1. Regulation of Organismal Proteostasis by Transcellular Chaperone Signaling 
Patricija van Oosten-Hawle, Robert S. Porter, Richard I. Morimoto 
Cell 
Volume 153, Issue 6, Pages (June 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Tissue-Specific Perturbation of Proteostasis Is Recognized across Multiple Tissues in a Cell-Nonautonomous Manner
(A and B) Confocal image of a young adult animal expressing the 2.5 kb hsp90 promoter region upstream of GFP (hsp90p::GFP) at 20°C. Pronounced expression was observed in multiple tissues, including pharyngeal muscle (ph), intestine (int), pharyngeal nerve ring (n), body wall muscle (bwm), and the excretory cell (ex). Scale bar, 100 μm. (B) 63× magnification of the head region. Expression is detected in the body wall muscle (bwm), the excretory cell (ex), the pharyngeal muscle (ph), pharyngeal nerve ring (n), and the intestine (int). Scale bar, 10 μm.
(C) Total mRNA levels of hsp90 in unc-54(e1301) animals relative to wild-type at 15°C. Bar graphs represent combined mean values of three independent experiments (means ± SEM). Bar graphs represent combined mean values of three independent experiments. Error bars represent ±SEM. **p < 0.01.
(D–K) hsp90p::GFP reporter expression in unc-54(e1301) animals compared to wild-type. Scale bars, 100 μm (D–K). (F and G) 100× image of wild-type expressing the hsp90 reporter in the excretory canal (ex) and body wall muscle (bwm). (J and K) hsp90 expression in intestinal cells, body wall muscle, and excretory canal in unc-54(e1301) animals. (J) 40× image. (K) 100× image. All fluorescent images were taken at equal exposure times.
See also Figure S1.
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4. Figure 2
Tissue-Specific Increased Levels of HSP90 Improves the Organismal Folding Environment of myosin ts Mutants
(A–C) Confocal images of young adult C. elegans animals overexpressing HSP90::GFP in the (A) body wall muscle (HSP90bwm), (B) the intestine (HSP90int), or (C) the neurons (HSP90neuro) (Scale bar, 100 μm), with 63× magnifications of selected regions (Scale bar, 10 μm).
(D) Western blot analysis of young adult animals overexpressing HSP90 using an anti-C. elegans HSP90 antibody. Levels of HSP90::GFP are normalized to the loading control (tubulin) and relative to endogenous HSP90.
(E) Percentage of young adult animals expressing ts myosin [unc-54 (e1301)] alone or in the presence of body wall muscle-specific HSP90::GFP overexpression [unc-54(ts),HSP90bwm], intestinal overexpression [unc-54(ts),HSP90int], or neuronal overexpression [unc-54(ts),HSP90neuro] showing movement after exposure to restrictive temperature (25°C) for 12–24 hr. n = 20 adult animals per strain per experiment. Bar graphs represent the combined results of three independent experiments. Error bars represent ±SEM. *p < 0.05; **p < 0.01.
(F) Confocal images of the body wall muscle of age-synchronized wild-type, unc-54(e1301), unc-54(e1301),HSP90bwm, unc-54(e1301), HSP90int, and unc-54(ts), HSP90neuro animals after exposure to restrictive temperature (25°C) for 12 hr, using myo-3::GFP for visualization. Scale bar, 20 μm.
See also Figure S2.
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5. Figure 3
Elevated Tissue-Specific HSP90 Levels Repress the HSR at an Organismal Level
(A) Thermosensitivity of young adult animals (n = 100) with indicated genotypes exposed to 35°C heat stress. *p < 0.05; **p < Bar graphs represent combined mean values of three independent experiments. Error bars represent ±SEM.
(B) Total mRNA levels of hsp70 (C12C8.1 and F44E5.4) and hsp16 (hsp-16.2) after heat shock (1 hr at 33°C) in young adult myo-2p::mCherry, hsf-1 (sy441) mutant, HSP90neuro, HSP90int, and HSP90bwm animals and upon RNAi-mediated GFP or hsp90 knockdown prior to heat shock in the transgenic HSP90 lines, relative to wild-type. Bar graphs represent combined mean values of three independent experiments. Error bars represent ±SEM.
