Heat shock did not lower the threshold for induction of Hsps by an Hsp90 inhibitor in motor neurons. Motor neurons were microinjected with a plasmid encoding green fluorescent protein (GFP) as a marker and treated with a concentration of 17-allylamino-17-demethoxygeldanamycin (17-AAG) that only induced Hsp70 in glial cells (0.5 μM), then incubated at either 37°C (non heat shocked) or 43°C (heat shocked) for 30 min, followed by 6 hours of recovery at 37°C/5% CO₂. (A and C) In the absence of heat shock, 0.5 μM 17-AAG induced Hsp70 in non-neuronal cells only. (B and D) Heat shock did not boost 17-AAG–induced Hsp70. Stronger glial immunolabeling was observed in heat shocked cultures as compared to non-heat shocked treatments, but this level was not significantly higher than can sometimes be observed by heat shock alone. Arrows indicate the position of motor neurons in corresponding micrographs double-immunolabeled for Hsp70. Scale bar: 50 μm

Aha1 overexpression failed to induce Hsp70 and Hsp40 in both heat shocked and non–heat shocked motor neurons. (A) Motor neurons had modest endogenous expression of Aha1 that was both cytoplasmic and nuclear. A few motor neurons were observed with cytoplasmic expression only (not shown). Heat shock had no effect on Aha1 expression in motor neurons (not shown). (B) Motor neurons were comicroinjected with plasmids encoding green fluorescent protein (GFP) and Aha1 and expression of both was assessed by immunocytochemistry 48 hours after injection (i, ii). Aha1 was expressed in both the cytoplasm and nucleus (ii). Neither Hsp70 (iii, iv) nor Hsp40 (v, vi) was induced by overexpression of Aha1 in unstressed motor neurons. Plasmid encoding Aha1 was microinjected alone for double-immunolabeling with Hsp40 because antibodies originated from mouse and rabbit, respectively (v, vi). (C) Motor neurons were comicroinjected with plasmids encoding GFP and Aha1, and expression of both was assessed by immunocytochemistry 48 hours after injection (i, ii). Motor neurons expressing Aha1 (at 48 hours) were incubated for 30 minutes at 43°C (heat shocked) followed by recovery incubation for six hours at 37°C/5% CO₂. Hsp70 (iii, iv) and Hsp40 (v, vi) were not induced by overexpression of Aha1 in combination with heat shock. Plasmid encoding Aha1 was microinjected alone for double-immunolabeling with Hsp40. Arrows indicate the position of GFP- or Aha1-expressing motor neurons coimmunolabeled for Hsp70 or Hsp40, respectively. All scale bars: 50 μm

Death-domain–associated protein (Daxx) overexpression failed to induce Hsp70 and Hsp40 in both heat shocked and non– heat shocked motor neurons. (A) Motor neurons endogenously express Daxx at low levels throughout the cytoplasm and nucleus, but expression is concentrated in focal nuclear regions that are likely promyelocytic leukemia oncogenic domains ([PODs], indicated by yellow arrows) (i). During 43°C heat shock, Daxx left PODs, but remained predominantly nuclear (ii). After recovery for 6 hours at 37°C/ 5% CO₂, Daxx was distributed homogenously throughout the cytoplasm and nucleus and positive-immunolabeling PODs were no longer detectable (iii). (B) Motor neurons were microinjected with plasmid encoding Daxx and, after 48 hours, were incubated for 30 minutes at either 43°C (heat shocked) or 37°C (non heat shocked), followed by recovery incubation for 6 hours at 37°C/5% CO₂. Overexpressed Daxx was exclusively nuclear in most motor neurons in the absence of heat shock (i), but usually was punctate and predominantly cytoplasmic after recovery from heat shock (iii). Some non–heat shocked motor neurons had cytoplasmic Daxx and a few retained Daxx in their nucleus after recovery from heat shock (not shown). Hsp70 was not induced in Daxx overexpressing motor neurons in either the absence (ii) or presence (iv) of heat shock. (C) Motor neurons were comicroinjected with plasmids encoding green fluorescent protein (GFP) and Daxx, and expression of both was assessed by immunocytochemistry 48 hours after injection (i, ii). Motor neurons expressing Daxx (at 48 hours) were incubated for 30 minutes at either 37°C (NHS) or 43°C (HS), followed by recovery incubation for 6 hours at 37°C/5% CO₂. Hsp40 was not induced by overexpression of Daxx either in the absence (iii, iv) or presence (v, vi) of heat shock. (D) Motor neurons were comicroinjected with plasmids encoding Daxx and wild-type heat shock transcription factor 1 (Hsf1^wt), and expression of both was assessed by immunocytochemistry 48 hours after injection (i, ii). Motor neurons expressing Daxx and Hsf1^wt were incubated for 30 minutes at either 37°C (NHS) or 43°C (HS), followed by recovery incubation for 6 hours at 37°C/5% CO₂. Hsp70 was not induced in motor neurons that were double-immunolabeled for Daxx in the absence (iii, iv) or presence (vii, viii) of heat shock. Results were confirmed in motor neurons immunolabeled for both Hsp70 and Hsf1 (v, vi and ix, x). White arrows indicate the position of double-immunolabeled motor neurons where not readily visible. All scale bars: 50 μm

