previously. P values are calculated by paired Student t test. *P < .05; **P < .01.

TG02 inhibits PI-induced HSF1 S326 phosphorylation and PI-induced HSR in myeloma cells. (A) MM.1s cells were cotreated with TG02 and either bortezomib (left) or carfilzomib (right). Protein lysates were collected at 9 hours for western blot analysis, and cells were analyzed at 24 hours for apoptosis. Apoptosis was measured by annexin V and PI staining and flow cytometry. Western blot data are representative of 4 independent experiments. (B) TG02 and PI combination leads to an additive effect on apoptosis in MM.1s cells. Experimental setup was as described in panel A. Apoptosis was measured as described previously. P values are calculated by paired Student t test. *P < .05; **P < .01. (C) TG02 inhibits bortezomib-induced HSF1 S326 phosphorylation and bortezomib-induced HSR in H929 cells and HSF1 S326 phosphorylation in U266 cells. H929 or U266 cells were cotreated with TG02 and bortezomib. Western blot data are representative of 4 independent experiments. (D) TG02 and PI combination leads to an additive effect on apoptosis in H929 cells but not U266 cells. Experimental setup was as described in panel C. Apoptosis was measured as described previously. P -values are calculated by paired Student t test. *P < .05; **P < .01. (E) TG02 inhibits carfilzomib-induced HSF1 S326 phosphorylation and bortezomib-induced HSR in patient samples. CD138+ (>90%: PS10001496; >75%: PS10001225-2) cells from freshly isolated patient samples were cotreated with TG02 and carfilzomib. Experimental design as previously. (F) CDK9 is not responsible for bortezomib-induced S326 phosphorylation, and its silencing does not sensitize cells to bortezomib-induced apoptosis. MM.1s cells were treated with a nonsilencing control (-), CDK9 (C), or HSF1 (H) siRNA for 24 hours followed by bortezomib treatment of either an additional 9 hours for western blot analysis (top) or 24 hours for flow cytometry analysis (bottom). Data are representative of 3 independent experiments. Apoptosis was measured as described previously. P values are calculated by paired Student t test. *P < .05; **P < .01.

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