(C) Differential interference contrast (DIC) Nomarski images of (i) wild-type, (v) HSP90bwm, (ix) HSP90int, and (xiii) HSP90neuro animals expressing the hsp70p::mCherry reporter. Expression of the hsp70p::mCherry reporter 7 hr after heat shock (33°C, 1 hr) in representative (ii) wild-type, (vi) HSP90bwm, (x) HSP90int, and (xiv) HSP90neuro animals. The yellow arrow in (vi), (x), and (xiv) indicates the pharyngeal myo-2::mCherry coinjection marker, present in all transgenic HSP90::GFP lines. (iii, vii, xi, and xv) 20× magnifications of the posterior region of (iii) wild-type and HSP90 overexpression lines (vii, xi, and xv), indicating hsp70 induction in spermatheca (sp), body wall muscle (m), and the intestine (i). (iv, viii, xii, and xvi) HSP90::GFP expression in true color. The yellow arrows in (xvi) indicate neuronal cells expressing HSP90::GFP. (i–xvi) Scale bar, 100 μm.
See also Figure S3.
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6. Figure 4
Tissue-Specific Knockdown of hsp90 Cell-Nonautonomously Induces the HSR
(A) Body wall muscle-, intestine-, and neuron-specific hsp90 RNAi induces basal levels of hsp70 (C12C8.1 and F44E5.4) expression at 20°C compared to control animals (sid-1). Wild-type animals allow import of dsRNA from surrounding tissues, leading to higher induction of organismal hsp70 than in the tissue-specific knockdown lines. Bar graphs represent combined mean values of three independent experiments. Error bars represent ±SEM. **p < 0.05.
(B) Thermosensitivity of young adult animals (n = 100) expressing the indicated tissue-specific hp-hsp90 construct exposed to 35°C heat stress. Bar graphs represent combined mean values of three independent experiments. Error bars represent ±SEM. **p < 0.01; *p < 0.05.
(C) Tissue-specific knockdown of hsp90 induces expression of the hsp70 reporter (hsp70p::mCherry) at 20°C. DIC images of synchronized young adult (i) control animals (sid-1), (v) hp-hsp90bwm, (ix) hp-hsp90int, and (xiii) hp-hsp90neuro animals expressing the hsp70 reporter. Expression of the hsp70p::mCherry in (ii) sid-1 control animals, (vi) hp-hsp90bwm, (x) hp-hsp90int, and (xiv) hp-hsp90neuro. (iii, vii, xi, and xv) 20× magnification of control (iii) and tissue-specific hsp90 knockdown lines (vii, xi, and xv) indicating expression of hsp70p::mCherry in the pharynx (ph), intestine (int), and body wall muscle (m). (iv, viii, xii, and xvi) Overlay of DIC Nomarski and hsp70p::mCherry (red). Scale bars, 100 μm.
See also Figure S4.
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7. Figure 5
Coordination of Cell-Nonautonomous hsp90 Expression Is Regulated Independent of Neural Control
(A) Tissue-selective increased levels of HSP90 upregulates expression of the transcriptional hsp90p::GFP reporter at normal conditions (20°C). DIC images of (i) wild-type, (v) HSP90bwm, (ix) HSP90int, and (xiii) HSP90neuro expressing the hsp90p::GFP reporter. hsp90 reporter expression in representative whole animals (ii, vi, x, and xiv) and magnified head region (iii, vii, xi, and xv) in (ii and iii) wild-type, (vi and vii) HSP90bwm, (x and xi) HSP90int, and (xiv and xv) HSP90neuro, indicating hsp90p::GFP in the pharynx (ph), intestine (int), body wall muscle (m), and excretory cell (ex). (iv, viii, xii, and xvi) HSP90::mCherry (red) expression in true color. Yellow arrows in (iii), (vii), (xi), and (xv) indicate hsp90p::GFP expression in the excretory cell. Scale bars, 100 μm.
(B) Schematic representation of the major modes of neurosecretion in C. elegans, regulated via either dense core vesicles (DCV) through unc-31 or via small core vesicles (SCV) through unc-13.