Wild-type heat shock transcription factor 1 (Hsf1^wt) suppresses HSP induction by Hsp90 inhibitors. (A) Motor neurons were microinjected with hsp70–green fluorescent protein (GFP) and were immediately treated with three different Hsp90 inhibitors. GFP expression was analyzed 24 hours after microinjection. hsp70-GFP plasmid was microinjected and cultures were treated with 0.01% dimethylsulfoxide (DMSO) vehicle as a negative control (i, ii) or comicroinjected with plasmid encoding constitutively active Hsf1 (Hsf1^act) as a positive control (iii, iv). Motor neurons injected with hsp70-GFP plasmid were treated with 0.1 μM geldanamycin (v, vi), 5 μM 17-allylamino-17-demethoxygeldanamycin (17-AAG) (vii, viii), and 5 μM radicicol (ix, x). Coordinate expression of Hsp70 was assessed by immunocytochemistry (ii, iv, vi, viii, x). Induction of both GFP and Hsp70 was observed with each chemical. Arrows indicate the position of a motor neuron treated with DMSO and immunolabeled for Hsp70. Scale bar: 50 μm. (B) Motor neurons were microinjected with plasmid encoding either GFP or Hsf1^wt and treated with 2.5 μM 17-AAG for 16 hours beginning 48 hours after injection. Hsp70 induction was detected in 80.7 ± 8.5% of GFP-expressing motor neurons and 63.7 ± 9.4% of these had moderate to robust Hsp70 immunolabeling (table). Only 25.5 ± 22.1% of motor neurons expressing Hsf1^wt had detectable Hsp70 in the cytoplasm and immunolabeling was very weak in all of these (*P < 0.005; t-test, data passed normality test). Hsp70 immunolabeling of representative motor neurons expressing GFP (i, ii) (moderate induction) or Hsf1^wt (iii, iv) (no induction). Nuclear labeling in (iv) is an artifact of double-immunolabeling with rat and mouse antibodies that is observed when Hsf1 expression is very high and was described previously (Batulan et al. 2006). Scale bar: 50 μm. (C) Motor neurons were microinjected with either hsp70-GFP plasmid alone or in combination with hsf1^wt and treated with 2.5 μM 17-AAG for 16 hours beginning 24 hours after injection. Average pixel intensity was calculated for GFP expression in all injected motor neurons identified with a dextran-tetramethylrhodamine marker and the mean of these values was reduced by approximately one-half in those expressing Hsf1^wt (*P < 0.005; Mann Whitney log rank test). Approximately one-third of motor neurons with hsp70-GFP alone had an average GFP intensity above a level that appeared qualitatively robust (average pixel intensity > 500), whereas only one motor neuron in the group injected with hsf1^wt had strong GFP expression. (D) Hsf1^−/− Mouse embryonic fibroblasts (MEFs) were transfected with hsf1^wt, hsf1^inact, or hsf1^act constructs. Beginning 48 hours after transfection, MEFs were heat shocked at 43°C for 1 hour, followed by recovery incubation for 6 hours at 37°C/5% CO₂. Expression of Hsf1 and Hsp70 was assessed, and only cells expressing Hsf1^wt and Hsf1^act expressed Hsp70 by immunocytochemistry. Arrows indicate dominant-negative, inactivatible Hsf1–(Hsf1^inact) expressing cells co-immunolabeled for Hsp70. Scale bar: 20 μm. (E) Hsf1^+/+ MEFs were mock-transfected or transfected with hsf1^wt and, after 48 hours, were heat shocked at 43°C for 1 hour, followed by recovery incubation for 6 hours at 37°C/5% CO₂. Mock-transfected MEFs positively labeled for endogenous Hsf1 (i) and Hsp70 (ii) following heat shock to confirm the presence of murine Hsf1. Expression of Hsf1^wt was nuclear (iii), as in motor neurons, but this did not prevent induction of Hsp70 when combined with heat shock (iv). Cytoplasmic labeling of Hsp70 outside of the regions where Hsf1^wt was expressed confirmed that this immunolabeling was not an artifact of rat and mouse antibody double-immunolabeling. Scale bar: 20 μm