(C) Total hsp90 mRNA levels of HSP90 overexpression lines in an unc-31 deletion mutant background (white bars) or crossed to an unc-13 deletion mutant (light gray bars) relative to control animals (dark gray bars). Bar graphs represent combined mean values of three independent experiments. Error bars represent ±SEM.
See also Figure S5.
Cell  , DOI: ( /j.cell )
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8. Figure 6
A PHA-4-Dependent Transcriptional Feedback Coordinates Cell-Nonautonomous hsp90 Expression
(A) Total mRNA levels of hsp-1 (constitutive hsp70), hs-inducible hsp70 (C12C8.1 and F44E5.4), small heat shock protein hsp16 (hsp-16.2), and hsp90 in HSP90 overexpression lines at 20°C relative to wild-type. The slightly increased hsp levels in the HSP90bwm may be indicative of the higher sensitivity of muscle cells to proteostatic perturbation, in line with the observations on tissue-specific hsp90 knockdown, where the HSR is primarily induced in the body wall muscle.
(B) Organismal hsp90 expression in HSP90 overexpression lines is independent of hsf-1. hsp90 mRNA levels in control (EV) and animals fed with hsf-1 RNAi. Whereas hsp90 expression in wild-type is hsf-1 dependent, RNAi-mediated knockdown of hsf-1 leaves organismal hsp90 levels in HSP90bwm, HSP90int, or HSP90neuro unchanged.
(C) pha-4 RNAi decreases elevated hsp90 expression in the overexpression lines.
(D) hsp90p::GFP reporter expression in myosin (ts, e1157) or paramyosin (ts, e1402) mutants is reduced during pha-4 RNAi when compared to control RNAi (EV). Scale bar, 50 μm.
(E) pha-4 activity is increased in the HSP90 overexpression lines. mRNA levels of pha-4-regulated genes sod-1, sod-2, sod-4, and sod-5 are induced in eat-2(ad1113) mutants, HSP90bwm, HSP90int, and HSP90neuro animals relative to wild-type. *p < 0.05; **p < 0.02.
(F) pha-4 mRNA expression levels in eat-2 and HSP90 overexpression lines are upreguated relative to the wild-type control. *p < 0.05.
(G) pha-4 activity in intestinal cells of eat-2(ad1113), HSP90bwm, HSP90int, and HSP90neuro animals relative to wild-type. **p < 0.02.
(H) pha-4 mRNA expression levels are induced in the intestines of eat-2 mutants and HSP90int (signaling tissue) but not in the intestines of HSP90bwm or HSP90neuro (recipient tissue). *p < n.s., not significant.
All bar graphs represent combined mean values of three independent experiments (three biological replicates). Error bars represent ±SEM. See also Figure S6.
Cell  , DOI: ( /j.cell )
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9. Figure 7
Model for the Cell-Nonautonomous Regulation of HSP90 Expression in C. elegans
An imbalance of the proteostasis network through the presence of metastable myosin increases the expression of HSP90 in muscle cells but also in different cell-types, such as the intestine. This is regulated by PHA-4 and communicated through transcellular chaperone signaling to other tissues, independent of neural activity. Increased pha-4 activity is required in the signaling and receiving tissue, but pha-4 may act from a distance to regulate gene expression in the receiving tissue via a downstream signaling cascade. The resulting highly abundant HSP90 levels in the entire animal are beneficial for myosin folding in unc-54(ts) mutants but can become detrimental during severe heat shock due to cell-nonautonomous repression of HSF-1 transcriptional activity. Likewise, a tissue-specific perturbation through reduced hsp90 levels (bottom) leads to induction of the HSR in the same and recipient tissues. Transduction of the response to the recipient tissue is also pha-4 dependent.
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10. Figure S1
Analysis of hsp90 Expression in Myosin ts and Paramyosin ts Mutants, Related to Figure 1
(A) Western Blot analysis of total protein extracts of young animals fed with hsp90 dsRNAi or a control (L4440) using anti-HSP90 antibody. Loading was assessed by probing with anti-tubulin antibody.
(B) Analysis of myosin (UNC-54) coimmunoprecipitation with CeHSP90::GFP expressed in the bodywall muscle. Animals expressing GFP in their bodywall muscle only (myo-3p::GFP; RW1596), wild-type animals expressing no GFP and animals expressing HSP90::GFP in their bodywall muscle were grown until young adulthood and an aliquot of worm cell extract (WCE) was loaded onto anti-GFP magnetic beads. Bound protein was eluted and subjected to western blot analysis using an anti-GFP antibody (Molecular Probes), an anti-CeHSP90 antibody, which recognizes both the endogenous and GFP tagged HSP90 bait protein with the higher molecular weight, and an anti-UNC-54 antibody. Tubulin was used as a loading control.
(C–F) hsp90p::GFP reporter expression in unc-54(e1157,ts) animals compared to wild-type at permissive temperature (15°C). (C and D) 20x images of hsp90p::GFP animals, (C) alone or (D) in the background of the unc-54(e1157) mutation. Scale bar, 100 μm. Higher expression of the hsp90p::GFP reporter can be observed in (E) bodywall muscle cells (bwm) as well as in (F) intestinal cells (int), the pharynx (ph) and the excretory cell (ex). Scale bar, 50 μm.
(G–L) hsp90p::GFP reporter expression in a paramyosin temperature sensitive mutant unc-15(e1402) at 15°C. hsp90 expression in (G and J) wild-type background or in (H–L) the presence of a unc-15 ts allele (e1402). Compared to wild-type animals, unc-15(ts,e1402) exhibit increased hsp90 reporter activity in (K) the bodywall muscle and (L) the pharynx (ph) as well as nonmuscle tissue such as intestine and excretory cell (ex). (G–I) Scale bar, 100 μm. (J–L) Scale bar, 50 μm.
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11. Figure S2
Characterization of Tissue-Specific HSP90 Overexpression, Related to Figure 2
(A) CeHSP90::GFP is functional in yeast. 5-FOA growth of a yeast strain expressing either yeast Hsp90 (Hsp82), C. elegans HSP90 or C. elegans HSP90::GFP as the sole source of HSP90.
(B) Growth analysis of yeast expressing CeHSP90 or CeHSP90::GFP at 30°C compared to a wild-type strain. See Extended Experimental Procedures.
(C) Age-synchronized representative animals expressing HSP90::GFP in either neurons, intestine or bodywall muscle and wild-type were grown at 20°C and imaged at day 3 after the first larval stage L1 ( = day 1 adults).
(D) Analysis of HSP90 overexpression lines throughout development compared to wild-type animals. See Experimental Procedures.
(E) Percentage of surviving progeny at 25°C, expressing unc-54(e1301,ts) alone or in the presence of bodywall muscle specific HSP90::GFP overexpression, intestine-specific HSP90::mCherry overexpression, or neuron-specific HSP90::GFP overexpression. Young adult animals were allowed to lay eggs at restrictive temperature and were removed after 2-3 hr. Transgenic progeny developing into L4 larvae or young adults after 48 hr at 25°C were scored.
(F) Confirmation of tissue-specific expression: GFP and hsp90 mRNA levels in the intestines of HSP90bwm, HSP90int and HSP90neuro animals, relative to wild-type. While animals expressing intestine-specific HSP90::GFP also harbor increased levels of GFP and endogenous hsp90 in their intestines, animals expressing neuron- or bodywall muscle-specific HSP90::GFP do not show increased GFP expression in the intestine. The increased endogenous hsp90 mRNA levels in the intestines of HSP90bwm and HSP90neuro confirm that increased tissue-specific HSP90 expression results in the upregulation of endogenous hsp90 in other tissues (see Figure 5).
Bar graphs represent combined mean values of three independent experiments. Error bars represent ±SEM.
Cell  , DOI: ( /j.cell )
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12. Figure S3
HSP90 Reduces HSF-1 DNA Binding Activity in a Cell-Nonautonomous Manner, Related to Figure 3
(A) EMSA of HSF-1 – HSE DNA binding activity following a HS. Lane 1 free dsDNA probe; lane 2 control nuclear protein extract from wild-type (NPE); lane 3 NPE from wild-type animals treated with a 4 hr 35°C HS. Lane 5 and 6: NPE from control and heat shocked HSP90bwm animals; Lane 8 and 9: NPE from control and heat shocked HSP90neuro animals. Lanes 4, 7 and 10: NPE from HS wild-type, HSP90bwm and HSP90neuro respectively, with equimolar amounts of cold and radioactively labeled HSE. Lanes 11-13: NPE from HS wild-type, HSP90bwm and HSP90neuro with labeled mutated HSE, respectively. Data shown are representative of replicate assays.
(B) Quantification of HSF-1 dsDNA binding activity in HSP90bwm and HSP90neuro relative to wild-type HSF-1 binding activity during HS. Values represent means and error bars represent ±SEM.
(C) Levels of hsf-1 mRNA expression in HSP90bwm or HSP90neuro at 20°C, relative to wild-type.
(D) hsp90 and GFP RNAi prior to HS rescues hsp70 induction in most tissues. Expression of the hsp70p::mCherry reporter 7 hr after HS in (i) HSP90bwm control or HSP90bwm fed with (ii) hsp90 dsRNA or (iii) GFP dsRNAi 24 hr prior to HS. (iv) Heatshocked HSP90int control, (v) HSP90int, hsp90 RNAi and (vi) HSP90int, GFP RNAi. (vii) Heatshocked HSP90neuro control and (viii) HSP90neuro,hsp90 RNAi and (ix) HSP90neuro, GFP RNAi.
(E) Quantification of hsp70p::mCherry fluorescence in the overexpression lines after HS on control or hsp90 or GFP RNAi relative to wild-type. 3 biological replicates, N = 15. Error bars represent ±SEM.
(F) hsp70 (C12C8.1) mRNA levels in intestinal cells of HSP90bwm, HSP90int and HSP90neuro animals that lack the hsp70p::mCherry reporter transgene relative to wild-type, isolated after HS.
(G) Overexpressed HSP90::mCherry is not taken up by coelomocytes. (i, iv, vii, x) Confocal images of GFP::RAB-5 expression in the coelomocyte of wild-type, HSP90bwm, HSP90int and HSP90neuro animals, respectively. (ii, v, viii, xi) HSP90::mCherry expression is not detected in the coelomocyte. (iii, vi, ix, xii) Merge of GFP and mCherry expression in the coeolomocyte. Scale bar, 20 μm.
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13. Figure S4
Tissue-Specific Knockdown of hsp90 Leads to Developmental Delays and Aberrant Phenotypes, Related to Figure 4
(A–F) Confirmation of tissue-specific hsp90 knockdown. (A) Expression of HSP90::mCherry in the bodywall muscle of HSP90bwm,sid-1 control animals alone or in the presence of hp-hsp90neuro, hp-hsp90bwm or hp-hsp90int. HSP90::mCherry fluorescence is decreased 2-fold in animals expressing the muscle-specific hsp90 hairpin RNAi, but not in hp-hsp90neuro or hp-hsp90int. (B) Quantification of HSP90::mCherry fluorescence in HSP90bwm,sid-1 animals expressing the different tissue-specific hp-hsp90 RNAi constructs relative to control animals (HSP90bwm,sid-1). Error bars represent ±SEM. (C) Expression of HSP90::mCherry in the intestine of HSP90int,sid-1 animals (control) alone or in the presence of tissue-specific hp-hsp90 RNAi. (D) Quantification of HSP90::mCherry fluorescence in the presence of hp-hsp90neuro, hp-hsp90bwm or hp-hsp90int relative to HSP90int,sid-1 control animals. Expression of the intestine-specific hp-hsp90 RNAi leads to a 5-fold reduction of HSP90::mCherry fluorescence in the intestine, while expression of neuron- or bodywall muscle-specific hairpin constructs have no effect. Error bars represent ±SEM. (E) HSP90::mCherry expression in the neurons of HSP90neuro,sid-1 control animals compared to HSP90neuro,sid-1 animals expressing an hp-hsp90 RNAi construct in the neurons, the intestine or the bodywall muscle. (F) Quantification of mCherry fluorescence in HSP90neuro,sid-1 animals expressing the different tissue-specific hp-hsp90 RNAi constructs relative to control animals (HSP90neuro,sid-1). Expression of the neuron-specific hp-hsp90 RNAi leads to a significant reduction of HSP90::mCherry fluorescence in neurons. Error bars represent ±SEM.
(G) Analysis of tissue-specific hsp90 knockdown lines throughout development compared to wild-type animals.
(H) Compared to wild-type animals (i, iv), hp-hsp90 RNAi in the bodywall muscle leads to defective development of the (ii) excretory canal (exc phenotype), (iii) the intestine and (v) the hermaphrodite tail. (vi) Young adult animals expressing hp-hsp90 in the intestine show abnormal development of the gonad.
(I) Expression of hsp70p::mCherry in wild-type animals after RNAi-mediated hsp90 knockdown. Animals were fed with hsp90 RNAi after L2 stage to avoid developmental affects. Systemic knockdown of hsp90 leads to induction of hsp70 in the pharynx (ph), the bodywall muscle (bwm), the vulval muscle (vm) and spermatheca (sp).
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14. Figure S5
hsp90 Expression in unc-31 and unc-13 Mutants, Related to Figure 5
DIC Nomarski images of (i) wild-type, (v) HSP90bwm, (ix) HSP90int and (xiii) HSP90neuro animals expressing the hsp90pr::GFP reporter. (ii, vi, x, and xiv) Expression of the hsp90p::GFP reporter in control animals, (iii, vii, xi, and xv) crossed into the unc-31 deletion background or (iv, viii, xii, xvi) crossed into the unc-13 deletion background. Scale bar, 50 μm.
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15. Figure S6
Transcellular Chaperone Signaling in the Tissue-Specific HSP90 Overexpression and Knockdown Lines Is PHA-4 Dependent, Related to Figure 6
(A) Expression of the hsp90p::GFP reporter in control and HSP90bwm, HSP90int and HSP90neuro fed with either control (empty vector; EV) or pha-4 RNAi.
(B and C) Model for the consequences of cell-nonautonomous regulation of HSP90 expression. (B) A tissue-specific imbalance of proteostasis through the expression of a ts allele of the HSP90 client myosin leads to upregulation of HSP90 expression via PHA-4 activity in muscle cells but also in nonmuscle tissue. This upregulation of HSP90 expression at the organismal level is beneficial for survival of myosin (ts) animals at the restrictive temperature. (C) A tissue-specific imbalance of proteostasis through local overexpression of HSP90 leads to a coordinated increase of HSP90 expression in multiple tissues regulated by PHA-4. Because the rise in organismal HSP90 expression is higher than when compared to myosin (ts) mutants, the increased organismal pool of HSP90 represses HSF-1 transcriptional activity and cell-nonautonomously inhibits the HSR. This leads to detrimental consequences for animal survival under heat stress conditions.
(D) Requirement of pha-4 in the tissue-specific hsp90 knockdown lines. (i-iii) Expression of the hsp70p::mCherry reporter in sid-1 animals harboring wild-type PHA-4 and expressing the tissue-specific hp-hsp90 RNAi constructs (hp-hsp90bwm, hp-hsp90int, hp-hsp90neuro) at 15°C (control). (iv-vi) pha-4(zu225);smg-1(cc546ts) double mutant worms containing the sid-1 mutation and expressing the tissue-specific hp-hsp90 RNAi constructs (hp-hsp90bwm, hp-hsp90int, hp-hsp90neuro) in addition to the hsp-70p::mCherry reporter were first grown at 25°C to inactivate smg-1 and allow the production of functional pha-4 until the end of the first larval stage (L1). pha-4 was inactivated by shifting animals to 15°C, which restores smg-1 function and results in degradation of the pha-4(zu225) allele. Images were taken at equal exposures times on the first day of adulthood.
(E) Quantification of hsp70p::mCherry fluorescence in the hp-hsp90 lines containing the pha-4 mutation (pha-4(zu225);smg-1) compared to control animals expressing wild-type pha-4. N = 15. Error bars represent ±SEM.
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